Fluorolog®-3 - Rose

Fluorolog®-3 - Rose
Fluorolog-3 v. 2.2 (10 Sep 2002)
Fluorolog®-3
Operation Manual
Rev. 2.2
July 2002
http://www.isainc.com
In the USA:
Jobin Yvon Inc.
3880 Park Avenue, Edison, NJ 08820
Tel: 1-732-494-8660
Fax: 1-732-549-5157
E-Mail: fluorescence@jyhoriba.com
1-800-533-5946
In France:
16-18, rue du Canal
91165 Longjumeau cedex
Tel: (33) 1/64.54.13.00
Fax: (33) 1/69.09.93.19
Japan: (81) 3/5823.0140
China: (86) 10/6836.6542
Germany: (49) 89/46.23.17-0
Italy: (39) 2/57.60.47.62
U.K.: (44) 208/2048142
Fluorolog-3 v. 2.2 (11 Jul 2002)
Copyright © 2002 by Jobin Yvon Inc.
All rights reserved. No part of this work may be
reproduced, stored, in a retrieval system, or
transmitted in any form by any means, including
electronic or mechanical, photocopying and
recording, without prior written permission from
Jobin Yvon Inc. Requests for permision should be
requested in writing.
Information in this manual is subject to change
without notice, and does not represent a commitment
on the part of the vendor.
July 2002
Part Number 81014
ii
Fluorolog-3 v. 2.2 (11 Jul 2002)
Table of Contents
0: Introduction ................................................................................................ 0-1
About the Fluorolog®-3............................................................................................................................ 0-1
Chapter overview .................................................................................................................................... 0-2
Symbols used in this manual .................................................................................................................. 0-4
1: Requirements & Installation ............................................................................ 1-1
Surface requirements ............................................................................................................................. 1-1
Environmental requirements................................................................................................................... 1-2
Electrical requirements ........................................................................................................................... 1-3
Installation............................................................................................................................................... 1-4
2: System Description ....................................................................................... 2-1
Overview ................................................................................................................................................. 2-1
Configurations......................................................................................................................................... 2-3
3: System Operation ......................................................................................... 3-1
Turning on the system ............................................................................................................................ 3-1
Checking system performance ............................................................................................................... 3-3
Notes on excitation and emission recalibration .................................................................................... 3-19
Useful materials for characterizing system and samples ..................................................................... 3-20
4: Optimizing Data Acquisition............................................................................. 4-1
Cuvette preparation ................................................................................................................................ 4-1
Sample preparation ................................................................................................................................ 4-2
Data collection techniques...................................................................................................................... 4-3
Correcting data ..................................................................................................................................... 4-18
5: System Maintenance ..................................................................................... 5-1
Lamp ....................................................................................................................................................... 5-1
Emission signal detector....................................................................................................................... 5-14
Reference signal detector..................................................................................................................... 5-15
Gratings ................................................................................................................................................ 5-16
Mirrors................................................................................................................................................... 5-20
Automated 4-position turret .................................................................................................................. 5-21
6: Components & Accessories ............................................................................. 6-1
Itemized list ............................................................................................................................................. 6-2
Model 1940 absorption/transmission accessory..................................................................................... 6-3
FL-1013 Liquid nitrogen Dewar assembly .............................................................................................. 6-6
Model 1908MOD scatter block assembly ............................................................................................... 6-7
Model 1908 standard lamp assembly..................................................................................................... 6-7
Sample cells............................................................................................................................................ 6-8
Model 1967 Photodiode reference detector ........................................................................................... 6-9
CCD detectors ...................................................................................................................................... 6-10
Model 1911F room temperature signal detector...................................................................................6-11
Model 1914F thermoelectrically cooled signal detector........................................................................ 6-12
FL-1030 thermoelectrically cooled near-IR photomultiplier tube ..........................................................6-13
F-3000 fiber optic mount....................................................................................................................... 6-14
Model 1938 cut-on filter ........................................................................................................................ 6-15
Model 1939 cut-on filter ........................................................................................................................ 6-15
Fl-1001 Front-face viewing option ........................................................................................................ 6-16
Gratings ................................................................................................................................................ 6-17
FL-1010 cut-on filter holder................................................................................................................... 6-18
FL-1011 four-position thermostatted cell holder ................................................................................... 6-19
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Fluorolog-3 v. 2.2 (11 Jul 2002)
FL-1012 dual-position thermostatted cell holder .................................................................................. 6-21
Model 1933 solid sample holder........................................................................................................... 6-23
FL-1039 xenon lamp housing ............................................................................................................... 6-25
FL-1040 dual lamp housing .................................................................................................................. 6-25
F-3005/6 autotitration injector............................................................................................................... 6-26
Models F-3001, F-3002, and F-3003 microscope interfaces ............................................................... 6-27
Model 1907 450-W xenon lamp............................................................................................................ 6-28
F-3004 sample heater/cooler Peltier thermocouple drive..................................................................... 6-29
Phosphorimeter accessory ................................................................................................................... 6-30
MicroMax microwell plate reader.......................................................................................................... 6-31
FL-1044 L-format polarizer & FL-1045 T-format polarizer.................................................................... 6-32
FL-1015 injector port............................................................................................................................. 6-33
F-1000/1 temperature bath................................................................................................................... 6-34
Model TRIG-15/25 external trigger accessory...................................................................................... 6-35
7: Troubleshooting........................................................................................... 7-1
Using diagnostic spectra ........................................................................................................................ 7-3
Further assistance…............................................................................................................................... 7-8
8: Introduction to lifetime measurements ............................................................... 8-1
Introduction ............................................................................................................................................. 8-1
Lifetime measurements .......................................................................................................................... 8-2
Types of lifetime scans ........................................................................................................................... 8-4
9: Xenon Lamp Information & Record of Use Form..................................................... 9-1
Xenon lamp record of use ...................................................................................................................... 9-3
10: Applications.............................................................................................10-1
Introduction ........................................................................................................................................... 10-1
Detecting sub-picomolar concentrations of fluorescein........................................................................ 10-3
Reduced-volume samples .................................................................................................................... 10-3
Fluorescence detection of highly scattering samples........................................................................... 10-4
Quantum-yield calculations .................................................................................................................. 10-4
Characterizing complex mixtures via synchronous scanning............................................................... 10-4
Operating in the IR region .................................................................................................................... 10-5
Phosphorescence for time-resolved data............................................................................................. 10-5
Low-temperature scans ........................................................................................................................ 10-6
Monitoring kinetic reactions using time-based fluorescence................................................................ 10-6
Front-face detection to enhance data collection for absorbent or solid samples ................................. 10-6
Polarization to detect trace quantities of biological probes .................................................................. 10-7
11: Producing Correction Factors ........................................................................11-1
Introduction ........................................................................................................................................... 11-1
Generating emission correction factors ................................................................................................ 11-2
Calculating emission correction factors ................................................................................................ 11-8
Calculating excitation correction factors ............................................................................................. 11-15
12: Determining the Plateau Voltage ....................................................................12-1
Introduction ........................................................................................................................................... 12-1
Overview of the procedure ................................................................................................................... 12-1
Procedure ............................................................................................................................................. 12-3
13: Reassemby Instructions...............................................................................13-1
Computer .............................................................................................................................................. 13-1
Spectrofluorometer assembly............................................................................................................... 13-2
Cable connections ................................................................................................................................ 13-3
Connecting power cables ..................................................................................................................... 13-7
14: Using TRIAX with the Fluorolog®-3..................................................................14-1
Introduction ........................................................................................................................................... 14-1
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Fluorolog-3 v. 2.2 (11 Jul 2002)
Hardware .............................................................................................................................................. 14-2
Software................................................................................................................................................ 14-4
Correcting data with the TRIAX .......................................................................................................... 14-35
TRIAX 320 Specifications ................................................................................................................... 14-38
Troubleshooting .................................................................................................................................. 14-39
15: Technical Specifications ............................................................................. 15-1
Spectrofluorometer system................................................................................................................... 15-2
Minimum computer requirements ......................................................................................................... 15-4
Software................................................................................................................................................ 15-4
16: Glossary ................................................................................................ 16-1
17: Bibliography............................................................................................ 17-1
18: Index..................................................................................................... 18-1
v
Fluorolog-3 v. 2.2 (11 Jul 2002)
Introduction
0: Introduction
About the Fluorolog®-3
The main parts of the Fluorolog®-3 spectrofluorometer system are:
• State-of-the-art optical components
• A personal computer
• DataMax for Windows™, the driving software.
This manual explains how to operate and maintain a Fluorolog®-3 spectrofluorometer.
The manual also describes measurements and tests essential to obtain accurate data. For
a complete discussion of the almost limitless power provided by DataMax, refer to the
DataMax Data Collection Handbook (contains data-acquisition information) and the
Grams/32® User’s Guide (contains post-processing instructions for data manipulation)
which accompany the system.
The combination of time-tested, performance-proven hardware with the powerful dataacquisition and manipulation software yields a system suitable for a wide variety of
applications. Equipped with expansion ports and slots, the Fluorolog®-3 can grow to
meet the changing needs of the user, it will provide years of dedicated service, and can
be updated easily to the Fluorolog®-Tau-3.
Note: Keep this and the
other reference manuals
near the system.
0-1
Fluorolog-3 v. 2.2 (11 Jul 2002)
Introduction
Chapter overview
1: Requirements & Installation
Power and environmental requirements;
select the best spot for the instrument.
2: System Description
Various Fluorolog®-3 configurations; their
features and benefits.
3: System Operation
Operation of the spectrofluorometer system,
and calibration instructions.
4: Optimizing Data Acquisition
Hints for improving the signal-to-noise ratio,
instructions for obtaining corrected data, and
other information useful for optimizing data
and ensuring reproducibility.
5: System Maintenance
Routine maintenance procedures such as
replacing the lamp.
6: Components & Accessories
Description and application of the
accessories available for the Fluorolog®-3.
7: Troubleshooting
Potential sources of problems, their most
probable causes, and possible solutions.
8: Introduction to Lifetime Measurements
Methods of determining the lifetime of a
sample using the Fluorolog®-Tau-3. The
Fluorolog®-Tau-3 is designed specifically for
phosphorescence applications, and does not
affect steady-state measurements.
9: Xenon Lamp Information & Record of
Use Form
Information about the xenon lamp, and a
form for recording the xenon-lamp usage.
10: Applications
Some interesting uses for the Fluorolog®-3.
11: Producing Correction Factors
How to correct for variation in sensitivity
across the spectral range.
12: Determining the Plateau Voltage
Finding the optimum voltage for the
detector.
13: Reassembly Instructions
How to reassemble the Fluorolog®-3 after it
has been moved.
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Fluorolog-3 v. 2.2 (11 Jul 2002)
Introduction
14: Using TRIAX with the Fluorolog®-3
Special instructions on using a TRIAX
imaging spectrometer with the Fluorolog®-3
system, including with a CCD detector.
15: Technical Specifications
Instrument specifications and computer
requirements.
16: Glossary
A list of some useful technical terms related
to fluorescence spectroscopy.
17: Bibliography
Important sources of information.
18: Index
0-3
Fluorolog-3 v. 2.2 (11 Jul 2002)
Introduction
Symbols used in this manual
Certain symbols are used throughout the text for special conditions when operating the
instruments:
Warning:
Note:
A hazardous condition exists, or danger exists that could damage
the equipment. Jobin Yvon Inc. is not responsible for damage
arising out of improper use of the equipment.
General information is given concerning operation of the
equipment.
0-4
Fluorolog-3 v. 2.2 (11 Jul 2002)
Requirements & Installation
1: Requirements & Installation
Surface requirements
•
•
A sturdy table- or bench-top.
Table size varies according to the
system configuration; an average size
of 38" × 60" (96.5 cm × 152.4 cm) is
usually sufficient.
Warning: Do not split the
system between two tables.
Using two tables can cause
instability, resulting in service
requirements or erroneous
data.
Table size for standard systems*
FL3-11
FL3-12
FL3-21
FL3-22
Length
144 cm
156 cm
173 cm
173 cm
Width
37 cm
53 cm
66 cm
66 cm
Height
43 cm
43 cm
43 cm
43 cm
* Custom configurations are available. See the System Description chapter.
1-1
Fluorolog-3 v. 2.2 (11 Jul 2002)
Requirements & Installation
Environmental requirements
•
•
•
Temperature 72 ± 5°F (22 ± 3°C)
Humidity level ~70%
No special ventilation.
Note: The standard xenon lamp
provided with the Fluorolog®-3 is
ozone-free. The lamp housing
contains an electrically powered
fan that removes the heat.
1-2
Fluorolog-3 v. 2.2 (11 Jul 2002)
Requirements & Installation
Electrical requirements
•
•
115 V, 20 A or 220 V, 20 A; factory-set.
As an extra measure of
caution, plug the xenon lamp
into a circuit separate from
the other components. This
guarantees that the electrical
surge from the lamp never
will interfere with the
computer or system.
Note: For the computer, Jobin
Yvon Inc. recommends using a
surge suppressor or an
uninterruptible power supply
(UPS) with a surge suppressor.
Make sure enough AC outlets are available for the
• Computer
• Printer (optional)
• Monitor
• Xenon lamp
• System controller (SAC)
Use three-prong plugs for proper grounding of the
system. If a two-prong adapter is used, for the
safety of the operator and to preserve the integrity
of the system, the adapter must be attached to the
wall outlet properly, according to the
manufacturer’s instructions. This provides a
positive connection to the electrical ground (earth),
ensuring that any stray or leakage current is
directed to earth ground.
1-3
Warning: A threeprong-to-twoprong adapter is
not recommended.
Fluorolog-3 v. 2.2 (11 Jul 2002)
Requirements & Installation
Installation
Warning: Customer installation
is not recommended. Special
tools and several critical
alignment verification
procedures are required.
Schedule the initial installation of a Fluorolog®-3 by calling the Spex® Fluorescence
Service Department at (732) 494-8660 × 160. Customers outside the United States
should contact a local representative. For up-to-the-minute information about products,
services, upgrades, frequently-asked questions, etc., visit our web site:
http://www.jyhoriba.com/fluor/fluor.htm
Subsequent assembly because of relocation either can be performed by a Jobin Yvon
Inc. engineer for a specified fee, or by the user. Re-assembly instructions and diagrams
are provided in Chapter 13: Reassembly Instructions.
1-4
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
2: System Description
Overview
General operation
All Fluorolog®-3 spectrofluorometers have common features:
a
b
c
d
e
f
A source of
radiation
produces
photons.
The beam of
light is
filtered by an
excitation
spectrometer
that allows a
single
wavelength
of light to
reach the
sample.
Controller
In the sample
compartment, the sample responds to the incoming radiation.
The resulting radiation is filtered by an emission spectrometer that feeds the
signal to a photomultiplier detector.
By stepping either or both spectrometers through a wavelength region, and
recording the variation in intensity as a function of wavelength, a spectrum
is produced.
The spectrofluorometer components (spectrometers, sample-compartment
module, accessories) are connected to a controller which, in turn, transfers
information to and from the computer. The computer may be attached to a
printer or plotter.
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Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
Basic components
Spectrometers
The Fluorolog®-3 comes equipped with either a single- or double-grating spectrometer in
the excitation and emission positions. Double-grating spectrometers offer a significant
increase in sensitivity, resolution and stray-light rejection.
Sample compartment
The standard sample-compartment module is a T-box, which provides efficient throughput
with a choice of standard right-angle emission collection or optional front-face emission
collection. The sample-compartment module comes equipped with a silicon photodiode
reference detector to monitor and compensate for variations in the xenon lamp output.
Accessories
Fluorolog®-3 spectrofluorometers offer sampling accessories to increase flexibility, and
extend their applications to techniques such as polarization measurements or
phosphorescence lifetimes.
2-2
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
Configurations
The different configurations and various accessories available for the Fluorolog®-3 system
allow you to customize a system specific for today’s needs, while the interchangeability of
the components and the inherent design enable the system to grow and change as new
applications arise.
Standard systems
The standard Fluorolog®-3 systems include a single- or double-grating monochromator in
the excitation and emission paths in an “L” configuration.
Source
Detector
Emission
spectrometer
Excitation
spectrometer
Sample compartment
module
Standard systems available:
Model
Source
Excitation
Spectrometer
Sample
Compartment
Module
Emission
Spectrometer
Detector
FL3-11
450-W Xe
Single
T-Box
Single
PMT
FL3-12
450-W Xe
Single
T-Box
Double
PMT
FL3-21
450-W Xe
Double
T-Box
Single
PMT
FL3-22
450-W Xe
Double
T-Box
Double
PMT
2-3
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
Fluorolog®-3 Model FL3-11
The Fluorolog®-3 Model FL3-11 is an economical system designed for routine fluorescence
measurements.
Source
Single-grating
Excitation Spectrometer
PMT
T-box Sample
Compartment Module
Single-grating
Emission Spectrometer
The standard model FL3-11 comes equipped with:
• 450-W light source
• single-grating excitation spectrometer
• single-grating emission spectrometer
• automatic slits
• room-temperature R928P detector
2-4
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
Fluorolog®-3 Model FL3-12
The Fluorolog®-3 model FL3-12 provides optimum performance for highly scattering
samples such as proteins, membranes, and solid samples.
Source
Single-grating
Excitation Spectrometer
PMT
Double-grating
Emission Spectrometer
T-box Sample
Compartment Module
Like the Model FL3-11, the Fluorolog®-3 Model FL3-12 has a single-grating excitation
spectrometer; but the optimum performance of the Model FL3-12 is as a result of a doublegrating emission spectrometer. Features of the Model FL3-12 are:
• 450-W light source
• single-grating excitation spectrometer
• double-grating emission spectrometer
• automatic slits
• room-temperature R928P detector
2-5
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
Fluorolog®-3 Model FL3-21
The Fluorolog®-3 model FL3-21 includes a double-grating spectrometer at the excitation
position.
Source
Double-grating
Excitation Spectrometer
PMT
T-box Sample
Compartment Module
Features of the Model FL3-21 are:
• 450-W light source
• double-grating excitation spectrometer
• single-grating emission spectrometer
• automatic slits
• room-temperature R928P detector
2-6
Single-grating
Emission Spectrometer
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
Fluorolog®-3 Model FL3-22
Because of the double-grating excitation and emission spectrometers, the Fluorolog®-3
model FL3-22 offers unsurpassed performance in resolution, sensitivity, and stray-light
rejection. This system is perfect for highly scattering samples like lipids and proteins, or
solids like powders, semiconductors, or phosphors.
Source
Double-grating
Excitation Spectrometer
PMT
T-box Sample
Compartment Module
Double-grating
Emission Spectrometer
Incorporating double-grating spectrometers in both the excitation and emission positions
places this model in a category by itself. Enhanced features, modular structure, and few
external controls are just a few of the reasons to consider a Fluorolog®-3 model FL3-22:
• 450-W light source
• double-grating excitation spectrometer
• double-grating emission spectrometer
• automatic slits
• room-temperature R928P detector
2-7
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
Standard options
The previously described systems represent the standard configurations. Each system,
however, can be customized by selecting different components. Available options are listed
below. For additional information, or for a list of the most recently developed products,
contact a Spex® Fluorescence Sales Representative.
Sources
•
•
•
Pulsed lamp
HgXe
Your laser
Detectors
•
•
•
Cooled PMT
IR
CCD
Refer to Chapter 6, Components and Accessories, for a list of accessories that will help you
further tailor a system to your application needs.
2-8
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
Custom configurations
With custom configurations, you can change the layout of the system to a T-format or add
the high-performance TRIAX 320M imaging spectrograph at the emission port of the
system. Using single-grating, double-grating, or the TRIAX 320M imaging spectrometer,
you can create a system for almost any application. Details on using a TRIAX are given in
Chapter 14: TRIAX Operation with the Fluorolog®-3. Some of the more popular
configurations are described on the following pages.
2-9
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
Fluorolog®-3 Model FL3-XXX
Systems with the T-configuration design were developed for T-format polarization or
anisotropy and dual-emission spectroscopy. Models FL3-XXX (where the Xs are the type of
spectrometer positioned at the excitation, first-emission, and second-emission positions,
respectively) are available in numerous configurations.
Dectector
Detector
Source
Excitation
Spectrometer
Emission
Spectrometer
Sample
Compartment
Module
Dectector
Emission
Spectrometer
Detector
Fluorolog®-3 model FL3-122
The T-configuration allows the Xe source and the signal detector to be positioned at rightangles to the sample, for a variety of experiments with results unequaled by other
configurations. The optical configuration of the model FL3-122 is shown below.
Source
T-box Sample
Compartment Module
Single-grating
Excitation Spectrometer
PMT
PMT
Double-grating
Emission Spectrometer
Double-grating
Emission Spectrometer
Optical layout of the Fluorolog®-3 Model FL3-122.
2-10
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
Fluorolog®-3 Model FL3-12-320M
The fully automated TRIAX 320M imaging spectrometer can be a part of a custom
Fluorolog®-3 configuration. The imaging spectrograph offers the latest advances in optical
design and automation.
Source
Excitation
Spectrometer
270M
320
M
Imaging
Spectrometer
Sample
Compartment
Module
Emission
Spectrometer
Fluorolog®-3 with a double-grating emission spectrometer and a TRIAX 320M
imaging spectrometer
The optical configuration of this system is shown below.
Source
T-box Sample
Compartment Module
Single-grating
Excitation Spectrometer
CCD
PMT
Double-grating
Emission Spectrometer
270M Imaging
320
Spectrometer
(Emission)
Custom optical layout. By switching the TRIAX 320M and double-grating
spectrometer, both right-angle and front-face collection are possible.
The unique optical layout of the TRIAX 320M eliminates rediffracted light. In addition,
with the single-grating excitation spectrometer in place, the TRIAX 320M imaging
spectrograph can be placed on either side of the sample-compartment module. When the
TRIAX 320M imaging spectrometer is used as the first emission spectrometer (not
pictured), both front-face and right-angle collection are possible.
2-11
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Description
Custom options
Each system can be customized further by selecting different options. Available options are
listed below. For additional information, or for a list of the newest options, contact a Spex®
Fluorescence Product Specialist.
Sources
•
•
•
Pulsed lamp
HgXe
Your laser
Detectors
•
•
•
Cooled PMT
IR
CCD
Refer to Chapter 6, Components and Accessories, to see accessories that will help you
further tailor a system to your needs.
2-12
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
3: System Operation
Turning on the system
1
Start the lamp.
The lamp must be turned on
prior to the Fluorolog®-3,
accessories, and peripheral
equipment.
a
b
On the back of the lamp
housing, turn on the switch
marked “power.”
Warning: When the lamp is
turned on, a large voltage is put
across the lamp, during which a
spike can feed back down the
electrical line. This spike can
cause damage to computer
equipment if the equipment is
operating at the time and on the
same power circuit as the
Fluorolog®-3, when the lamp is
started.
Just above the “power” switch, turn on the “main lamp” switch.
2
Start the fluorometer accessories.
3
Start the SpectrAcq.
Turn on any automated accessories (e.g., Temperature Bath, MicroMax,
etc.) used with the Fluorolog®-3.
a
b
Make sure the boot disk is in the floppy
drive.
Push in the power button to start.
Immediately below the power button, the
green LED indicator lamp should turn on.
Note: The boot disk should access
for ~ 60 s to load system drivers.
3-1
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
4
Start the peripheral devices.
5
Start the computer and software.
Turn on all peripheral devices such as printers and plotter (i.e., all devices
other than the computer).
a
b
c
Turn on the computer.
In Windows™, click on the DataMax icon to start Instrument Control
Center. The Layout Selection window appears.
In the Layout Selection window, select
the layout to use.
The Fluorolog®-3 spends ~ 1 min
initializing, then the Instrument Control
Center appears:
d
Note: The layout filename
will vary, depending on the
instrument configuration.
Choose one of the four available applications:
Run Experiment
Real Time Display
Visual Instrument Setup
Constant Wavelength Analysis
If there are difficulties in starting the software or initializing the Fluorolog®-3, please refer to
Chapter 7: Troubleshooting for suggestions.
3-2
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
Checking system performance
Introduction
Upon installation and as part of routine maintenance checks, examine the performance of
the Fluorolog®-3. Jobin Yvon Inc. recommends checking the system calibration before each
day of use with the system. Scans of the xenon-lamp output and the Raman-scatter band of
water are sufficient to verify system calibration, repeatability, and throughput.
• Calibration is the procedure whereby the drive of each spectrometer is reference to
a known spectral feature.
• Repeatability is the ability of the system to produce consistent spectra.
• Throughput is the amount of signal passing through and detected by the system.
The throughput is correlated to the signal-to-noise ratio and sensitivity of the
system.
The Fluorolog®-3 is an autocalibrating spectrofluorometer. This means the system initializes its monchromator’s drives, locates the home position of the each drive, and assigns a
wavelength value to this position from a calibration file. While the system usually maintains
calibration by this method, it is wise to check the calibration prior to the day’s session with
the instrument. For the calibration checks detailed here, a single-sample mount or automated
sample changer should be the only sample-compartment accessories used.
The scans shown herein are examples. A Performance Test Report for your new instrument
is included with the documentation. Use the Performance Test Report to validate the spectral shape and relative intensity taken during the calibration checks.
These scans are described for systems with the default 1200-grooves/mm gratings and an
R928P PMT emission detector with coverage from the UV to high visible. If the monochromators contain gratings with groove densities other than 1200 grooves/mm, with different wavelength sensitivity, or with a different emission detector, please consult the Performance Test Report for appropriate scan parameters.
3-3
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
Excitation calibration check
This calibration check verifies the wavelength calibration of your excitation monochromator, using the reference photodiode located before the sample compartment. It is an excitation scan of the xenon lamp’s output, and should be the first check performed.
1
2
Secure the lid of the sample chamber in place.
On the Run Experiment toolbar, select the
Experiment button.
This opens the Emission Acquisition dialog box:
3
Click on the Exp
Type… button.
This opens the
Experiment Type
box:
4
Select
dialog
Choose Excitation
Acquisition.
Click OK to close the
Select Experiment Type dialog box. The Emission Acquisition window
3-4
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
converts to Excitation Acquistion. (For calibration and calibration
verification, always adjust the excitation spectrometer first.)
5
Set the scan parameters for the xenon lamp
scan:
a
In the Excitation Acquisition dialog box:
Name the experimental parameters lamp.exp,
Start the scan at 220 nm,
Step the wavelength by 0.5 nm,
Set the emission to 650 nm,
Perform 1 scan.
Use a filename lamp.spc,
End the scan at 600 nm,
Use an Integration Time of 0.1 s.
Describe the file as the spectral profile of the xenon lamp.
3-5
Fluorolog-3 v. 2.2 (11 Jul 2002)
b
System Operation
Click on Signals...
This opens the Signals
dialog box.
Enter R (reference), then
click OK to close the box.
c
Click on Slits...
This opens the Slits
dialog box.
Set
the
excitation
entrance and exit slits
to 0.5 mm.
Set
the
emission
entrance and exit slits
to 0 mm (closed).
Click OK to close the
window.
If the slits’ units are not
in millimeters (mm), then open Visual Instrument Setup, select Options,
then Units, and set the slits’ units to mm. Use the reference (R) detector
channel.
d
Click Run to execute the scan.
3-6
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
Lamp scan for single-monochromator systems:
structure ~ 450 nm
calibration peak
at 467 nm
broadband
Xenon-lamp scan for an FL3-11 spectrofluorometer.
Lamp scan for double-monochromator systems:
On a double-monochromator system, the 467-nm line is no longer the most intense peak
within the spectrum. Take care to isolate the 467-nm calibration line from other peaks.
Xenon-lamp scan for a double-monochromator spectrofluorometer.
3-7
Fluorolog-3 v. 2.2 (11 Jul 2002)
6
System Operation
Find the calibration peak for the xenon-lamp
spectrum.
The 467-nm peak is used for excitation calibration. The intensity of this
spectrum should be noted for reference, although it is not used for
instrument specifications.
Is the peak at
467 ± 0.5 nm?
Yes
Instrument is within
specification.
Go to emission calibration check.
No
Instrument is not within
specification.
Recalibrate excitation
monochromator.
3-8
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
Excitation monochromator recalibration
Re-calibration of the Fluorolog®-3 is performed by moving to the position of the observed
peak, going into Visual Instrument Setup, and telling the software the correct position at
which this peak should be. The software will save this change in position to the calibration
file.
1
2
3
Note the wavelength where the 467-nm peak
was observed.
Open the Real
Time Display.
Set the excitation
monochromator to
the position where
the peak was
observed.
Hit the Tab key on the
keyboard to set the
monochromator to the
entered value.
4
Close the Real
Time Display.
3-9
Fluorolog-3 v. 2.2 (11 Jul 2002)
5
System Operation
Open the Visual Instrument Setup dialog box:
Note: The Visual
Instrument Setup
will vary, depending on the instrument configuration.
6
Click on the
grating for the
excitation
monochromator.
The Grating/Turret dialog box appears.
7
Click on the
Calibrate...
button.
This opens the Enter Correct Position
dialog box:
8
9
10
11
Enter the actual xenonlamp peak, 467 nm.
Click OK.
The excitation monochromator should
now be calibrated.
Click Close to close the Grating/Turret dialog
box.
Close Visual Instrument Setup.
3-10
Fluorolog-3 v. 2.2 (11 Jul 2002)
12
System Operation
Confirm that the excitation monochromator is
calibrated by running another lamp scan in Run
Experiment.
This time the peak should occur at 467 ± 0.5 nm.
3-11
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
Emission calibration check
Note: The emission calibration of the instrument is directly affected by the
calibration of the excitation monochromator.
This calibration check verifies the wavelength calibration of the emission monochromator
with the emission photomultiplier tube. It is an emission scan of the Raman-scatter band of
water performed in right-angle mode. This check should be performed after the xenon-lamp
scan. When completed, the performance of the system has been verified.
The water sample should be research-quality, triple-distilled or de-ionized water. HPLCgrade (18-MΩ spec.) or equivalent water is suggested for the Raman scan. Impure samples
of water will cause elevated background levels as well as distorted spectra with (perhaps)
some unwelcome peaks.
Use a 4-mL quartz cuvette.
1
Note: Avoid glass or acrylic cuvettes: they
Insert the
may exhibit UV fluorescence or filtering effects.
water
sample into the sample compartment.
With an automated sample changer, note the position number in which the
sample cell is placed.
2
Make sure the lid of the sample chamber is
securely in place.
In the Run Experiment toolbar, choose the
Experiment button.
3
This opens the Emission Acquisition dialog box.
a
Click on Exp Type… to
open the Select
Experiment Type dialog
box:
Choose
Emission
Acquisition. Click OK to
close the box.
3-12
Fluorolog-3 v. 2.2 (11 Jul 2002)
b
System Operation
In the Emission Acquisition dialog box:
Name the experimental setup water.exp,
Start the scan at 365 nm,
Step the wavelength by 0.5 nm,
Set the excitation to 350 nm,
Perform 1 scan.
Use a filename water.spc,
End the scan at 450 nm,
Use an Integration Time of 0.5 s.
Describe the file as a water Raman-emission spectrum.
c
Click on the Signals...
button to open the Signals
dialog box:
Set the signal (S) detector.
Click OK to close the box.
3-13
Fluorolog-3 v. 2.2 (11 Jul 2002)
d
System Operation
Click on the
Slits... button to
open the Slits
dialog box:
Set the slits to 5
nm. Click OK to
close the box.
If your slits’ units
are
not
in
bandpass
units
(nm), then open
Visual
Instrument Setup, select Options, then Units, and set the slits’ units to nm.
If you have a Front-Face/Right-Angle accessory, set it to the RA position.
e
Click Run to execute the scan.
Your spectrum should resemble the following:
For a new instrument,
intensity > 450 000
counts s–1.
water
Raman
peak at
397 nm
3-14
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
The Rayleigh scatter band
or excitation band, which is
about 10 times the intensity
of the Raman band, occurs
at 350 nm (the excitation
wavelength). The scan
begins at 365 in order to
avoid detecting the Rayleigh
band.
4
Note: Observed throughput (and hence peak
intensity) is affected by lamp age and alignment, slit settings, and sample purity. As the
xenon lamp ages, the throughput of the system will decline slowly. Therefore, low water
Raman peak intensity may indicate a need
to replace the xenon lamp.
Find the
peak of the water Raman band.
Yes
Is the peak at
397 ± 0.5 nm?
Instrument is within
specification.
Calibration is complete.
Yes
No
Instrument is not within
specification.
Has the excitation
monochromator
been calibrated?
Continue on to
Emission recalibration.
No
Go back to Excitation
calibration.
3-15
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
Emission recalibration
Calibration for the Fluorolog®-3 is performed by:
• Moving to the position of the observed peak,
• Going to the Visual Instrument Setup application, and
• Telling the software the correct position for this peak.
The software will save this change in position to the calibration file.
1
2
3
Note where the 397-nm peak was observed.
Open the Real Time
Display.
Reset the emission
monochromator to the
position where the peak
was observed.
Hit Tab on the keyboard to set the
monochromator to the entered
value.
4
Close the Real Time
Display.
3-16
Fluorolog-3 v. 2.2 (11 Jul 2002)
5
System Operation
Open the Visual Instrument Setup dialog box:
Note: The Visual
Instrument Setup
will vary, depending on the
instrument configuration.
6
Click on the
grating for
the
emission
monochromator.
The Grating/Turret
box appears:
7
dialog
Click on the
Calibrate... button.
This opens the Enter Correct
Position dialog box:
8
9
Enter 397 nm, the
actual peak position
using 350-nm
excitation.
Click OK to close the dialog box.
10
Click OK to close the Grating/Turret dialog box.
The emission monochromator now should be calibrated.
3-17
Fluorolog-3 v. 2.2 (11 Jul 2002)
11
12
System Operation
Close Visual Instrument Setup.
Confirm that the emission monochromator is
calibrated, by running another water Raman
scan in Run Experiment.
This time the peak should occur at 397 ± 0.5 nm.
3-18
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
Notes on excitation and emission calibration
•
•
•
•
Two experiments, lamp.exp and water.exp, have been defined and saved.
They can be run, after the system is switched on each day, to check the calibration
and performance of the Fluorolog®-3.
Jobin Yvon Inc. recommends that the number of hours of xenon-lamp use be
recorded in a log (see sample sheet in Chapter 9: Xenon Lamp Information &
Record of Use Form).
Additionally, you may want to record the water-Raman intensity daily or weekly.
The lamp is rated for 1800–2000 h, but if the Raman intensity starts to drop, you
may wish to change the lamp sooner.
3-19
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Operation
Useful materials for characterizing system
and samples
The following are materials that Jobin Yvon Inc. has found useful in determining system
sensitivity or as standards for lifetime measurements.
Substance
CAS Number
Purpose
Emission
Wavelength
Range (nm)
Lifetime (ns)
Anthracene (99+%, zone-refined)
120-12-7
Excitation and emission
spectral characterization
380–480
4.1 (in MeOH)
9-CA (97%), or 9Anthracenecarbonitrile
1210-12-4
Single-exponential lifetime standard
380–500
11.8 (in
MeOH)
Europium(III) chloride hexahydrate
(99.9%)
13759-92-7
Phosphorescence emission and decay standard
580–700
1.40 × 105
Fluorescein (99%)
2321-07-5
Lifetime and sensitivity
standard
490–630
4.02 (in pH ≥
11)
D-glycogen
9005-79-2
Light-scattering standard
LDS 750, or Styryl 7
114720-33-1
Single-exponential lifetime standard
LUDOX®, or colloidal silica
7631-86-9
Light-scattering standard
(Me)2POPOP, or 1,4-bis-2-(4-methyl5-phenyloxazolyl)-benzene
3073-87-8
β-NADH (β-nicotinamide adenine
dinucleotide)
0
680–700
0.248 (in
MeOH, λexc =
568 nm)
0
Single-exponential lifetime standard
390–560
1.45 (in
EtOH)
606-68-8 or
104809-32-7
Single-exponential lifetime standard
390–600
0.38 ± 0.05 (in
pH = 7.5)
POPOP (99+%), or 1,4-bis(5phenyloxazol-2-yl)] benzene
1806-34-4
Single-exponential lifetime standard
370–540
1.32 (in
MeOH)
PPD (97%), or 2,5-diphenyl-1,3,4oxadiazole
725-12-2
Single-exponential lifetime standard
310–440
1.20 (in
EtOH)
PPO (99%), or 2,5-diphenyloxazole
92-71-7
Single-exponential lifetime standard
330–480
1.40 (in
EtOH)
Rose Bengal (90%), or 4,5,6,7tetrachloro-2′,4′,5′,7′tetraiodofluorescein
p-Terphenyl (99+%)
632-69-9
Single-exponential lifetime standard
560–680
0.98 ± 0.10
92-94-4
Single-exponential lifetime standard
310–410
1.05 (in
EtOH)
Water (18-MΩ, de-ionized, tripledistilled)
7732-18-5
water Raman sensitivity
test
3-20
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
4: Optimizing Data Acquisition
Spectra can be enhanced by optimization of data acquisition. This chapter lists some methods of optimizing sample preparation, spectrofluorometer setup, and data correction to get
higher-quality data.
Cuvette preparation
1
2
Note: Sample cells should
be cleaned thoroughly before use to minimize background contributions.
Empty all contents
from the cuvettes.
Fully immerse and
soak the cuvettes for 24 h in 50% nitric acid.
This cleans the cuvettes’ inner and outer surfaces.
Warning: Nitric acid is a dangerous substance. When using
nitric acid, wear safety goggles, face shield, and acid-resistant
gloves. Certain compounds, such as glycerol, can form explosive materials when mixed with nitric acid.
3
4
Rinse with deionized water.
Clean the
cuvettes in the
Warning: Soaking the cuvettes for a long
cleaning
period causes etching of the cuvette sursolution with a
face, which results in light-scattering
when the cuvettes are used.
test-tube
brush.
Use Alconox® or equivalent detergent as a cleaning solution.
5
6
7
Rinse the cuvettes with deionized water.
Soak the cuvettes in concentrated nitric acid.
Rinse them with deionized water before use.
4-1
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Sample preparation
The typical fluorescence or phosphorescence sample is a solution analyzed in a standard cuvette. The cuvette itself may contain materials that fluoresce. To prevent interference, Jobin
Yvon Inc. recommends using non-fluorescing fused-silica cuvettes that have been cleaned
as described above.
Small-volume samples
If only a small sample volume is available, and the intensity of the fluorescence signal is
sufficient, dilute the sample and analyze it in a 4-mL cuvette. If fluorescence is weak or if
trace elements are to be determined, Jobin Yvon Inc. recommends using a capillary cell such
as our 50-µL or 250-µL optional micro-sample capillary cells, which are specifically designed for a small volume. A 1-mL cell (5 mm × 5 mm cross-section) is also available.
Solid samples
Solid samples usually are mounted in the Model 1933 Solid Sample Holder, with the fluorescence collected from the front surface of the sample (see Components and Accessories).
The mounting method depends on the form of the sample.
• Thin films and cell monolayers on coverslips can be placed in the holder directly.
• Minerals, crystals, vitamins, paint chips, and similar samples usually are ground
into a homogeneous powder. The powder is packed into the depression of the Solid
Sample Holder. For very fine powder, or powder that resists packing (and therefore
falls out when the holder is put into its vertical position), the powder can be held in
place with a thin quartz coverslip, or blended with potassium bromide for better
cohesion.
Dissolved solids
Solid samples, such as crystals, sometimes are dissolved in a solvent and analyzed in solution. Solvents, however, may contain organic impurities that fluoresce and mask the signal
of interest. Therefore use high-quality, HPLC-grade solvents. If background fluorescence
persists, recrystallize the sample to eliminate organic impurities, and then dissolve it in an
appropriate solvent for analysis.
Biological samples
For reproducible results, some samples may require additional treatment. For example, proteins, cell membranes, and cells in solution need constant stirring to prevent settling. Other
samples are temperature-sensitive and must be heated or cooled to ensure reproducibility in
emission signals. (A thermostatted cell holder with a magnetic stirrer is available: See Components and Accessories).
4-2
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Data collection techniques
Selecting the collection method
The two basic collection methods are right-angle and front-face. In right-angle detection, the
fluorescence is collected at 90° to the incident exciting beam. Right-angle detection is used
primarily for clear solutions. Front-face detection is used for optically dense solutions such
as hemoglobin and for solid samples. In front-face detection, fluorescence is collected off
the front surface of the cuvette or the solid sample. Inner-filter and re-absorption characteristics of opaque samples preclude right-angle detection. The optional front-face accessory for
cuvettes is required for solutions. For solid samples, use the Model 1933 Solid Sample
Holder.
Determining optimal wavelengths
The optimum excitation and emission wavelengths are known for many samples. If you are
running a sample whose wavelength positions are unknown, you must determine these
wavelengths to obtain the best possible results when you run the sample.
The method used to determine the excitation and emission wavelengths consists of first running an emission scan and observing the peak emission value. Once this value has been obtained, it is necessary to conduct an excitation scan using the peak emission value determined by running an emission scan.
4-3
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Preliminary emission maximum
1
Verify that all system components are on.
Note: If you have not checked instrument performance, Jobin
Yvon Inc. recommends acquiring a lamp spectrum and a water Raman spectrum, as outlined in the previous chapter, before proceeding.
2
3
4
Place the sample in
the sample
compartment.
Enter the Real Time
Display:
Turn on the high
voltage to the signal
detector.
Note: Make sure the lid
is completely closed.
Note: The default voltages will be used if the
value is not changed.
5
Set the excitation
and emission slits to
a bandpass of 5.0
nm.
After the value is entered, hit the Tab key on the keyboard to set the slits.
6
Close the Real Time Display.
The objective is to acquire a preliminary emission scan using an
approximate excitation wavelength. Once the emission peak is determined,
this value will be used to obtain the optimal excitation spectrum.
4-4
Fluorolog-3 v. 2.2 (10 Sep 2002)
7
Optimizing Data Acquisition
On the Run Experiment toolbar, choose the
Experiment button:
This opens the Emission Acquisition dialog box:
8
Click on Exp Type….
9
Choose
Emission
Acquisition.
This opens the
Select
Experiment Type
dialog box:
Click OK to
close the Select
Experiment Type
dialog box.
4-5
Fluorolog-3 v. 2.2 (10 Sep 2002)
10
Optimizing Data Acquisition
Specify the data acquisition parameters.
If unsure, try 300 nm,
where many organic
samples absorb light.
Add 15 nm to the excitation wavelength.
Try 550 nm.
Try 2 nm for a
rapid scan.
a
Try 0.1 second for
a rapid scan.
Click on Signals... to open
the Signals dialog box:
b
Click on Slits... to
open the Slits dialog
box:
Set the slits
to 5 nm.
Select S (signal) detector.
Click OK to close this box.
4-6
Fluorolog-3 v. 2.2 (10 Sep 2002)
11
12
13
14
Optimizing Data Acquisition
Enter the data file name and experiment file
name, and any comments desired to distinguish
this experiment from others.
Click Run to execute the experiment.
With the preliminary emission spectrum on the
screen, note the greatest intensity.
Either
Record the wavelength at which this occurs. This is your emission
maximum.
Note: If the signal level exceeds 2 × 106 cps, decrease the slit
widths by 50%. If you do not see an obvious peak, increase
the excitation wavelength and the starting and ending wavelengths by 25 nm, and acquire another emission scan.
Or
Repeat this procedure until you find an emission peak.
4-7
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Excitation scan
The next step is to use the recently discovered emission maximum to determine the optimal
excitation wavelength for the sample. The procedure is very similar to that outlined above.
1
On the Run Experiment toolbar, choose the
Experiment button:
As before, this opens the Emission Acquisition dialog box.
2
Click Exp Type.
3
Choose Excitation Acquisition.
4
Specify the data acquisition parameters:
This opens the Select Experiment Type dialog box.
Click OK to close the Select Experiment Type dialog box..
Try 250 nm.
Use the emission maximum from the previous procedure minus 15 nm. That is, if the emission maximum was 450 nm, use 435 nm.
Use the emission maximum
from the previous procedure.
5
Click on the Signals... button.
This opens the Signals dialog box:
4-8
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Enter S/R (Signal/Reference).
Using S/R produces a
spectrum
corrected
for
variations in lamp intensity
with respect to time.
Click OK to close the Signals
dialog box.
6
Enter the remaining
parameters and, if
desired, the data file
name and the
experiment file
name, and any
comments desired that will distinguish this
experiment from others.
Click Run to execute the experiment.
7
The resulting spectrum displays intensity versus wavelength, and shows the
maximum excitation wavelength.
Optimized excitation and emission spectra of a 1 × 10–8-M anthracene solution are shown
below. Because the acquisition modes were different for the excitation and emission scans,
the data intensity had to be normalized. After normalization, notice that the excitation and
emission scans are virtually mirror images of one another.
Intensity (106 counts s–1)
4
3
Emission
Excitation
2
1
0
300
350
400
450
500
Wavelength (nm)
Normalized excitation and emission spectra of a 1 × 10–8-M anthracene solution.
4-9
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Measuring the G factor
Note: Measure the G facThe grating factor, or G factor, ought to be included
tor before a polarization
anytime polarization measurements are taken. The
experiment.
G factor corrects for variations in polarization
wavelength-response for the emission optics and
detectors. A pre-calculated G factor may be used when all other experimental parameters are
constant. In other cases, the system can measure the G factor automatically before an experimental run.
Constant Wavelength Analysis
Use Constant Wavelength Analysis to determine polarization at particular excitation/emission wavelength-pairs.
Use Polarization Modes checkbox activates acquisition parameters for polarizers. This brings up the Polarizer Settings
dialog box for polarization, anisotropy,
and raw polarization modes.
Proceed to Acquisitions opens the New
Polarization Sample window:
4-10
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Either enter the G factor or have it measured automatically during the scan.
Note: For weak signals, enter the G
factor, rather than measure it automatically. This may improve the S/N.
Run Experiment
For general experimental use of polarizers, use Run Experiment. Be sure that a layout including polarizers is loaded. When selecting the type of experiment, the Polarization
checkbox must be active. A typical Emission Acquisition dialog box is as follows:
Note the G Factor field that appears.
• To measure the G factor automatically, set the field to 0.
• To use a pre-determined G factor, enter it here.
4-11
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Faster scans with polarizers can be taken with Polar Scan…. Here, the G factor is entered
in the Quick Polarization window:
Note: For detailed information on the G factor, see the Polarizers
Operation Manual.
4-12
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Improving the signal-to-noise ratio
Because of various hardware or software conditions, occasionally it is necessary to optimize
the results of an experiment.
The quality of acquired data is determined largely by the signal-to-noise (S/N) ratio. This is
true especially for weakly fluorescing samples with low quantum yields. The signal-to-noise
ratio can be improved by:
• Using the appropriate integration time,
• Scanning a region several times and averaging the results,
• Changing the bandpass by adjusting the slit widths, and
• Mathematically smoothing the data.
The sections that follow discuss the alternatives for improving the S/N ratio and the advantages and disadvantages of each.
4-13
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Determining the optimum integration time
The length of time during which photons are counted and averaged for each data point is referred to as the integration time. An unwanted portion of this signal comes from noise and
dark counts (distortion inherent in the signal detector and its electronics when high voltage is
applied). By increasing the integration time, the signal is averaged longer, resulting in a better S/N ratio. This ratio is enhanced by a factor of t1/2, where t is the multiplicative increase
in integration time. For example, doubling the integration time from 1 s to 2 s increases the
S/N ratio by over 40%, as shown below:
For an integration time of 1 second,
S / N = t 1/ 2
= 11/ 2
=1
For an integration time of 2 seconds,
S / N = t 1/ 2
= 21/ 2
.
≈ 1414
or approximately 42%. Because S/N determines the noise level in a spectrum, use of the appropriate integration time is important for high-quality results.
To discover the appropriate integration time:
a
b
Find the maximum fluorescence intensity by acquiring a preliminary scan, using
an integration time of 0.1 s and a bandpass of 5 nm.
From this preliminary scan, note the maximum intensity, and select the
appropriate integration time from the table below.
Signal intensity
(counts per second)
1000 to 5000
5001 to 50 000
50 001 to 500 000
500 001 to 4 000 000
Integration time can be set either through
Run Experiment for a specific experiment,
Real Time Display to view the effects of
different integration times on a spectrum, or
Visual Instrument Setup to establish a
default for all experiments. Refer to the
software manual to learn more about setting
the integration time.
4-14
Estimated integration time
(seconds)
2.0
1.0
0.1
0.05
Note: This table is only a
guide. The optimum integration time for other
measurements, such as
time-base, polarization,
phosphorescence lifetimes,
and anisotropy, may be different.
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Scanning a sample multiple times
Scanning a sample more than once, and averaging the scans together enhances the S/N ratio.
In general, the S/N ratio improves by n1/2, where n is the number of scans.
To scan a sample multiple times, in an experiment dialog box, specify the number of scans
in the Number of Scans field in Run Experiment. Refer to the software manual for detailed
instructions regarding the data-entry fields of each type of experiment dialog box.
4-15
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Selecting the appropriate bandpass
The bandpass (wavelength spread) affects the resolution of your spectra. If the bandpass is
too broad, narrow peaks separated by a small change in wavelength may be unresolved. For
example, for two 2-nm peaks 5 nm apart, and a bandpass of 10 nm, one broad peak, instead
of two well-defined ones, will be visible.
By adjusting the slit widths, you can control the intensity and bandpass of the light. The slits
of the excitation spectrometer determine the amount of light that will pass through the excitation spectrometer to the sample. The emission spectrometer slits control the amount of
fluorescence recorded by the signal detector.
Bandpass can be calculated using the following formula:
bandpass (nm) = slit width (mm) × dispersion (nm/mm)
The dispersion of the Fluorolog®-3 spectrofluorometer system depends upon the type of
monochromator and the groove spacing of the gratings. For example, a Fluorolog®-3 with a
single-grating monochromator and 1200 groove/mm gratings has a dispersion of 4.0
nm/mm, while a Fluorolog®-3 with a double-grating monochromator and 1200 groove/mm
gratings has a dispersion value of 2.0 nm/mm. Below is a table listing various monochromators, installed gratings, and their respective dispersions.
Monochromator
Grating groove-density
(grooves/mm)
Dispersion of the monochromator
Double
1200
600
300
2.1
4.2
8.4
Single
1200
600
300
4.2
8.4
16.8
4-16
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Smoothing data
Smoothing the data improves the appearance of your spectrum. Smoothing, as are most
post-processing features, is handled by GRAMS/32®. By selecting Arithmetic from the
main menu of Run Experiment, you can choose FFT (fast-Fourier transform), Binomial, or
Savitsky-Golay smoothing. Automatic smoothing and the degree to which smoothing occurs can be achieved by entering the Peaks/Settings dialog box and typing the relevant information. Refer to the GRAMS/32® User’s Guide for additional information regarding
smoothing data.
An additional option, Zap, for removing outliers, also appears under the Arithmetic menu.
The GRAMS/32® User’s Guide covers this option thoroughly.
In general, start with a 9- or 11-point smooth for a time-base measurement. To select the
proper number of points for wavelength scan types, first locate the area that requires
smoothing—usually this is a peak. Determine the number of data points used to make up the
peak and then smooth the data using the number of points closest to this number. To avoid
artificially enhancing the data, use the appropriate number of points to smooth the data. For
example, selection of too large a number results in the background being smoothed into the
peak.
4-17
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
Correcting data
Introduction
Collecting accurate information about the fluorescent or phosphorescent properties of a
sample depends upon several factors:
• Equipment specifications
• Sample characteristics
• Timing considerations.
To ensure that the spectra collected indicate the actual properties of the sample and not external conditions, data often must be corrected. To correct data means to subtract information from the data not directly related to the properties of the sample. Gratings, detectors,
and other spectrometer components have response characteristics that vary as a function of
wavelength. These characteristics may be superimposed on
spectra, thereby yielding a potentially misleading trace. For
accurate intensity comparisons, such as those required for
Note: The excitaquantum-yield determinations, response characteristics must
tion range is 240–
be eliminated. Supplied with the instrument are sets of
600 nm; the emisexcitation and emission correction factors to eliminate
1
sion range is
response characteristics. These files , xcorrect and
290–850 nm.
mcorrect, are included with the software; copy them to
your hard disk.
Items that may be convoluted into a
spectrum
Ways to remove these artifacts
Fluctuations caused by the light source
Monitoring lamp output using the
reference detector, R, and using the
signal ratio S/R to correct lamp profile
or temporal fluctuations
Influence of the sample holder
Running a blank scan (which is then
subtracted from the sample scan)
Using radiometric correction factors
System hardware (e.g., optics, detectors).
To use radiometric correction factors, either:
• Select the ones supplied with the program, or
• Select a set generated at your facility during or after acquisition, discussed in the
following section. Acquiring radiometric correction factors is explained in Chaper
11: Producing Correction Factors.
Blank Subtraction and Dark Offset functions are described in the DataMax manual.
1
Filenames include a three-letter extension. For the sake of clarity, we have omitted the extensions in this section. Refer to the
software manual for specifics regarding extensions.
4-18
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
During acquisition
Data can be acquired either as raw data or as corrected data. A spectrum composed of raw
data exhibits the effects of system parameters, while a corrected spectrum displays only the
properties related to the sample. To automatically acquire corrected emission data, enter
mcorrect in the Correction factor file field in the Run Experiment dialog box. To acquire
corrected excitation data, enter xcorrect.
Note: Before applying correction factors, Jobin Yvon
recommends subtracting the dark counts (because dark
counts are not wavelength-dependent), and the spectrum of the blank, from the data. Refer to the software
manual for specific instructions.
4-19
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
After acquisition
To apply the correction factors after the data have been acquired, multiply the data file by
the appropriate correction factor file (mcorrect or xcorrect).
1
Make sure the trace to be corrected is active in
the Run Experiment window.
2
3
Select Arithmetic from the toolbar.
Select Functions.
4
Next to Function, select
Multiply.
Next to Operand, choose
Term File*K.
5
6
This opens the Math Functions dialog
box:
Click on the Select
button.
This opens the Select New Term File:
dialog box:
7
Select the
appropriate
file
(mcorrect or
xcorrect).
8
Click OK to
close the
Select New
Term File:
4-20
Fluorolog-3 v. 2.2 (10 Sep 2002)
Optimizing Data Acquisition
dialog box.
The name of the file appears in the Term File area.
9
Click Apply.
The trace that appears on the screen is a result of the mathematical operation, giving a corrected spectrum.
4-21
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
5: System Maintenance
The Fluorolog®-3 spectrofluorometer requires very little maintenance. The outside panels
may be wiped with a damp cloth to remove dust and fingerprints. The lamp is the only component that has to be replaced routinely. Regular examination of lamp and water-Raman
spectra serves as an early indicator of the system’s integrity. (These two tests are described
in Chapter 3, System Operation.)
Xenon lamp
Obtaining good spectral results depends upon the xenon lamp. After 1700–2000 hours of
use for the 450-W xenon lamp, the lamp output decreases significantly, indicating that the
lamp should be replaced. Replacing the lamp within the specified time may prevent system
failure. Jobin Yvon Inc. advises to keep a laboratory notebook near the Fluorolog®-3 to record lamp usage. Each time the lamp is turned on, it constitutes one full hour of use; therefore, Jobin Yvon Inc. suggests leaving the lamp on between brief periods of inactivity. Record the hours of use on the form in Chapter 9: Xenon Lamp Information and Record of Use
Form.
Required tools
Tool
Size
Purpose
Allen wrench
5/32"
Screws for lamp power cables
Allen wrench
5/64"
Height adjustment; centering
adjustment
Allen wrench
7/64"
Lamp support arm
Phillips screwdriver
Medium
Screws for top cover of
housing
5-1
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
Replacement
The replacement xenon lamp is packed in
the manufacturer’s box and must be
installed in the lamp housing. Read all the
packing material including instructions and
precautions before attempting to insert the
lamp into the lamp housing.
Warning: Do not remove the protective cover from the replacement xenon lamp until instructed to do so.
Warning: Xenon lamps are an explosion hazard. Make sure the power
is off and all AC power is disconnected from the system. Read and
follow the cautions presented below.
!
Hazards
!
Xenon arc lamps are an explosion hazard. Wear goggles and protective clothing
when opening the lamp housing and when handling the lamp.
!
The lamp power supply should not be connected to an AC power line while handling
lamp leads. Lethal high voltages may exist.
!
The lamp will remain extremely hot for approximately one-half hour after it has been
turned off. Do not touch the lamp or the metal unit until the lamp has cooled.
!
Never look directly at the xenon arc or its reflection. Intense radiation can permanently damage eyes.
!
Do not touch the focusing lens, back scatter mirror, or the surface of the lamp. Fingerprints will be burned onto these surfaces when the lamp is ignited.
The following instructions are divided into three distinct procedures—each of which may be
performed as a stand-alone operation:
• Open the lamp housing
• Remove the existing xenon lamp
• Install a new xenon lamp
5-2
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
To open the lamp housing:
1
Remove the 10 Phillips-head screws on top of
the lamp housing.
2
Lift off the cover.
This exposes the lamp assembly:
Upper
bracket
Lamp
Lower
bracket
Mirror
Top view of the lamp housing with cover removed.
5-3
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
To remove an existing lamp,
1
Remove the 5/32" cap screws from the upper
and lower brackets, freeing the positive and
negative power leads.
upper bracket
7/64" cap screw
5/32" cap screws
2
Remove the 7/64" cap screw from the upper
bracket.
3
4
5
Remove the upper bracket.
6
Lock the cover into place.
Remove the lamp.
Place the protective cover
around the xenon lamp.
Dispose of the spent xenon lamp, following all safety
precautions and regulations.
5-4
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
To insert a xenon lamp
1
Be sure the cover of the lamp housing is
removed.
Note: Jobin Yvon Inc. provides new bulbs with leads and
connectors of the proper length and size. Other manufacturers’ lamps may require extra adjustment before installation.
2
Pay attention to the polarity of the xenon lamp.
Lamp lead
Positive end
Large electrode
Small electrode
Negative end
Lamp lead
Diagram of lamp emphasizing polarity.
3
Seat the xenon lamp in the bottom bracket.
Make sure that the lamp is inserted all the way into the aluminum bracket. The
negative end (marked with a “–“) should be downward.
4
Place the square aluminum upper bracket over
the positive terminal of the bulb.
5-5
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
The positive end is marked with a “+”, and should be pointing upward.
5
Insert and tighten the 7/64" cap screw on the
upper bracket.
6
Secure the upper lamp lead and upper power
cable to the upper bracket,
Power
cable
using a 5/32" cap screw.
7
Secure the lower lamp lead
and lower power cable to the
lower bracket, using a 5/32"
cap screw.
8
Tighten these two 5/32" cap
screws on the upper and
lower brackets.
9
Lamp lead
Pull the excess power cables away from the
lamp assembly beneath the baffle.
Pull excess cable beneath internal
housing away from lamp assembly
Baffle
Note: Make sure
the cables do
not block the exit
slit of the lamp
housing.
Do not block exit slit
Lower bracket
Diagram of lamp in place.
10
Replace the cover of the lamp housing and
secure with the ten Phillips-head screws.
5-6
Fluorolog-3 v. 2.2 (11 Jul 2002)
11
System Maintenance
Plug the power cord from the lamp power supply
into an outlet with the proper line voltage.
Note: After installation, the lamp
should burn in for 24 hours. After
the burn-in, the lamp’s position
may be adjusted to optimize the
signal intensity.
Warning: DO NOT operate
this system from an ungrounded AC power
source.
5-7
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
Adjustment
Once the lamp is installed (and after a 24-hour burn-in period), it may need an adjustment to
maximize the sensitivity of the Fluorolog®-3. To do this, Jobin Yvon Inc. recommends running a water Raman scan to check wavelength accuracy, and then monitoring the peak intensity while adjusting the vertical height of the lamp:
1
Insert a water-filled cuvette in the sample
compartment.
2
Take a water Raman scan with the following
parameters:
Excitation wavelength 350 nm
365 nm to 450 nm
Scan range
Increment
0.5 nm
Integration time
Slits
1.0 s
1.25 mm (single-grating spectrometer)
2.50 mm (double-grating spectrometer)
Acquisition mode
S
The water Raman peak intensity for
spectrofluorometer systems are as follows:
Model
Intensity
FL3-11
>450 000 cps
FL3-12
>450 000 cps
FL3-21
>450 000 cps
FL3-22
>450 000 cps
the
various
Expected intensity for various systems.
5-8
Fluorolog®-3
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
Start
Calibrate the emission monochromator. Go to System
Operation.
Is the water Raman peak at 397
± 0.5 nm?
No
Yes
Lamp is
properly
adjusted.
3
4
Yes
Is the peak intensity as listed in the
above table?
No
Continue
with step
3 below.
Enter the Real Time Display application.
Set the excitation and emission
spectrometers to 350 nm and
397 nm, respectively.
The Raman band’s emission appears at 397 nm
when water is excited using 350 nm.
5
Make sure HV1 is on and set to
950 V.
6
Open the programmable
excitation shutter.
7
View the incoming data in acquisition mode S
(signal detector).
Watch the signal intensity as you
adjust the vertical position of the
lamp. Therefore, position the monitor
to see the changes while standing by
the lamp housing.
5-9
Note: If the lamp is not at
the best vertical position,
correctly positioning the
lamp will increase the signal significantly.
Fluorolog-3 v. 2.2 (11 Jul 2002)
8
System Maintenance
Insert a 5/64" Allen wrench inside the middle
port on the top of the lamp housing.
Focus
Horizontal
adjustment
Vertical
adjustment
Note: To reach the
set screw, you may
have to fish around
with
the
Allen
wrench.
The Allen wrench is ready to adjust the vertical position of the lamp.
9
View the trace and make vertical adjustments by
rotating the wrench either left or right to increase
the signal.
10
11
Return to the Run Experiment application.
Re-scan the water Raman spectrum.
This should result in a clean scan at maximum intensity.
Note: If there is a problem, you may have to adjust the horizontal position of the lamp. This is rarely necessary, and should
only be performed as a last resort.
5-10
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
12
Record the date that the new lamp was
installed, as well as its maximum intensity.
13
Save the protective cover of the new bulb, for
when the bulb must be replaced in the future.
5-11
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
Lamp housing
Clean the window in front of the lamp housing once a year, or more frequently if needed.
Note: Dust reduces the excitation light transmission, resulting in
lower system sensitivity.
1
2
Remove 10
Phillips-head
screws on the
lamp
housing’s
cover.
Baffle
Window is
visible
through here
Lift off the
cover of the
lamp housing.
Mounting screws
for baffle
3
Remove the 2
screws that
hold the baffle
in place inside
the housing.
4
5
Carefully lift out the baffle from the housing.
6
Clean the window
with lens tissue and
methanol.
If desired, remove
the 2 cap screws
holding the window in
place.
Cap screws on interior wall of lamp housing.
5-12
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
7
8
If the cap screws were removed, replace them.
9
Re-install the cover, and secure it with the 10
Phillips-head screws.
Replace the baffle, and re-install the 2 screws
holding it in place.
5-13
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
Emission signal detector
There is a wide range of photomultipliers available to optimize different wavelength regions. When the R928P photomultiplier is replaced with a different detector, the emission
correction factors must be updated (see Chapter 11: Producing Correction Factors).
Note: Fingerprints on the glass
of the photomultiplier increase
the dark count of the tube.
Immediately remove fingerprints from the photomultiplier
by rinsing with methanol and
wiping with lens tissue.
Warning: Lethal high voltages are
applied to the photomultiplier cathode. Turn off all instrument power
before installing or removing a photomultiplier. Never expose a photomultiplier tube to room light while
the high voltage is turned on.
To install a new R928P photomultiplier,
1
2
Be sure that the instrument power is turned off.
3
Remove the cables and wires attached to the
tube.
4
5
Carefully lift the tube from its base socket.
6
Plug the photomultiplier into the base socket.
Be sure that a liquid-N2-cooled PMT is warmed
to room temperature.
Remove the protective cap from the new
photomultiplier.
Make sure the photomultiplier is positioned all the way down on the socket.
7
Carefully slide the photomultiplier into the base
socket.
8
Connect the cables.
This is outlined in the “Cable Connections” section in Chapter 13: Re-assembly
Instructions.
5-14
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
Reference signal detector
The reference signal detector is a state-of-the art silicon diode that requires no routine maintenance.
5-15
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
Gratings
The excitation and emission spectrometer gratings are 1200 grooves/mm, and are blazed at
330 nm and 500 nm, respectively. If an application requires that the system be optimized for
a particular region, the gratings can be changed by following a simple procedure.
Required tools
Tool
Size
Purpose
Phillips screwdriver
medium
Top cover.
Lid support
--
Supports the lid in an open position.
Replacement
Gratings require no routine maintenance. To use a grating with different specifications than
those installed in the system, replace the existing ones.
Warning: Never touch the
diffraction surface of a
grating.
Jobin Yvon Inc. recommends that you never attempt
to clean off any dust on the gratings and mirrors. The
effect of cleaning may cause more problems than the
dust. The surface is easily marred and cannot be
cleaned the way a mirror can.
Warning: Do not loosen
the mirror mounts.
Mirrors in the spectrometers have been aligned at the
factory. If mirror realignment is necessary, contact
the Spex® Fluorescence Service Department.
Typically, mirror realignment requires special targets
and fixtures, and a laser.
5-16
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
Remove the existing grating
1
2
3
4
Close the slits in the emission spectrometer.
Turn off all instrument power.
Remove the Phillips-head screws holding the lid
on the
monochromator.
Warning: A circuit board and critical
connections are attached to the lid of
the spectrometer. DO NOT attempt to
lift the lid completely off.
Lift the lid slowly
until you feel
resistance.
This is the maximum length of the cables attached to the lid.
5
Insert the lid support at the rear of the
monochromator.
6
Place the lid in the support.
Mounting pin
Thumbscrew
Grating
Grating stand
Grating motor
Overhead view of the grating assembly.
5-17
Fluorolog-3 v. 2.2 (11 Jul 2002)
7
System Maintenance
Release the grating from the stand.
a
Loosen the thumbwheel screw securing the grating to the grating stand.
Back view of grating assembly.
b
Pull the grating away from the stand.
5-18
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
To insert a new grating,
1
Place the 3 mounting pins on the back of the
grating into the matching mounting slots on the
grating stand.
2
Tighten the thumbscrew on the back of the
grating stand.
3
Lift the lid of the spectrometer off of the lid
mount.
4
5
6
Remove the lid mount.
Place the lid on the spectrometer.
Secure the lid with the Phillips screws.
No further adjustments or alignments are necessary.
5-19
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
Mirrors
Mirrors are aligned at the factory and usually do not need realignment. Only the Model
1692M Selection Mirror (an option) can be adjusted easily to optimize the fluorescence signal from the sample. This mirror should be adjusted periodically if low signal is caused by
poor alignment at the slit position.
Warning: Adjust the mirrors only if a laser is available to check the alignment. Misalignment will
destroy the similarity of the right-angle and frontface light paths in the single-beam sampling
module. (Front-face capability is an option in the
T-Box.)
Improperly moving any of the mirrors in the spectrometers will reduce resolution capability
and refocusing will be required. If the system becomes misaligned, contact the Spex® Fluorescence Service Department to arrange for a service visit.
5-20
Fluorolog-3 v. 2.2 (11 Jul 2002)
System Maintenance
Automated 4-position turret
If a circulating bath is used to regulate the turret’s temperature, periodically replace the fluids in the bath.
Note: Over time, bacteria can grow in the
temperature bath, or the water can become hard.
5-21
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
6: Components & Accessories
The Fluorolog®-3 can be configured to obtain optimum results for a variety of applications. The basic system, regardless of configuration, consists of slits, detectors, a xenon
lamp with power supply, and a sampling module. These items can be combined in different ways to provide maximum benefits.
The following list represents all the accessories and components (including those mentioned above), in alphabetical order, available for the Fluorolog®-3 spectrofluorometers.
A brief description of each is included in the following sections. Like the list presented
below, the descriptions that follow are alphabetized, except where logical order dictates
otherwise.
For additional information or product literature on any of these items, contact your local Jobin Yvon Inc. Sales Representative.
6-1
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Itemized List
Fluorolog®-3 Accessories
Item
Model
Accessory, Absorption/Transmission
Adapter, Micro Cell (See Cell, Micro)
Adapter, Micro Cell (See Cell, Micro)
Assembly, Liquid Nitrogen Dewar
Assembly, Scatter Block
Assembly, Standard Lamp
Cell, HPLC Flow
Cell, Micro
Cell, Micro
Cell, Quartz
Cell, Sample
Cell, Sample (Reduced Volume)
Detector, Reference Photodiode
Detector, Signal CCD
Detector, Signal: Room Temperature Photomultiplier
Detector, Signal: Thermoelectrically Cooled Housing
Detector, Signal: Thermoelectrically Cooled Near-IR PMT
Dewar, Liquid Nitrogen (See Assembly, Liquid Nitrogen Dewar)
Fiber Optic Mount
Filter, Cut-On (1" × 2")
Filter, Cut-On (2" × 2")
Front-Face Viewing Option
Gratings
Holder, Filter
Holder, Four-Position Variable Temp. Control w/ Magnetic Stirrer
Holder, Dual-Position Variable Temp. Control w/ Magnetic Stirrer
Holder, Solid Sample
Housing, Xenon Lamp
Housing, Dual Lamp
Injector, Autotitration
Interface, Microscope Fiber-Optic (Nikon)
Interface, Microscope Fiber-Optic (Olympus)
Interface, Microscope Fiber-Optic (Zeiss)
Lamp, Xenon 450-W
Peltier Drive, Sample Heater/Cooler
Phosphorimeter Accessory
Plate Reader
Polarizer, L-Format
Polarizer, T-Format
Port, Injector
Temperature Bath
Trigger Accessory, External
1940
1923A
1924A
FL-1013
1908MOD
1908
1955
1923
1924
1925
1920
QC-SK
1967
various
1911F
1914F
FL-1030
1932D
F-3000
1938
1939
FL-1001
various
FL-1010
FL-1011
FL-1012
1933
FL-1039
FL-1040
F-3005/6
F-3001
F-3002
F-3003
1907
F-3004
FL-1042
MicroMax
FL-1044
FL-1045
FL-1015
F-1000/1
TRIG-15/25
6-2
Page
6-3
6-6
6-7
6-7
6-8
6-8
6-8
6-8
6-8
6-8
6-9
6-10
6-11
6-12
6-13
6-14
6-15
6-15
6-16
6-17
6-18
6-19
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6-25
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6-27
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6-28
6-29
6-30
6-31
6-32
6-32
6-33
6-34
6-35
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Model 1940 Absorption/Transmission
Accessory
The Model 1940 Absorption/Transmission Accessory slightly displaces the sample
from its normal position and directs the transmitted light into the collection optics with
a mirror mounted at 45°.
Model 1940 Absorption/Transmission Assembly.
Operation of the Model 1940 Accessory requires two steps:
a
b
Installation
Optimization of the emission and reference signals
Installation
1
2
3
Remove the sample holder currently in place.
Position the Model 1940 on the posts.
Tighten the two thumbscrews.
Before you acquire transmission or absorption spectra, the emission and reference signals must be optimized.
Signal optimization
1
Select right-angle detection.
Turn the selection knob to RA.
6-3
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
2
Place the cuvette containing the blank in the
sample holder.
3
4
5
Set the emission spectrometer position to 0 nm.
Select acquisition mode S/R.
Run an excitation scan over the absorption
range of the sample.
Note: If uncertain of the absorption range, enter a range of 250 nm to
400 nm, and scan the excitation spectrometer. Detailed instructions
for setting parameters are contained in the software manual. The
S/R acquisition mode compensates for fluctuations in lamp intensity,
and wavelength dependency of the lamp and excitation spectrometer.
6
Note the emission signal at maximum
transmission of the blank.
7
Adjust the slits to
maximize the
signal without
saturating the
detector.
Note: The linear range of the
R928P detector operated in
the photon-counting mode is
2 million cps.
8
Similarly, locate the maximum reference signal
of the lamp spectrum.
9
Set the excitation spectrometer to that position.
Warning: Only when both emission and reference signals are within
prescribed limits should you proceed with the measurements.
6-4
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
10
Adjust the high voltage HV2, as needed, so that
the reference signal is ~ 1 µA (saturation of the
reference detector is 10 µA).
11
Acquire transmission spectra.
Either
a
b
c
Position the emission spectrometer at 0 nm, and use the S/R acquisition
mode.
Acquire an excitation scan of the blank.
Acquire and excitation scan of the sample.
Or
When the spectrometers are synchronously scanned using an offset of 0 nm, the
emission slits should be opened five times as wide as the slits of the excitation
spectrometer.
d
e
12
Acquire a synchronous scan
with an offset of 0 nm over the
absorption range of the blank.
Acquire a synchronous scan
with an offset of 0 nm over the
absorption range of the
sample.
Note: If the detectors are
saturated, close the slits a
little or install a neutraldensity filter.
Calculate the absorption.
The absorption spectrum can be determined from the following equation:
A = log(S/R)blank – log(S/R)sample
6-5
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
FL-1013 Liquid Nitrogen Dewar Assembly
For phosphorescence or delayed fluorescence measurements, samples are often frozen
at liquid-nitrogen temperature (77 K) to preserve the fragile triplet state. The sample is
placed in the quartz cell and slowly immersed in the liquid-nitrogen-filled Dewar flask.
The white Teflon® cone in the bottom of the Dewar flask keeps the quartz sample-tube
centered in the Dewar flask. The Teflon® cover on the top of the Dewar flask holds any
excess liquid nitrogen that bubbled out of the assembly. A pedestal holds the Dewar
flask in the sampling module. A special stove-pipe sample cover replaces the standard
sample lid, so that liquid nitrogen can be added to the Dewar flask as needed. The Dewar flask holds liquid nitrogen for at least 30 min with minimal outside condensation
and bubbling.
FL-1013 Liquid Nitrogen Dewar Assembly.
Included in the FL-1013 Liquid Nitrogen Dewar Assembly, the Dewar flask
can be purchased as a spare. The bottom
portion, which sits directly in the light
path, is constructed of fused silica.
6-6
Note: If condensation appears on the outside of the
Dewar flask, it must be
re-evacuated.
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Model 1908MOD Scatter Block Assembly
The Scatter Block Assembly includes a
white scatter block assembly and a
clamp holder for use with a usersupplied standard lamp and regulated
power supply.
Model 1908MOD Scatter Block Assembly.
Model 1908 Standard Lamp Assembly
The Model 1908 Standard Lamp Assembly is a
complete correction factor kit used to generate
radiometric emission correction factors for the
spectrofluorometer systems. The assembly
includes:
• an NIST-traceable, calibrated, 200-W quartz
tungsten-halogen lamp with irradiance values.
• Regulated 6.5-A power supply with lamp
holder
• 1908MOD Scatter Block Assembly (see
above)
Emission correction factors compensate for the
response of the photomultiplier detector as well as
the wavelength dependency of the gratings in the
emission spectrometer. Emission correction
factors should be updated periodically and
whenever different gratings or a new signal
detector is installed. The procedure for generating
new correction factors is contained in Chapter 11.
6-7
Model 1908 Standard Lamp
Assembly.
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Sample Cells
Model 1955 HPLC Flow Cell
With a sample capacity of 20 µL, this non-fluorescing fused silica cell is ideal for online monitoring of fluorescent samples. The cell maintains high sensitivity because it
has a large aperture for collecting the excitation light to the sample and fluorescence
emission from the sample. The flat sides allow maximum throughput while keeping the
scattering of the incident radiation to a minimum. The cell fits in a standard cell holder.
Model 1923 Micro Cell (with Model 1923A Adapter)
This non-fluorescing fused silica cylindrical cell holds 50 µL. This cell will not accept a
magnetic stirrer. The 1923A Adapter is required to be able to mount in a standard 10
mm by 10 mm cell holder.
Model 1924 Micro Cell (with Model 1924A Adapter)
This non-fluorescing fused silica cylindrical cell holds 250 µL. A magnetic stirrer cannot be used with this cell. The 1924A Adapter is required to enable mounting in the
standard 10-mm-×-10-mm cell holder.
Model 1925 Quartz Cuvette
With a 4-mL volume, this cell measures 10 mm × 10 mm in cross-section, and comes
with a Teflon® stopper to contain volatile liquids.
Model 1920 Sample Cell
This 2-mL to 4-mL non-fluorescing fused silica cell, capable of accepting a magnetic
stirrer, has a 10-mm path length and includes a white Teflon® cap that prevents sample
evaporation.
Model QC-SK Reduced Volume Sample Cell
This non-fluorescing fused silica cell is selected for samples with a maximum volume
of 1 mL. The square cross-section of the sample cavity is 5 mm. The precise imaging
capability of the excitation light focused onto the sample allows for high sensitivity.
The adapter and a “flea” magnetic stirrer are included.
6-8
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Model 1967 Photodiode Reference
Detector
The Photodiode Reference Assembly monitors the xenon lamp up to 1 µm. This accessory is standard in the Fluorolog®-3 package, but a photomultiplier can be substituted
instead (necessary for phosphorescence measurements).
Model 1967 Photodiode Reference Assembly.
6-9
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
CCD Detectors
For multichannel spectral acquisition, many charge-coupled devices are available to
suit the researcher’s needs. Both air-cooled and liquid-nitrogen-cooled CCDs can be inserted into the Fluorolog®-3. Available options include extended-UV and near-IR detection, various pixel sizes and arrays, and maximum-coverage options. Contact a local
Sales Representative for details and specific model numbers.
6-10
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Model 1911F Room Temperature Signal
Detector
The Fluorolog®-3 includes a room temperature R928P emission signal detector. This
detector is mounted to the emission spectrometer and operated in the photon-counting
mode.
Model 1911F Room-Temperature Signal Detector.
The dark count is specified at <1000 cps, and the R928P delivers useful output from
190 nm to 860 nm.
6-11
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Model 1914F Thermoelectrically Cooled
Signal Detector
As a rule, cooling a detector improves the S/N ratio by reducing the inherent dark count
or noise. For the standard signal detector, Model 1911F (described above), cooling reduces the dark count from 1000 cps to 20 cps; the useful wavelength band (190–860
nm) remains the same.
The Thermoelectrically Cooled Signal Detector consists of the housing, power supply,
Model 1630 Field Lens Adapter, a black flange, and a silver adapter plate. To cool, a
water line and drain are attached to the tubing extending from the housing, and roomtemperature water is circulated through the housing.
Model 1914F Thermoelectrically Cooled Signal Detector R928P.
6-12
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
FL-1030 Thermoelectrically Cooled NearIR Photomultiplier Tube
For spectral measurements extending into the near-infrared, the FL-1030 Thermoelectrically Cooled PMT is perfect. Included in the FL-1030 is the thermoelectrically
cooled housing. The InGaAs detector has a spectral range from 250 nm all the way to
1050 nm. Required for this item is the DM302 Photon Counting Module.
6-13
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
F-3000 Fiber Optic Mount
Now you can study marine environments, skin and hair, or other large samples in situ!
For those users who want to examine samples unable to be inserted into the sample
compartment, the F-3000 Fiber Optic Mount (plus fiber-optic bundles) allows remote
sensing of fluorescence. The F-3000 couples to the T-box; light is focused from the excitation spectrometer onto the fiber-optic bundle, and then directed to the sample. Fluorescence emission from the sample is directed back through the bundle and into the
front-face collection port in the sample compartment. Randomized fiber optic bundles
ranging in length from 1 meter to 5 meters are available. Contact the local Jobin Yvon
Inc. Sales Representative for details.
F-3000 Fiber Optic Mount.
6-14
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Model 1938 Cut-On Filter
The Model 1938 Cut-On Filter Set consists of 5 filters with dimensions of 1" × 2". To
properly position
the filter, the
Model FL-1010
Filter Holder is
required.
Cut-on filters are
used to eliminate
second-order effects of the gratings. For example, if sample excitation is at 300 nm, a
second-order peak occurs at 600 nm. If the emission spectrum extends from 400 nm to
650 nm, a sharp spike occurs at 600 nm. This peak is the second-order peak of the excitation spectrometer. To remove this unwanted peak in the emission spectrum, place a
350-nm filter in the emission slot. Cut-on filters typically are used for phosphorescence
measurements, where second-order effects are likely to be found.
The single-beam and T-box sampling compartments have three slots that can hold the
Model FL-1010 filter holder. One slot is in the excitation light path and the other two
are the emission light path positions. To eliminate second-order effects from an excitation spectrum, install the filter holder and the appropriate cut-on filter in the excitation
light path.
Model 1939 Cut-On Filter
The Model 1939 Cut-On Filter set consists of five 2" × 2" filters with cut-on wavelengths of 350 nm, 399 nm, 450 nm, 500 nm, and 550 nm. To properly position the filter, the FL-1010 Filter Holder is required.
Cut-on filters are used to eliminate second-order effects of the gratings. For example, if
sample excitation is at 300 nm, a second-order peak occurs at 600 nm. If the emission
spectrum extends from 400 nm to 650 nm, a sharp spike occurs at 600 nm. This peak is
the second-order peak of the excitation spectrometer. To remove this unwanted peak in
the emission spectrum, place a 350-nm filter in the emission slot. Cut-on filters are
typically used for phosphorescence measurements, where second-order effects are
likely to be found.
The single-beam and T-box sampling compartments have three slots that can hold the
FL-1010 filter holder. One slot is in the excitation light path and the other two are the
emission light path positions. To eliminate second-order effects from an excitation
spectrum, install the filter holder and the appropriate cut-on filter in the excitation light
path.
6-15
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
FL-1001 Front-Face Viewing Option
Designed to examine fluorescence from the surface of solid samples, the FL-1001
Front-Face Viewing Option includes a swing-away mirror. This allows the researcher
to change from front-face and right-angle data collection instantly. In the front-face collection mode, the viewing angle is 22.5°. The FL-1001 is ideal for such samples as pellets, powders, inks, monolayers, dyes, and various solids.
FL-1001 Front-Face Viewing Option.
6-16
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Gratings
Many gratings are available to replace the standard model in the Fluorolog®-3. Rulings
with the following specifications are available:
• 300 grooves/mm
• 600 grooves/mm
• 1200 grooves/mm
In addition, a number of different blazes can be purchased:
• 250 nm
• 330 nm
• 500 nm
• 750 nm
• 1000 nm
All gratings are classically ruled, and measure 50 mm × 50 mm. For details and model
numbers, contact a Jobin Yvon Inc. Sales Representative.
Grating.
6-17
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
FL-1010 Cut-On Filter Holder
Cut-on filters are used to eliminate second-order effects of the gratings. The singlebeam and T-box sampling compartments have three slots that can hold the FL-1010 Filter Holder. Refer to either Model 1939 Cut-On Filter or Model 1938 Cut-On Filter for a
detailed description of the placement of the filter holder and the interaction of the cuton filters and the holder.
6-18
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
FL-1011 Four-Position Thermostatted Cell
Holder
The FL-1011 Four-Position Thermostatted Cell Holder keeps a sample at a constant
temperature from –20°C to +80°C. The temperature is maintained by an ethyleneglycol–water mixture pumped through from an external circulating temperature bath
(not included). The holder also includes a magnetic stirrer, for mixing turbid or viscous
samples. Also required is the FM-2003 Sample Compartment Accessory.
FL-1011 Four-Position Thermostatted Cell Holder.
Installation
1
2
3
4
Remove the compartment gap-bed.
Position the FL-1011 gap-bed drawer.
Tighten with four screws.
Attach the ¼" tubing to the brass inlets on the
bottom of
the holder.
Warning: Failure to clamp these hoses securely
may result in flooding and damage to the optics
and electronics of the instrument.
6-19
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Use
1
Place the sample in a 10 mm × 10 mm cuvette
and insert a magnetic stirring bar.
(The stirring bar is available from Bel-Art Products, Pequannock, NJ)
2
Place a cuvette in
each holder.
Note: While the four-position
model maintains the temperature of all four samples, only
one sample is mixed at a
time.
3
Allow the
samples to reach
the desired
temperature.
4
5
Turn on the magnetic stirrer.
Select the
appropriate mixing
speed.
The speed at which the sample
should be mixed depends on the
viscosity of the sample.
6
Note: Selecting too high a
speed may create a vortex,
which could affect the reproducibility of the measurement.
Run your experiment as usual.
Either right-angle or front-face detection can be used.
7
Place the next cuvette in the sample position by
lifting up the knob and rotating the holder.
Be sure to press down, to lock the cuvette into the proper position.
6-20
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
FL-1012 Dual-Position Thermostatted Cell
Holder
The FL-1012 Dual-Position Thermostatted Cell Holder keeps a sample at a constant
temperature from –20°C to +80°C. The temperature is maintained by an ethyleneglycol–water mixture pumped through from an external circulating temperature bath
(not included). The holder also includes a magnetic stirrer, enabling mixing of turbid or
viscous samples. Also required is the FM-2003 Sample Compartment Accessory.
FL-1012 Dual-Position Thermostatted Cell Holder.
Installation
1
2
3
4
Remove the present holder from the posts.
Replace with the FL-1012.
Tighten the two thumbscrews
Attach the ¼" tubing to the brass inlets on the
bottom of
the
Warning: Failure to clamp these hoses securely
holder.
may result in flooding and damage to the optics
and electronics of the instrument.
6-21
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Use
1
Place your sample in a 10 mm × 10 mm cuvette
and insert a magnetic stirring bar.
(The stirring bar is available from Bel-Art Products, Pequannock, NJ)
2
Place a cuvette in
each holder.
Note: While the two-position
model maintains the temperature of both samples, only one
sample is mixed at a time.
3
Allow the sample
to reach the
desired
temperature.
4
5
Turn on the magnetic stirrer.
Select the
appropriate speed.
The speed at which the sample
should be mixed depends on the
viscosity of the sample.
6
Run your experiment
as usual.
Note: Selecting too high a
speed may create a vortex,
which could affect the reproducibility of the measurement.
Either right-angle or front-face detection can be used.
6-22
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Model 1933 Solid Sample Holder
The Model 1933 Solid Sample Holder is designed for samples such as thin films, powders, pellets, microscope slides, and fibers. The holder consists of a base with graduated
dial, upon which a bracket, a spring clip, and a sample block rest.
Model 1933 Solid Sample Holder with sample block removed.
Installation
1
2
3
Remove the present holder.
Position the base on the posts.
Tighten the two thumbscrews.
For pellets, crystals, creams, gels, powders, and
similar materials:
1
2
Fill the well of the block.
Place a quartz coverslip or Teflon® film over the
well.
This holds the sample in place when
vertically positioned.
3
Note: When the sample is
perpendicular the light is
collected at an angle of
22.5°. This orientation
minimizes stray and reflected light off the surface of the sample.
Carefully insert the
block between the
bracket and spring
clip, so that the
sample is perpendicular to the excitation light.
6-23
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
For samples such as thin films, microscope slides,
fibers, or other materials:
1
Place the material on the block on the side
opposite that of the well.
2
Insert the block between the bracket and spring
clip.
The sample should be perpendicular to the excitation light and fluorescence
collected using front-face detection.
3
Select front-face
detection by
turning the knob
on the top panel
of the sample
compartment to
FF (front-face).
Note: In the T-box sample compartment, the Model 1692M Selection Mirror is an option. If
front-face measurements are to
be acquired, this mirror must be
installed in the sample compartment.
Note: Before scanning a solid sample, Jobin Yvon Inc. recommends running a water Raman scan in a cuvette with front-face
detection. This ensures accurate alignment of the Model 1692M
Selection Mirror.
6-24
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
FL-1039 Xenon Lamp Housing
The FL-1039 is the standard lamp housing for the 450-W Xenon Lamp. The power
supply is included internally.
FL-1040 Dual Lamp Housing
The FL-1040 Dual Lamp Housing contains both the standard continuous 450-W xenon
lamp and a UV xenon flash tube. A swing mirror selects which of the two sources excites the sample. This housing with pulsed lamp is useful for studies of phosphorescence lifetimes and decay analysis. The power supply is internal to the housing.
FL-1040 Dual Lamp Housing.
6-25
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
F-3005/6 Autotitration Injector
For controlled, automatic injection of aliquots into the sample of your choice, the F3005/6 Autotitration Injector is just the thing, available in both 110-V (F-3005) and
220-V (F-3006) models. The F-3005/6 comes with dual syringes, for complete control
over dispensing and aspirating volumes of liquids into and out of the sample cell. A
mix function is included. With the injector come 18-gauge Teflon® tubing and two syringes (1 mL and 250 µL). The syringes are interchangeable; aliquot size is controllable
to 0.1% of total syringe volume.
F-3005/6 AutoTitrator Injector.
6-26
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Models F-3001, F-3002, and F-3003
Microscope Fiber-Optic Interfaces
The Microscope Fiber-Optic Interface eases the use of the Fluorolog®-3 systems for
fluorescence-microscopy measurements. Interfaces are available for Nikon, Olympus,
and Zeiss microscopes.
Models F-3001, F-3002, F-3003 Microscope Interfaces
6-27
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Model 1907 450-W Xenon Lamp
The Model 1907 450-W xenon lamp delivers light from 240 nm to 850 nm for sample
excitation. The lamp has an approximate life of 2000 hours, and is ozone-free. The
lamp is designed to fit into the FL-1039 Xenon Lamp Housing and the FL-1040 Dual
Lamp Housing.
450-W xenon lamp, removed from the protective case.
6-28
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
F-3004 Sample Heater/Cooler Peltier
Thermocouple Drive
For rapid control of the sample’s temperature in the Fluorolog®-3’s sample compartment, choose the F-3004 Peltier Drive. Instead of messy fluids, the Peltier device heats
and cools the sample thermoelectrically and fast! The temperature range is –10°C to
+120°C. To prevent condensation of moisture on chilled cuvettes, an injection port for
dry nitrogen gas is provided. All software is included, along with a controller and stirring mechanism.
F-3004 Sample Heater/Cooler Peltier Thermocouple Drive.
6-29
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Phosphorimeter Accessory
The phosphorimeter adds a programmable, pulsed excitation source and selectable signal gating from the signal photomultiplier tube. This provides time-discrimination capability to sort out the lifetimes of simultaneous, competing luminescence emissions.
Because the duration of each exciting pulse from the phosphorimeter is very short
(~3 µs), lamp interference during acquisition of decay curves is minimized. This allows
the researcher to follow the decay of samples an order-of-magnitude faster than can be
achieved with conventional systems that depend on mechanical choppers.
To run phosphorescence experiments, the
FL-1040 dual-lamp housing is required, as
well as the FL-1042 phosphorescence upgrade, which adds a controller and electronics. To use the phosphorimeter, turn the external knob on the lamp housing counterclockwise to Alternate Lamp:
This action rotates a mirror inside, directing light from the flash tube to the sample:
CW lamp
Mirror
Pulsed lamp
External knob rotates internal mirror.
6-30
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
MicroMax Microwell Plate Reader
The MicroMax Microwell Titer-Plate Reader allows multiple samples to be scanned in
one experiment. The MicroMax is controlled through the DataMax software via a serial
port to the host computer. The titer plate moves beneath a stationary optical beam, and
fluorescence measurements are collected with top-reading geometry. Thus, any titer
plates—even disposable ones—may be used. Up to 384-well plates may be inserted
into the MicroMax, with a scan speed < 1 min for all plates. Various scan types are
possible:
• Single-Point Analysis
• Excitation
• Emission
• Time-Base
• Synchronous
• Multigroup
Signals are transmitted between the Fluorolog®-3 and the MicroMax via fiber-optic
bundles.
MicroMax Microwell-Plate Reader.
6-31
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
FL-1044 L-Format Polarizer & FL-1045 TFormat Polarizer
For L-format spectrofluorometers, the FL-1044 dual polarizer is ideal. The kit includes
two polarizers, to be placed at the entrance and the exit of the T-box. The polarizers are
fully automated, and are adjustable to within 1° rotation. Insertion and removal from
the optical path is controlled by the computer. For T-format spectroscopy, researchers
should order the FL-1045 third polarization unit in addition to the FL-1044.
Polarizer.
6-32
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
FL-1015 Injector Port
For the study of reaction kinetics, such as Ca2+ measurements, the FL-1015 Injector
Port is ideal. This accessory allows additions of small volumes via a syringe or pipette
to the sample cell without removing the lid of the sample compartment. With the injector in place, a lock-tight seal is achieved, prevented both light and air from reaching the
sample. The Injector Port is recommended for use with the TRIG-15/25 Trigger
Box/Event Marker.
FL-1015 Injection Port.
The Injector Port will accommodate most pipettes and syringes, with an injection hole
diameter of 0.125" (3.2 mm). A cap is included to cover the port when not in use.
6-33
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
F-1000/1 Temperature Bath
For studies of samples whose properties are temperature-dependent, use the F-1000/1
Temperature Bath. The controller circulates fluids externally, with tubes leading to the
sample chamber. The temperature range is from –25°C to +150°C. Sensor and all cables are included with the F-1000/1. The Temperature Bath is available in a 110-V (F1000) and 220-V (F-1001) version.
F-1000/1 Temperature Bath.
6-34
Fluorolog-3 v. 2.2 (10 Sep 2002)
Components & Accessories
Model TRIG-15/25 External Trigger
Accessory
The TRIG-15/25 accessory permits the fluorescence system to be operated with almost
any external trigger stimulus. Data acquisition can be synchronized with external
events, either automatically following a voltage pulse (minimum 3 V above ground), or
manually by pushing a button on a trigger-release cable. Multiple trigger events are recorded and stored with the associated data file. A TTL trigger output also is provided,
for activating external devices, such as a stopped-flow unit. The front panel has four
sets of banana-jack inputs for two independent trigger inputs, Trigger 1 and Trigger 2.
Model TRIG-15/25 External Trigger Accessory.
There are two sets of jacks for each of these two trigger inputs: an upper set, for manual
switch inputs, and a lower set, for pulsed voltage inputs. These two input types can be
used simultaneously, but any one event is ignored while the interface is activated by
another.
6-35
Fluorolog-3 v. 2.2 (31 Jul 2002)
Troubleshooting
7: Troubleshooting
The Fluorolog®-3 spectrofluorometer system has been designed to operate reliably and
predictably. Should a problem occur, examine the chart below, and try the steps listed
on the following pages.
Problem
Light is not reaching
the sample.
Possible Cause
Excitation shutter closed.
Remedy
Using the software, open the shutter.
Slits are not open to the
proper width.
Adjust the slits.
Lamp is not turned on
Turn on lamp by pressing lamp rocker switch,
and then the Start button on the xenon-lamp
power-supply front panel.
Check and recalibrate monochromator.
Monochromator is miscalibrated.
Signal intensity is low.
No change in signal
intensity.
No signal.
Erratic signal.
Sample turret is not in correct position.
Using the software, set the position and open
the cover to verify the position.
Lamp is not aligned or
focused.
Align and focus the lamp.
Slits are not open to proper
width.
Adjust the slits.
Shutter(s) is(are) not completely open.
Open the shutter(s).
Lamp power supply is set
to the wrong current rating.
Call the Spex® Fluorescence Service Department. (450-W Xe lamp current = 25 A.)
High voltage is improperly
set.
Enter proper voltage: Default HV1 = 950 V
Polarizer is in the light
path.
In Visual Instrument Setup, move the polarizer out of the light path.
Lamp is too old.
Shutter(s) closed.
Replace lamp. (450-W lamp has lamp lifetime
1500–2000 h.)
Open all shutters.
Spectrometers are set to
wrong wavelength.
Select appropriate wavelength based on excitation and emission of sample.
Detectors are saturated
Adjust slits and/or voltages. (Signal detector
has linearity to 1.5 × 106 cps when operated in
photon-counting mode.)
Detectors are saturated.
Reduce slit settings.
High voltage is off.
Turn on high voltage through the software.
Lamp is not on.
Turn on lamp.
Light leaks.
Check dark value to determine.
[continued on next page]
7-1
Fluorolog-3 v. 2.2 (31 Jul 2002)
Troubleshooting
Boot disk corrupted.
Use backup boot disk. If no backup boot disk
is available, call Fluorescence Service Department.
Cables are improperly connected.
Check communications cables’ connections.
Wrong settings for COM
ports.
Check
file.
Computer I/O controller or
SAC I/O controller is failing.
Replace I/O controller: Call Fluorescence
Service Department.
Hardware Init. error
appears.
Broken IR sensor in monochromator.
Replace IR sensor: Call Fluorescence Service
Department.
Error converting slit
units appears.
Datamax\isa_ini\df
lt.set file is corrupt.
Delete
datamax\isa_ini\dflt.set
file. When system is restarted, the system will
recreate this file.
Sample turret is not
operating.
Not turned on.
Verify that the rocker switch on the front
panel of the Model 1976 Accessory Controller
is in the “1” (On) position.
Software is not enabled.
Check status.
Cables are improperly connected.
Check cable connections.
isamain.ini is improperly set
In
the
windows
isamain.ini
Communication problems between computer and instrument.
Instrument Control
Center icon is not on
desktop, but appears
in taskbar
datamax\isa_ini\sac.ini
directory,
open
Change the coordinates listed to
Left = 524
Top = 174
Right = 824
Bottom = 282
If problem persists, change isamain.ini’s
properties into a read-only file.
Error “no trace view
object” appears
isascan.set or isascan.vw are corrupted
7-2
Delete isascan.set and isascan.vw
from datamax directory. (They will be recreated automatically.)
Fluorolog-3 v. 2.2 (31 Jul 2002)
Troubleshooting
Using diagnostic spectra
Often the spectrum reveals information regarding the hardware or software parameters
that should be adjusted. The following spectra occur with explanations regarding problems leading to their appearance.
Note: Not all spectra shown in this section were produced using the
Fluorolog®-3. The spectra are presented to show different possible
system or sample problems, and may not reflect the superior performance of the Fluorolog®-3.
Lamp scan
Running a lamp scan verifies system integrity and indicates whether the correct parameters for the best possible trace are being used. The following spectrum shows the
trace resulting from a lamp scan run with a known good lamp.
0.35
0.3
467 nm
Intensity
0.25
0.2
0.15
0.1
0.05
0
250
300
350
400
450
500
550
Wavelength (nm)
Scan of good quality 450-W xenon lamp in Fluorolog-3
with single excitation monochromator.
7-3
600
Fluorolog-3 v. 2.2 (31 Jul 2002)
Troubleshooting
The following lamp scan spectrum shows poor resolution in the area around the peak.
0.4473
Intensity (counts s–1)
Xenon-lamp peaks
are unresolved
0.3355
0.2236
0.1118
0
300
400
500
600
Wavelength (nm)
700
800
Lamp scan of 150-W Xe lamp. Note poor resolution in the area near the
467-nm peak.
This lack of spectral resolution appears because the slit widths are set too wide. To resolve this problem, narrow the slit widths.
7-4
Fluorolog-3 v. 2.2 (31 Jul 2002)
Troubleshooting
Water Raman spectra
Contaminated water
Running a water Raman scan helps identify abnormalities as a result of accessory problems or miscalibration. The following spectrum is normal:
5
397 nm
Intensity (10 counts/s)
4
5
3
2
1
0
365
380
395
410
425
440
455
Wavelength (nm)
Clean water Raman scan.
Below is a normal water Raman spectrum superimposed on one that exhibits a problem.
In this instance, the water was contaminated, resulting in a high background.
4
3
5
Intensity (10 counts/s)
5
Contaminated water
(note high background)
2
1
Clean water
0
365
380
395
410
425
440
455
W a v e le ngth (nm)
C o n ta m in a te d w a te r in a w a te r R a m a n s c a n .
If a spectrum similar to this is obtained after running a water Raman scan,
1
Rotate the cuvette 90° and rerun the scan.
7-5
Fluorolog-3 v. 2.2 (31 Jul 2002)
Troubleshooting
If the problem goes away, then the problem was due to the cuvette surface. Clean or use
a different cuvette.
Or
1
2
Clean the cuvette.
Fill with fresh, double-distilled, deionized water.
If the problem goes away, then the problem was due to contaminated water.
Light not striking cuvette
The following graph shows a normal water-Raman scan with a superimposed problem
scan.
5
Clean water
3
5
Intensity (10 counts/s)
4
2
Low signal
1
0
365
380
395
410
425
440
455
Wavelength (nm)
Low intensity during a Raman scan.
Here the problem is low intensity of the water signal when compared with the superimposed typical water Raman scan. To resolve this problem:
1
Make sure the cuvette is filled to the proper
level.
Light should fall on the sample, and the meniscus should not be in the light
path.
2
Make sure that the excitation and emission slits
are set to the proper widths.
3
Verify that the detector is set to the proper
voltage.
7-6
Fluorolog-3 v. 2.2 (31 Jul 2002)
Troubleshooting
Stray light
In the following diagram, notice the high level of stray-light below 380 nm in the water
Raman spectrum.
50
S
40
5
Intensity (10 counts/s)
60
Nor
mal
30
20
10
0
365
380
395
410
425
440
455
Wavelength (nm)
High stray light in a water Raman scan.
To correct this problem,
1
Inspect the cuvette surface for fingerprints and
scratches.
2
3
Clean the cuvette or use a new one.
4
Verify that the excitation spectrometer is at the
correct position.
Verify that the excitation and emission slits are
set correctly for a water Raman scan.
7-7
Fluorolog-3 v. 2.2 (31 Jul 2002)
Troubleshooting
Further assistance...
Read all software and accessory manuals before contacting the Spex® Fluorescence
Service Department. Often the manuals show the problem’s cause and a method of solution. Technical support is available for both hardware and software troubleshooting.
Before contacting the service department, however, complete the following steps.
1
2
3
4
5
6
7
8
If this is the first time the problem has occurred, try turning off the system and
accessories. After a cool-down period, turn everything back on.
Make sure all accessories are properly configured, and turned on as needed.
Following the instructions in Chapter 3, System Operation, run a lamp scan and
a water Raman scan to make sure the system is properly calibrated. Print the
spectrum for each and note the peak intensities.
Check this chapter to see if the problem is discussed.
Visit our web site at www.isainc.com/fluor/fluor.htm to see if the question is
addressed in the Systems or FAQs sections of the site.
Try to duplicate the problem and write down the steps required to do so. The
service engineers will try to do the same with a test system. Depending on the
the problem, a service visit may not be required.
If an error dialog box appears in DataMax, write down the exact error
displayed.
In DataMax, in the Instrument Control Center toolbar, choose Help. Under
Help, choose About Instrument Control Center. This opens the About
Instrument Control Center window. The version of the software is listed here.
7-8
Fluorolog-3 v. 2.2 (31 Jul 2002)
9
Troubleshooting
In Run Experiment toolbar, open the About DataMax window. Make a note of
the software’s and instrument’s serial numbers, and instrument configuration,
including all accessories.
If the problem persists or is unlisted, call the Spex® Fluorescence Service Department
at (732) 494-8660 × 160.
7-9
Fluorolog-3 v.2.2 (10 Sep 2002)
Introduction to Lifetime Measurements
8: Introduction to Lifetime
Measurements
Introduction
Although the Fluorolog®-3 spectroscopy system is fully capable of performing lifetime
measurements, it was designed to excel in the area of fluorescence measurements. Operating within a lifetime range of 10 ps to 10 µs and a frequency range of 0.2 to 310
MHz, the Fluorolog®-Tau-3 specifically addresses the needs of phosphorescence measurements.
To design and build the Fluorolog®-Tau-3, Jobin Yvon Inc. started with the proven performance of a Fluorolog®-3, the most sensitive, steady-state spectrofluorometer in the
world, and added unique optical and electronic components for frequency-domain
measurements. From picoseconds to microseconds, the Fluorolog®-Tau-3 performs reliably and delivers reproducible results time after time.
The proven sensitivity and resolving power of the Fluorolog®-3 and the high-quality
Jobin-Yvon-Inc. optics provide the precise collimation to take maximum advantage of
the system’s CW source, thereby ensuring accurate results. In addition, software is
equipped with a user-friendly interface, intuitive commands, a powerful postacquisition module, and comprehensive on-line help.
DataMax, the software included with the Fluorolog®-Tau-3, allows you to acquire
steady-state as well as lifetime measurements. DataMax controls the spectrometers,
programmable excitation shutter, automated four-position sample changer, slits, and
high voltage to the signal and reference detectors. The software acquires, processes,
and manages data.
The lifetime application is integrated seamlessly into the steady-state software. Researchers who upgrade a Fluorolog®-3 to a Fluorolog®-Tau-3 will find the DataMax
lifetime software easy to use. Researchers using a Fluorolog®-Tau-3 instrument for the
first time will likewise find it easy to use.
8-1
Fluorolog-3 v.2.2 (10 Sep 2002)
Introduction to Lifetime Measurements
Lifetime measurements
The lifetime software is based on acquisition of frequency-domain lifetime measurements. In this technique, the excitation light is sinusoidally modulated. The emission
from the sample is a forced response to the excitation, and therefore is modulated at the
same frequency as the excitation light. The excited state has a finite lifetime. This
means that the modulated emission relative to the excitation is offset by a phase angle,
φ, and is demodulated. The following figure shows various parameters used within the
frequency domain for lifetime measurements.
Excitation
φ
Intensity
ACx
ACf
Emission
1
DCx
0
180
360
Degrees
DCf
540
Parameters used in frequency-domain measurements.
The figure shown above illustrates a sinusoidal excitation with a circular frequency, ω,
an amplitude, ACx, and offset by a DC voltage, DCx. The sample responds with an
identical circular frequency, ω, damped amplitude, ACf, and offset by a DC voltage,
DCf. In a fluorescence-lifetime measurement, the phase angle, φ, and demodulation factor, m, are measured at each frequency and used to calculate the phase lifetime (τp) and
modulation lifetime (τm). The above figure shows that the relative amplitude of the
emission (ACf/ DCf) is smaller than that of the excitation (ACx/DCx). The demodulation
factor, m, is ACf/ DCf divided by ACx/DCx.
The equations used to calculate the phase (τp) and modulation (τm) of lifetime measurements are:
8-2
Fluorolog-3 v.2.2 (10 Sep 2002)
Introduction to Lifetime Measurements
tan φ = ωτ p
τ p = ω −1 tan φ
c
m = 1 + ω 2τ 2m
τ m = ω −1
h
−1/ 2
FG 1 − 1IJ
Hm K
8-3
2
=
1/ 2
AC f / DC f
AC x / DC x
Fluorolog-3 v.2.2 (10 Sep 2002)
Introduction to Lifetime Measurements
Types of lifetime scans
The type of scan defines which measurement will be acquired. In lifetime operation,
four scan types are available:
• Lifetime
• Lifetime-resolved
• Dynamic depolarization
• Time-resolved.
Recalling an experiment allows the user to retrieve the experimental parameters. Each
scan type is defined below.
Lifetime acquisition
The lifetime acquisition type of scan determines accurate lifetimes from simple singlecomponent systems as well as complex heterogeneous systems. The measurement records the phase shift and modulation at specified frequencies for an unknown sample
relative to a reference standard.
Lifetime-resolved acquisition
A lifetime-resolved acquisition scan resolves up to three components of overlapping
spectra based on differences in the fluorescence lifetimes. More complex systems can
result in improved resolution of one or more spectra but complete resolution requires
additional manipulation of data acquisition parameters such as excitation wavelength.
An application using lifetime-resolved-acquisition scans can spectrally resolve tyrosine
and tryptophan emission spectra from a protein containing both residues. To improve
resolution, simply measure the lifetime and obtain the spectral characteristics of the individual components prior to conducting a lifetime-resolved acquisition scan. Generally, this technique works best if a factor of at least 1.5 exists between the lifetimes being resolved spectrally.
Anisotropy-decay acquisition
An anisotropy-decay acquisition experiment choice allows the study of rotational properties of fluorescent molecules and probes. As the fluorophore rotates, a change in the
polarization occurs. Monitoring this change provides information about the excited
state properties of the sample. The anisotropy is affected by Brownian rotation, energy
transfer, re-absorption, re-emission and light scattering. Applications involve studying
asymmetric complex molecules, environmental perturbations, binding, hinderedrotation phase transitions, and internal viscosities of bilayers.
Time-resolved acquisition
Time-resolved acquisition scans measure the change in the spectral characteristics of
the sample during the lifetime of the excited state. The measurement consists of determining the frequency response of the sample over a specified emission range. Applications involve solvent relaxation of the excited state and excimer formation.
8-4
Fluorolog-3 v. 2.2 (10 Sep 2002)
Xenon Lamp Information
9: Xenon Lamp Information &
Record of Use Form
Xenon lamps typically are used in fluorescence instruments because they provide a
continuous output from 240 nm to 600 nm. In the Fluorolog®-3 spectrofluorometers,
the standard xenon lamp is ozone-free. The xenon-lamp spectrum exhibits a
characteristic peak around 467 nm, which can be used to indicate whether the excitation
spectrometer is properly calibrated.
As the xenon lamp ages, water Raman spectra have a progressively lower peak
intensity. During its lifetime, the lamp stabilizes at approximately 60% of its original
intensity. Keep a record of the time the lamp is in use on the form provided on the
following page. From this record, you will be able to determine when the lamp is near
the end of its lifetime. The maximum lifetime of the 450-W lamp is 2000 hours.
9-1
Fluorolog-3 v. 2.2 (10 Sep 2002)
Xenon Lamp Information
Xenon Lamp Record of Use
Page _____ of _______
In Service
Date
Operator
Current
Date
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
/ /
Time
On
Time
Off
Total Hours
9-3
Total Time
(Hours/Min.)
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
Fluorolog-3 v. 2.2 (11 Jul 2002)
Applications
10: Applications
Introduction
The Fluorolog® series of spectrofluorometers have earned the reputation for being the
most sensitive in the world. Because the Fluorolog®-3 provides superior optical and
electronic components combined with a comprehensive selection of accessories, it provides the highest sensitivity and selectivity for all types of samples.
Jobin Yvon Inc. realizes the importance of accurate and reproducible data. Therefore,
the systems are calibrated and tested prior to shipment to a customer site and then again
after installation. Performance spectra indicate the operational parameters and ensure
that the system is operating within specifications. Fluorolog®-3 systems employ the
most sophisticated data-correction techniques to yield accurate and reproducible fluorescence spectra.
For example, fluorescence emission spectra are affected by the response characteristics
of the spectrometer and optical components such as gratings and detectors. To compensate for these responses, radiometric correction factors are individually determined for
each Fluorolog®-3 system and are supplied with the DataMax software. The user can
update these correction factors periodically if necessary. Simple point-and-click operations or user-controllable default system parameters allow real-time or post-processing
correction of spectral data.
To correct for variations in the intensity of the excitation source, Fluorolog®-3 spectrofluorometers also monitor the excitation beam with a wavelength-independent reference detector. The raw emission data can then be automatically ratioed to intensity information generated by the reference detector. The effect of this correction can be seen
in the upper plot on the next page.
10-1
Fluorolog-3 v. 2.2 (11 Jul 2002)
Applications
0.3
Intensity
0.225
0.150
0.075
0
250
300
350
Wavelength (nm)
400
250
300
350
Wavelength (nm)
400
0.41
Intensity
0.308
0.205
0.103
0
Spectra of azulene. Upper plot: corrected (solid line) and uncorrected (broken line)
excitation spectra. Lower plot: an absorption spectrum.
The broken line in the upper plot traces the uncorrected excitation spectrum of an azulene sample acquired on a Fluorolog® system without reference-detector ratioing. The
solid line represents the same spectrum automatically corrected for the wavelengthdependence of excitation-source intensity. The lower plot shows the absorption spectrum of azulene, acquired on the same spectrofluorometer system. The corrected excitation spectrum shows strong similarity and fine structure compared with the corresponding absorption spectrum.
The Fluorolog® series of spectrofluorometers have carved a niche in the scientific
community by consistently demonstrating the capability to perform extraordinary tasks.
Some of the remarkable features of the system are outlined below.
10-2
Fluorolog-3 v. 2.2 (11 Jul 2002)
Applications
Detecting sub-picomolar concentrations of
fluorescein
Instrument sensitivity is often expressed in terms of the limit of detection of a standard
substance. The superior sensitivity of Fluorolog®-3 systems is demonstrated by their
ability to detect sub-picomolar concentrations of standard substances such as fluorescein. Often, other instruments perform this task by extrapolating from much higher
concentrations. The Fluorolog®-3, however, not only detects femtomolar concentrations
of fluorescein, but directly measures the true emission spectrum of 50-femtomolar fluorescein using an integration of only 1 second—a clear demonstration of superior performance.
Reduced-volume samples
Intensity (× 106 counts s–1)
Because the samples required to produce spectral data may be expensive or obtained
only in limited quantities, the precise imaging quality and photon-counting sensitivity
of the Fluorolog®-3 spectrofluorometers are invaluable assets. The following diagram
compares the fluorescence emission of 20-nM resorufin acquired using the 20-µL and
4-mL cells. Notice that the signal level is maintained with either cell.
20 nM resorufin in
20-µL flowcell
20 nM resorufin in 4mL flowcell
600
650
Wavelength (nm)
700
Comparison of the fluorescence emissions of 20-nM resorufin using a 20-µL cell
and a 4-mL cell.
10-3
Fluorolog-3 v. 2.2 (11 Jul 2002)
Applications
Fluorescence detection of highly scattering
samples
Qualitative and quantitative determinations normally are difficult to ascertain from
highly scattering samples. Typically, fluorescence signals are dwarfed by stray or scattered light from the sample. The flexibility of the Fluorolog®-3 systems, however, allows the introduction of a double-grating emission spectrometer, thereby improving the
system’s stray-light rejection. With a single-grating emission spectrometer, scattered
light from the sample often finds its way through the exit along with the selected band
of emitted light. However, when the band is re-dispersed by the double-grating spectrometer, most stray light is stripped away as the band passes through the exit slit to the
emission detector.
Quantum-yield calculations
Among the many features unique to Fluorolog®-3 systems, is the capacity to measure
fluorescence-related parameters required to calculate the quantum yield for samples in
solution. This includes recording excitation-source intensity and calculating the area
under the corrected emission spectra.
Characterizing complex mixtures via
synchronous scanning
The fluorescence spectrum of a complex mixture often contains overlapping spectral
features representative of the mixture and revealing no indications of the contents of the
sample. A spectrum of this nature is all but useless. Synchronous scanning offers a solution to this problem.
Simultaneous scanning of the excitation and emission spectrometers with a constant
offset between them yields an intensity proportional to the product of the emission and
excitation intensities. This resulting spectrum often can be analyzed readily.
The figure below shows the emission scan and the synchronous scan of a mixture of
polynuclear aromatic hydrocarbons.
10-4
Fluorolog-3 v. 2.2 (11 Jul 2002)
Applications
1.0
Intensity (× 106 counts s–1)
Perylene
436 nm
7.5
Anthracene
382 nm
5
2,3-Benzofluorene
342 nm
Fluorene
303 nm
2.5
0
Acenaphthene
323 nm
300
400
Wavelength (nm)
500
Emission (solid line) and synchronous (broken line) scans of a mixture of
polynuclear aromatic hydrocarbons.
The solid line in the figure above is the emission spectrum acquired on a Fluorolog®
system (single-grating monochromators) with constant wavelength excitation. As
shown by the broken line, when the sample is scanned synchronously, five individual
components are resolved into unique, sharp peaks indicative of the individual compounds.
Operating in the IR region
The Fluorolog®-3 series of spectrofluorometers can be equipped to operate in the infrared region of the spectrum, thereby opening up totally new areas of applications for
fluorescence spectroscopy. Typically, an IR spectrofluorometer requires components
significantly different from those found in conventional instruments. Because the most
widely used photomultiplier detector is insensitive above 860 nm, an IR spectrofluorometer must be equipped with a red-sensitive photomultiplier, or a solid-state detector
whose response is effective far into the IR region. With a photomultiplier sensitive to 1
µm, only minimal system modification is necessary. A phase-sensitive lock-in amplifier
and a light chopper are required.
Phosphorescence for time-resolved data
Both fluorescence and phosphorescence spectra are photon emissions that occur when
molecules return from an excited electronic state to the ground state. The nature of the
excited state distinguishes the two: fluorescence is associated with relaxation from a
singlet excited state, while phosphorescence is associated with relaxation from a triplet
excited state. Fluorescence usually occurs within a few nanoseconds after excitation.
10-5
Fluorolog-3 v. 2.2 (11 Jul 2002)
Applications
Because triplet transitions are “forbidden” quantum-mechanically, the average phosphorescence decay times are generally longer, ranging from a few microseconds to several seconds. Thus, phosphorescence offers a longer observation period for monitoring
reactions, looking at environmental effects on a sample, or following changes in the
hydrodynamic characteristics of macromolecular systems.
In phosphorescence experiments using the Fluorolog®-3 and the FL-1042 Phosphorimeter Assembly, the sample is excited by a pulsed light source. Acquisition of the
emission signal is synchronized to the pulse, with user-specified delay and sampling
times, to produce time-resolved spectral data. With an appropriate choice of delay time,
the user may select only the luminescence of interest.
Time-resolved data-acquisition also makes it possible to acquire phosphorescence decay curves and compute lifetimes of lanthanides such as europium and terbium, as well
as the biological probe eosin.
Low-temperature scans
One way to protect a sample from molecular collisions that can quench luminescence is
by isolating the sample in a rigid matrix. Thus, cooling with liquid nitrogen enhances
the phenomenon of fluorescence, even for seemingly dormant samples. In addition, the
superior resolution of a Fluorolog®-3 double-grating spectrometer system optimizes
measurements under these conditions.
Monitoring kinetic reactions using timebased fluorescence
By setting the wavelengths at the excitation and emission peaks of a sample, the
Fluorolog®-3 systems can monitor fluorescence as a function of time. This permits the
use of Fluorolog®-3 systems in reaction-rate determinations, which monitor the formation or breakdown of a fluorescing species. Reaction-rate determinations are highly selective. Because only changes in intensity are considered, the method is not affected by
interference from continuous background signals or steady-state scatter.
Front-face detection to enhance data
collection for absorbent or solid samples
Fluorescence typically is collected at right angles (90°) from transmitted or scattered
light. Yet right-angle viewing is inappropriate for some samples. Imprinted paper, for
example, reflects light, which interferes with accurate data collection. In highly absorbent samples like hemoglobin or milk, most of the emitted light is reabsorbed before
the fluorescence can be measured. A significant design feature of the Fluorolog®-3 Single-Beam and T-Box spectrofluorometers is that they offer a choice between conventional right-angle or front-face fluorescence detection. Front-face viewing is ideal for
solid, turbid or highly absorbent samples such as pellets, powders, and monolayers on
10-6
Fluorolog-3 v. 2.2 (11 Jul 2002)
Applications
microscope slides. A swing-away mirror is positioned to allow collection of sample
luminescence at 90° to the excitation beam, or front-face at 22.5°. In front-face viewing, the fluorescence is collected from the sample’s surface.
Polarization to detect trace quantities of
biological probes
Used in conjunction with the large number of fluorescent dyes suitable for biological
research, fluorescence spectroscopy has greatly expanded our understanding of metabolic processes on the molecular level. The Fluorolog®-3 design offers unparalleled
sensitivity for such work.
Fluorescence polarization offers a safe, sensitive immunoassay technique—
fluoroimmunoassay (FIA). This method has none of the licensing and waste-disposal
problems associated with radioimmunoassay (RIA). Immunoassay methods, which are
based on competitive antibody-binding reactions, require the ability to distinguish between bound and unbound species. This selectivity is inherent in fluorescence polarization techniques.
In fluorescence polarization, the excitation beam is passed through a polarizing prism,
and the emitted luminescence is analyzed with another polarizer alternately oriented
parallel and perpendicular to the excitation polarization. The measured polarization depends on the rotation of the molecules between absorption and emission. Because the
measurement reflects changes in rotation, polarization can be used to distinguish between free molecules and the larger, slower antibody-bound molecules in immunoassays. The use of polarizer prisms instead of film polarizers ensures that the researcher is
guaranteed full spectral coverage from the ultraviolet to the visible. Over time, film polarizers tend to become photobleached, especially if exposed to UV light.
The applications for the Fluorolog®-3 spectrofluorometers are almost endless. By simply changing accessories, adding or removing a hardware component, or accessing the
proper software controls, the user can ensure that the system continues to grow or
change as application needs change. The modular construction and interchangeable accessories make the Fluorolog®-3 even more attractive to most industries.
10-7
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
11: Producing Correction
Factors
Introduction
Collecting accurate information about the fluorescent or phosphorescent properties of a
sample depends upon several factors: equipment, sample, and timing. To ensure that
the spectra are indicative of the actual sample properties and not of external conditions,
data often must be corrected. To correct data means to remove information from the
data not directly related to the properties of the sample. Specifically, several items that
may appear mixed into a spectrum are:
• Fluctuations caused by the light source.
• Influence of the sample holder.
• System features.
Corrections are made for each of these potential problems by using radiometric correction factors, running a blank scan (which is then subtracted from the sample scan), and
using the software’s Auto Zero function (which closes the programmable excitation
shutter, records the background dark counts for 10 s, and automatically subtracts this
value from the data as they are acquired). Blank and Auto Zero functions are described
in the system’s software manual.
Gratings, detectors and other spectrometer components have response characteristics
that are functions of wavelength. These characteristics are superimposed on spectra,
and may yield a potentially misleading trace. For accurate intensity comparisons, such
as those required for quantum-yield determinations, spectrometer-response characteristics must be eliminated.
Supplied with your instrument are sets of excitation and emission correction factors designed to eliminate response characteristics. These files1, xcorrect and mcorrect,
are included with the software and should be copied to the hard disk. These files must
be in the same directory as the data to be corrected. The excitation correction range is
from 220–600 nm, and the correction range for emission spectra is from 300–850 nm.
Instructions for use of correction factors are in Chapter 4. This chapter only describes
the steps for generating correction factors. Perform this procedure only when the gratings or detectors have been replaced with those of different specifications than the
original hardware.
1
Filenames include a three-letter extension. For the sake of clarity, the extensions are omitted in this manual. Refer to the software manual for specifics regarding extensions.
11-1
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
Generating emission correction factors
Required kits
Emission correction factors should be updated periodically or whenever different gratings or signal detectors are installed. The correction factors can be updated either at the
user’s location, or by a representative from the Spex® Fluorescence Service Department. To arrange for a visit and a fee estimate, call our service department. To update
the correction factors without a service visit, follow the instructions below.
One way to generate correction factors for your instrument is to scan the spectrum of a
standard lamp. Because the actual irradiance values of the standard lamp as a function
of wavelength are known, dividing the irradiance values by the lamp spectrum results
in a set of relative correction values. These values can then be applied to the raw fluorescence data. The emission correction factor file mcorrect was acquired in this
manner.
To generate emission correction factors, several items are needed: a standard lamp, appropriate holders, and a scatter assembly. Jobin Yvon Inc. offers two kits: the
Model 1908 Standard Lamp Accessory, and the Model 1908MOD Scatter Assembly.
The Model 1908 is a complete correction factor kit, while the Model 1908MOD Scatter
Assembly is provided for users who already have a calibrated standard lamp and a constant-current source.
The Model 1908 Standard Lamp Assembly is a complete correction factor kit, which
includes the following items:
• 200-watt quartz tungsten-halogen filament lamp with irradiance values
• Constant Current Power Supply with lamp holder
• 1908MOD Scatter Assembly
•
•
•
The Model 1908MOD Scatter Assembly includes:
Lamp Mount Assembly and Mask with Square Center
Scatter Block with neutral-density filter and reflectance plate
11-2
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
Generation
1
Open the Real Time
Display.
2
Turn off the high
voltage to the
emission
photomultiplier tube.
3
4
Close the slits.
Place the 1908MOD
Scatter Block
Assembly in the
sample chamber, so that
light is directed toward the
right angle.
Looking down, the scatter plate should be
toward the left.
5
Place the mask over the
sample compartment.
The square hole should be vertically
centered over the white scatter plate. Secure
the mask with black tape.
11-3
Fluorolog-3 v. 2.2 (31 Jul 2002)
6
Attach the 2 wire ends of the standard lamp to
the lamp holder
posts.
a
b
c
7
Producing Correction Factors
The positive (+) lead goes to
the positive (red) side of the
holder.
The negative (–)
lead goes to the
negative (black)
side of the holder.
Warning: Do not touch the
lamp. Use cotton gloves or
lens paper.
Nipple points upward
The nipple of the
lamp should point
upward.
Fix the lamp holder to the top of the sample
compartment with double-sided tape.
Make sure the the filament is vertically centered over the fixture.
View looking downward from the standard lamp to the mask. Dotted lines
(in perspective) indicate how the lamp should be over the square hole.
11-4
Fluorolog-3 v. 2.2 (31 Jul 2002)
8
Producing Correction Factors
Connect the 2 wire leads from the constantcurrent power supply to the lamp holder.
Attach the red wire to the red clip and the black wire to the black clip, located
on the sides of the lamp holder.
9
Turn on the constant current power supply. Wait
until the current ramp function is 6.5 A.
This may take up to
two minutes. For
valid
irradiance
values, the lamp
current must be
maintained at 6.5 A.
Warning: Avoid looking directly into the
lamp’s radiation. Wear protective eyeglasses to shield against ultraviolet light.
10
Turn off the
room lights.
11
Set the emission spectrometer to 520 nm, and
both emission slits to a 5 nm bandpass.
12
Turn on high voltage to the signal detector
(HV1).
13
Observe the intensity of the signal detector.
Note: The signal level should not exceed 3 × 106 cps—the linear
range of the R928P detector—when operated in the photoncounting mode of detection. If necessary, open or close the
emission slits to adjust the signal intensity.
A good emission correction factor file depends on ample signal at both high and
low points of the lamp spectrum.
14
Set the emission spectrometer to 290 nm.
This is the wavelength at which the standard lamp has its lowest light output.
15
Check the signal: there should be sufficient
intensity above the dark counts.
11-5
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
Determine the dark counts
1
Place the sample lid over the mask to block light
to the detector.
2
In Run Experiment, select the Experiment button.
This opens the Emission Acquisition dialog box.
3
Type in the following parameters in the Emission
Acquisition dialog box:
Number of Scans
Start
End
Increment
Integration Time
Excitation Position
1
290 nm
850 nm
5 nm
1s
350 nm
4
5
6
Run the standard lamp spectrum.
7
Run another scan using the same parameters,
and name this file blank.
Name this file stlamp.
Place the lid over the mask to block the light
between the standard lamp and the scatter
fixture.
This data file is a straight line with low intensity.
8
9
Choose Arithmetic from the toolbar.
10
Name this file stdlamp2.
Using the Arithmetic menu, subtract blank from
stdlamp.
Below are the blank and lamp spectra. Notice that the blank is almost nonexistent. Because of the low intensity of the blank file, the blank-subtracted file,
stdlamp2, will resemble the stdlamp file.
11-6
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
Intensity (× 106 counts s–1)
1.2
stdlamp
0.9
0.6
0.3
blank
0
300
400
500
600
Wavelength (nm)
700
800
Blank and lamp spectra.
Your spectrum should appear similar to the one pictured above. Its actual
appearance, however, depends on the configuration of your Fluorolog®-3
system. The lamp scan was acquired with gratings in the emission spectrometer
blazed in the visible region and an R928P red-sensitive photomultiplier as the
detector. Different gratings or detectors may alter the shape of the lamp
spectrum.
Note: Obtaining emission correction factors for the region between
250 nm and 300 nm is possible. Because the gratings are extremely inefficient in this range and the standard lamp output is
low, generating these factors is somewhat more involved.
11-7
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
Calculating emission correction factors
Introduction
Irradiance values for a standard lamp, packaged with the lamp, are usually expressed in
10–6 W·cm–2·nm. With photon-counting systems like the Fluorolog®-3 spectrofluorometers, however, data usually are collected in units of photons·s–1·cm–2·nm. To convert the
units, multiply each irradiance value by the wavelength at which it is valid. (The data
will still be off by a factor of c, but normalizing the correction factors compensates for
this.)
For more information about the theory and application of radiometric correction, consult Accuracy in Spectrophotometry and Luminescence Measurements, Mavrodineau,
Schultz, and Menis, NBS Spec. Publ. 378 (1973), especially p. 137, “Absolute Spectrofluorometry,” by W.H. Melhuish.
Load the irradiance values
1
In Run Experiment, choose the
Experiment button
This opens the Emission Acquisition dialog box.
2
Click Exp Type.
This opens the Select Experiment Type dialog box:
11-8
Fluorolog-3 v. 2.2 (31 Jul 2002)
3
Producing Correction Factors
Choose
Emission
Acquisition.
Click OK to close the
Select
Experiment
Type dialog box.
4
Enter the
following parameters:
Start the scan at 300 nm.
End the scan at 850 nm.
Increment the
wavelength by
50 nm each
step.
Use an integration
time of 0.1 s.
End the scan at 850 nm.
11-9
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
a
b
Click Signals... to
open the Signals
dialog box:
Click HV to
open the High
Voltage dialog
box:
Turn off the voltage to the detectors.
Select S (signal) detector.
Click OK to close this
box.
5
6
By turning off the high voltage, a file which
has 0 intensity for each point will be
created. The irradiance values will be
entered for each 0 point.
Run the scan with these parameters.
Name the file CRE.
After performing the irradiance value multiplication and displaying the file CRE
on the screen, enter each value as follows:
Note: Remember to multiply each irradiance value by the wavelength at which it is valid prior to attempting to enter its value. The
units will be photons·s–1·cm–2·nm.
7
8
Select View from the toolbar.
Under View, choose Table View.
A table appears, displaying the wavelengths and the value of the intensity at
each wavelength. The table may be displayed showing only the last value at the
last wavelength. Click on the value (the number will become highlighted), and
use the arrow keys to scroll up and down.
11-10
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
Because this file was created with the high voltage off, each intensity value is
zero. The irradiance values calculated in a previous step will be entered into the
intensity locations for each wavelength.
9
Click on the value to be changed. From the
keyboard, type in the calculated irradiance
value.
As soon as you begin to
type, the Edit Table dialog
box will appear:
Click on the up and down
arrows to move within a
column, or click on the left
and right arrows to move
between columns. After all of the irradiance values have been entered,
10
Click on OK.
The CRE file has the irradiance values incremented every 50 nm. To calculate
the actual correction factors, the irradiance file needs to have a 5-nm increment.
11
Using the instructions presented above, create
another file.
(Keep the high voltage off.) This time, however, enter 5 nm for the Increment
instead of 50 nm. Name this file IRR.
When this scan is complete, the scan range will be from 290–850 nm with an
increment of 5 nm. All data points will have intensity values of 0 cps.
12
Using the Arithmetic menu, add the constant 1
to the IRR file and resave it as IRR.
13
Multiply IRR by CRE and resave it as IRR.
This causes the original IRR file to be overwritten. The irradiance values will
be spaced every 5 nm as opposed to every 50 nm. The IRR file should look
similar to this:
11-11
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
4000
Intensity
3000
2000
1000
0
250
350
450
550
650
750
850
Wavelength (nm)
IRR file.
Now you have the two files: IRR and stdlamp2. These files are required to
calculate the emission correction factors for the Fluorolog®-3 system.
11-12
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
Calculate the correction factors
1
Using the Arithmetic menu in Run Experiment,
divide IRR by stdlamp2, and name the
resulting file mcorrect.
Note: Naming the file mcorrect will overwrite the
mcorrect file supplied with the software.
Normalize the mcorrect file
1
Display the mcorrect file and find the minimum
signal intensity.
2
Using the Arithmetic menu, divide the
mcorrect file by this minimum signal intensity.
3
Save this new file as mcorrect.
(That is, overwrite the existing mcorrect file). This normalizes the correction
factor file so that the minimum intensity of mcorrect will be 1 count s–1.
mcorrect contains the emission correction factors for the system. The
correction-factor file should look similar to this:
250
Intensity
200
150
100
50
0
250
350
450
550
Wavelength (nm)
mcorrect File.
11-13
650
750
850
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
The correction factors shown above were acquired for a Fluorolog®-3 system
with 500-nm blazed gratings in the emission spectrometer and a red-sensitive
R928P photomultiplier detector.
Once the emission correction factors have been found, determination of the excitation
correction factors may be necessary. The following procedures describe how to obtain
excitation correction factors using the photomultiplier and the photodiode. Follow the
procedure that applies to your configuration.
11-14
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
Calculating excitation correction factors
The photodiode reference detector handles the bulk of excitation correction from 240–
600 nm when a ratio acquisition mode is selected (e.g., S/R for single-beam and T-box
sampling modules). More accurate measurements require that compensation be applied
for the difference in optical path between the detector and the sample. This can be accomplished by a simple excitation scan with rhodamine-B placed in the sample position.
1
Fill a cuvette with a solution of rhodamine-B.
Use 8 g L–1 of laser-grade rhodamine-B in 1,2-propanediol.
2
Place the cuvette in the sample compartment.
For single-beam sampling modules, place the cuvette in the standard cell holder
and select right-angle detection.
3
Enter the Real
Time Display.
4
Set the excitation
and emission
monochromators
to 467 nm and 630
nm, respectively.
The largest lamp peak occurs
at 467 nm.
5
Set the two slits
on the excitation
spectrometer to
0.5 mm.
6
Make sure the
shutter is open.
7
Set the excitation monochromator to 560 nm
and the emission monochromator to 630 nm.
8
Adjust the slits on the emission monochromator.
11-15
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
The slit width discovered in
this step will be used to run the
scan.
Note: Obtain a signal intensity of
no greater than 1.5 × 106 cps.
9
With the Real Time
Display still running, open Run Experiment.
10
11
In Run Experiment, choose Collect.
Under Collect, choose Experiment.
This opens the Emission Acquisition dialog box:
12
Click Exp
Type.
This opens the Select
Experiment
Type
dialog box:
13
Choose
Excitation
Acquisition,
then OK.
This closes the Select Experiment Type window, and then resets the Emission
Acquisition dialog box to Excitation Acquisition.
11-16
Fluorolog-3 v. 2.2 (31 Jul 2002)
14
Producing Correction Factors
Click Signals....
This opens the Signals dialog
box:
15
Enter S/R as the
Selected Signal.
Click OK to close the Signals
box.
16
Click Slits....
This opens the Slits dialog box:
17
Use the XFER
button in the Real
Time Display to
move the slit widths
to Slits.
Click OK to close the Slits
window.
18
Click HV.
This opens the High Voltage dialog box:
19
Use the XFER button
the high voltages to High Voltage.
to move
Click OK to close the High Voltage window.
20
Enter the remaining parameters in the Excitation
Acquisition window:
11-17
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
Start the scan at 240 nm.
End the scan at 600 nm.
Increment by 5 nm
after each data point.
Set the Integration
Time to 5 s.
Perform one scan.
Note: Be sure the Auto Zero function is on.
21
22
23
Click Run to execute the scan.
Save the file
as
xcorrect.
Note: Doing so replaces the xcorrect file
that was shipped with the system.
If no zeroes are shown, invert the data.
In Run Experiment, use the Arithmetic menu to divide the spectrum into 1.
24
Normalize the data.
11-18
Fluorolog-3 v. 2.2 (31 Jul 2002)
Producing Correction Factors
Find the minimum data point and divide the file by that value.
25
Save the normalized file as xcorrect.
This overwrites the existing excitation correction-factor file.
To acquire corrected data for an experiment, enter the name of this file in the CORRECTION factor file field in the Data Acquisition Parameters dialog box. The CORRECTION factor file must be in the same directory as the data to be acquired and corrected.
11-19
Fluorolog-3 v. 2.2 (11 Jul 2002)
Determining the Plateau Voltage
12: Determining the Plateau
Voltage
Introduction
The plateau voltage is the voltage at which the signal intensity is maximized, while the
dark-count intensity is minimized. The emission signal detector requires a voltage input to
operate. The operating voltage determines the sensitivity of the detector. To achieve the
highest sensitivity possible, the plateau voltage determination should be performed whenever a new detector is installed.
Approximately 30 min are needed to acquire the data and 15 min to display the data in the
form of two data files.
Note: In the standard configuration, the software supplies a maximum
voltage of 1200 V to the detector(s). Certain applications (IR fluorescence measurements, for example) require detectors with operating
voltages near 1450 V. In these cases, the standard high-voltage board
should be replaced with an optional board that increases the highvoltage output to 2000 V.
Overview of the procedure
1 Compose a table with dark counts and
corresponding signal intensities at incremental
voltages.
2
Construct a graph. Note the point at which the
signal intensity is maximized and the dark-count
intensity is minimized.
This is the plateau voltage, the point at which the highest sensitivity of the
detector is achieved.
To record the values, construct or photocopy the following table:
12-1
Fluorolog-3 v. 2.2 (11 Jul 2002)
Determining the Plateau Voltage
High Voltage
(Volts)
Signal
(cps)
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
12-2
Dark Counts
(cps)
Fluorolog-3 v. 2.2 (11 Jul 2002)
Determining the Plateau Voltage
Procedure
Set up the instrument
1
Turn on the xenon lamp, Fluorolog®-3 system,
peripherals, and computer.
2
3
Enter the DataMax software.
Run a xenon-lamp spectrum and the water
Raman scans.
Do water Raman scans both right-angle and front-face to ensure that the system
is functioning properly.
4
5
Enter the Real Time Display.
6
Insert a cuvette filled with distilled water in the
sample holder.
7
Select right-angle detection.
Set the slits of the excitation and emission
spectrometers to a bandpass of 5 nm.
Turn the selection mirror knob located on top of the sampling module to RA.
8
9
Set the Integration Time to 2.0 s.
10
Display the High Voltage dialog box by clicking
on the icon.
11
Turn on HV1 (signal detector or S voltage).
Position the excitation and emission
spectrometers to 350 nm and 397 nm,
respectively. Click on the check boxes to
activate them.
Record the signal from 650 V to 1200 V.
12-3
Fluorolog-3 v. 2.2 (11 Jul 2002)
Determining the Plateau Voltage
1
Open the programmable excitation shutter by
clicking on the shutter icon.
2
3
Enter 650 V in the S voltage text box.
Click on the right arrow on the
Continuous area.
This records data in Prompt-Step mode (see the DataMax
manual for details).
4
Record the signal in cps at 650 V in a table such
as the one shown on a previous page.
This intensity will be the same value as the intensity at the peak of the water
Raman scan acquired, using right-angle detection and the specific high voltage.
5
Close the shutter by sliding it to the right.
This closes the manual shutter on the variable exit slit of the emission
spectrometer.
6
Record the dark counts at 650 V.
Write this value next to the signal intensity in your table.
7
8
Open the shutter by sliding it to the left.
9
Repeat steps 3 through 8,
Change the high voltage of the signal detector
from 650 V to 700 V.
increasing the signal detector voltage by 50 V each time and recording the
signal intensity and the corresponding dark counts.
The sample data below were acquired using a Fluorolog® Model FL212
equipped with a 450-W xenon lamp and an uncooled signal detector.
12-4
Fluorolog-3 v. 2.2 (11 Jul 2002)
Determining the Plateau Voltage
High Voltage
(Volts)
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
Signal
(cps)
8.7 × 104
1.28 × 105
1.52 × 105
1.68 × 105
1.74 × 105
1.80 × 105
1.81 × 105
1.96 × 105
1.96 × 105
2.05 × 105
2.12 × 105
2.25 × 105
Dark Counts
(cps)
150
260
350
480
420
450
490
530
640
560
640
710
Determine the plateau voltage
From the data obtained, construct two graphs:
Signal versus Voltage, and Dark Counts versus
Voltage.
Graphs of the above data are shown here, stacked into one combined graph.
Signal (105 counts/s)
2.5
2
1.5
1
800
0.5
Dark Counts versus Voltage
700
Dark Counts (counts/s)
1
600
500
400
300
200
100
0
600
700
800
12-5
900
Voltage (V)
1000
1100
1200
Fluorolog-3 v. 2.2 (11 Jul 2002)
Determining the Plateau Voltage
From these graphs, at ~1000 V there is a plateau both in the signal and dark
counts. Therefore, the point at which there is maximum voltage with minimum
dark counts is 1000 V—the plateau voltage for this detector.
12-6
Fluorolog-3 v. 2.2 (11 Jul 2002)
Reassembly Instructions
13: Reassembly Instructions
The Fluorolog®-3 system consists of four
main components:
• Personal computer
• Color monitor
• Expanded keyboard
• Fluorolog®-3 spectrofluorometer
Warning: Jobin Yvon Inc. does
not recommend reassembly by
the user. Contact the Spex®
Fluorescent Service Department for reassembly. Moving
the Fluorolog®-3 system may
degrade performance, and invalidates all warranties by Jobin
Yvon Inc.
An optional printer may also be
included. The Fluorolog®-3
spectrofluorometer is a combination of
discrete modules and components. These
components include excitation and
emission monochromators, sample compartment, xenon light source and a System Controller (SAC).
Occasionally, because of relocating the instrument, reassembling the system is necessary. The following instructions are provided as a guide.
Computer
The computer must be set-up and operating before the spectrofluorometer system can
be connected.
1
Connect the
Note: The computer’s COM1 port will be
computer’s
connected to the System Controller in a
components
later step. Make sure this port is available.
according to
the instructions with the computer.
2
Plug the power cables
from the monitor, the
main unit (CPU), and
the printer or plotter
into outlets with the
proper line voltage.
Connect later
to SAC
Warning: Do not turn on the
computer or the peripherals.
13-1
Fluorolog-3 v. 2.2 (11 Jul 2002)
Reassembly Instructions
Spectrofluorometer assembly
Once the computer has been assembled, the external components of the Fluorolog®-3
spectrofluorometer must be connected. The modules of the Fluorolog®-3 fit together in
a seamless configuration. Each module and the lamp housing has alignment studs,
alignment receptacles, and D-connectors on the outside of the housing:
Stud
Stud receptacle
Male D-connector
1
Female D-connector
Assemble the spectrofluorometer system’s
modules into the proper configuration.
That is, attach the spectrometers to the sample compartment, and the lamp
housing to the excitation monochromator.
Source
Detector
Emission
spectrometer
Excitation
spectrometer
Sample compartment
module
Note: See Chapter 2, System Description, for configurations.
Concerning
the lamp housing, xenon
sources, and cautions,
see Chapter 5, System
Maintenance.
Example of a Fluorolog®-3 configuration.
13-2
Fluorolog-3 v. 2.2 (11 Jul 2002)
Reassembly Instructions
Cable connections
These
connectors
connect
the
spectrofluorometer
components with the
System Controller (SAC)
and
the
computer
system.
BNC Connector
D-shell Connector
MHV Connector
SHV Connector
4-Pin Circular Connector
The jacks on the rear of the SAC are shown to
the right:
Fan
Note: The actual layout of connections on the rear of the SAC may
vary, depending on the system’s
configuration.
Trigger
180F
COM 1
Input S
Input T
Input R
Input A
HV1
HV2
HV3
HV4
Rear of SAC
13-3
Fluorolog-3 v. 2.2 (11 Jul 2002)
Reassembly Instructions
The cable diagram below shows the path of each cable. During installation, refer to the
schematic to ensure proper system interconnections.
AC
mains
AC
mains
AC
mains
Lamp
housing
System
controller
(SAC)
Mono drive
Excitation
spectrometer
In
Sample
compartment
module
DM302
module
Out
R
HV1
S
HV2
Power
Emission
spectrometer
Signal
detector
Note: May be a
single- or doubleended cable.
13-4
Note: Use HV2 for the second
emission spectrometer.
Fluorolog-3 v. 2.2 (11 Jul 2002)
Reassembly Instructions
Connecting the reference detector to the SAC
The Fluorolog®-3 spectrofluorometer system comes standard with a silicon-photodiode
reference detector inside the sample-compartment module. The connections to the reference detector are on the outside front of the sample compartment module. The sample
compartment module (reference detector) is joined with the SAC via a split cable; that
is, a cable with three ends.
1
Find the double-headed
end of the split cable
#33979A.
2
Plug the end with the 7-pin
D-shell connector into the
D-shell jack on the front of
the sample compartment
module. Plug the BNC plug
into the BNC jack next to
the D-shell jack.
3
BNC connector
D-shell connector
Plug the remaining D-shell
end of the cable into the INPUT R connector on
the back panel of the SAC.
DM302 PC module and signal-detector connections
1
Plug the end of the high-voltage cable #34040
with the MHV connector into the signal detector.
2
Connect the other end to the HV1 SHV
connector on the SAC.
3
The DM302 PC module has three plugs labeled
SIGNAL OUT, POWER and IN. Plug the short
cable #30645 with the BNC connector from the
signal detector to the IN connector on the
DM302.
The DM302 is joined to the SAC via the split cable #33977.
13-5
Fluorolog-3 v. 2.2 (11 Jul 2002)
Reassembly Instructions
4
Take the end of the #33977 cable that has two
connectors.
5
Plug the end with the 4-pin circular connector
into the POWER jack of the DM302, and the
BNC plug into the OUT jack of the DM302.
6
Plug the remaining end of the #33977 cable into
the INPUT S connector on the back panel of the
SAC.
Sample compartment module
1
25-pin–9-pin cable
Insert the 25-pin–9-pin cable
#400108 between the
sample compartment and
the jack labeled “mono
drive” on the SAC.
Note: With special options (e.g.,
trigger accessory), there may be
an additional cable connection.
Refer to the accessory’s instructions.
Lamp power supply
The lamp power supply is integrated into the lamp housing. No additional connections
are required.
Computer to SAC
The spectrofluorometer system is controlled via the SAC through the computer.
1
Connect the 9-pin–9-pin connector cable
between COM1 of the computer and COM1 of
the SAC.
13-6
Fluorolog-3 v. 2.2 (11 Jul 2002)
Reassembly Instructions
Connecting power cables
Several components in the system have AC power cables. These items include:
• SAC
• Computer (and peripherals)
• Lamp
• System Accessories (such as MicroMax®, temperature bath, etc.)
1
•
•
Plug the AC components into a
properly rated receptacle or
power strip.
Calibrate the system before conducting an experiment.
Refer to the appropriate chapter for calibration instructions.
To properly operate the equipment, have a complete understanding of the software.
Read the software manuals to gain an understanding of the system interaction, and to
discover the best methods to unleash the power of DataMax.
13-7
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
14: TRIAX operation with the
Fluorolog®-3
Introduction
Some users choose the TRIAX series of imaging spectrometers as a building block in a
Fluorolog®-3 spectrofluorometer. Using a TRIAX imaging spectrometer on the emission side of the sample mount offers the option of detection with a CCD, to create an
image of the dispersed fluorescence for subsequent analysis. This chapter discusses
special hardware connections and software operation specific to TRIAX users, especially with regard to CCD detectors.
Fluorolog®-3 TRIAX users should note these major differences between the TRIAX
and standard monochromators:
• Four ports generally are available, rather than two on a standard monochromator.
Most TRIAX spectrometers used with the Fluorolog®-3 have two entrance ports
(“lateral”, i.e., on the side, and “axial”, i.e., on the back) and two exit ports (lateral
and axial). Usually the lateral entrance port is attached to the sample compartment.
Lateral entrance port (with
baseplate)
Axial exit port
with CCD
Axial entrance
Lateral exit port with PMT
•
•
A DataMax-controlled flip mirror is available to
choose between entrance ports and exit ports. Be sure
the flip mirror is set to the proper position before
running an experiment.
Three gratings can be mounted on the rotatable grating
turret. Choosing which grating to use is under
DataMax’s control.
14-1
Flip mirror in
axial position
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Hardware
Electrical
power
Warning: Some users may wish to remove the monochromator and replace it with a TRIAX. Always switch
off the power to the monochromator and TRIAX before
removal of these accessories from or addition to a system. Failure to shut off the power to the TRIAX and
monochromator can damage the SpectrAcq.
Have a free
AC (mains)
outlet available
for the TRIAX. A 24-V adapter and power supply plugs into the AC circuit, and connects to the back of the TRIAX. The power supply contains a voltage converter that
automatically adjusts to the input AC voltage.
Internal workings
Multiple gratings
Generally, gratings with different groove-densities are
installed, but gratings with different blaze-angles are also
possible for custom applications. Rotation to a new grating is
discussed in Visual Instrument Setup below. After installing
a new grating on the turret, its groove density must be
entered in Visual Instrument Setup, and its calibration must
be performed in Visual Instrument Setup. Both of these
procedures are described later in this section.
Top view of TRIAX
grating turret.
Turret removal
2
3
4
Grasp turret
Warning: Never touch
the face of a grating!
by the
thumbscrew
on top.
Lift the turret up and out of the instrument.
Leave one shimwasher on the shaft.
Turret installation
5
Reverse steps 1 to 3.
6
Rotate turret until it
rests in a depression.
The turret is self-indexing, so the
initial position does not matter.
When switched on, the turret rotates to the correct position.
14-2
Warning: Leave clearance under the collar when replacing
the top shim-washer. This ensures free rotation of the turret.
The turret rises several mm
when it switches gratings. If
the collar is too tight, it causes
the motor to grind. This may
damage the drive mechanism.
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Automated slits
The TRIAX has automated adjustable slits at the entrance (2 mm maximum) and exit (7
mm maximum) ports. The slit width may be adjusted in steps of 0.0125 mm. For a 0.6mm slit on the TRIAX 320, dispersion is 1.58 mm.
Connections to system
Below is a sketch of the special cable connections required between a TRIAX spectrometer, the CCD detector, and the system. Details of these connections are given on
the following pages. Other Fluorolog®-3 connections are not shown.
Baseplate adapter
RS-232 cable
Fluorolog®-3
sample
compartment
TRIAX
BNC
cable
CCD
37-pin
cable
GPIB cable
CCD Controller
SpectrAcq
Host PC
Attachment to sample compartment
Fluorolog®-3 users typically choose the
lateral entrance port as the path for luminescence from the sample compartment into the
TRIAX. To attach the TRIAX to the sample
compartment, a baseplate adapter must be
inserted between the sample compartment
and the TRIAX.
Baseplate attached to lateral
entrance port.
14-3
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Communication between TRIAX and SpectrAcq
A cable runs between the TRIAX and SpectrAcq for proper
communication. To the right is a photograph of the cable
attachment to the TRIAX.
Cables to CCD detector
A coaxial cable runs from the terminal on the back of the TRIAX
to the CCD. This controls the CCD shutter. The large 37-pin
cable (bound together with the coaxial cable) carries data between
the CCD controller and the CCD chip inside the detector.
CCD detector
Cable from
controller
to CCD
CCD
detector
CCD controller
A GPIB cable connects between the
CCD controller and the host
computer.
Cable between
controller and
CCD chip
GPIB cable
from controller
to host PC
Back of CCD controller
14-4
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Software
Visual Instrument Setup
The TRIAX appears as a
standard monochromator
icon within Visual
Instrument Setup’s
instrument layout.
Moving the flip
mirrors
For those TRIAX
accessories with dual
entrance and exit ports,
the flip mirrors to choose
which entrance and exit
ports are controlled in
DataMax.
1
Click on
the mirror
icon.
The Entrance or Exit Mirror dialog box
appears.
• Lateral = side port
• Axial = rear port
2
3
4
Click Change to move
the mirror to the other port.
Allow several seconds for the mirror to move.
Click Close.
The Mirror dialog box closes.
14-5
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Rotating the turret to
a different grating
1
Click the
grating
icon.
The Grating/Turret
window opens.
2
Click the
desired
grating’s
radio
button.
Allow several
seconds for the
turret to rotate to the new position.
3
Click Close.
Installing a new grating on the turret
1
2
3
4
Follow instructions in the TRIAX Series User
Manual on replacing the grating.
Click Change….
The New Value dialog box appears.
Enter the new grating’s
groove density.
Click OK.
14-6
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
The New Value dialog box closes.
5
Run a scan with the grating, to find a known
peak.
Jobin Yvon Inc. suggests running a xenon-lamp scan to find the 467-nm peak,
or a water-Raman scan to find the 397-nm peak. This procedure is similar to
that given in Chapter 3.
6
Note the position of the found peak.
7
Open the Real Time
Display.
Enter the observed
peak position.
8
As an example, we assume a lamp scan was performed, and the 467-nm peak
was observed at 480 nm.
Don’t forget to hit the Enter key.
Here, we entered 480 nm, the
position where the 467-nm peak
was seen.
9
10
Close the Real Time
Display.
Click Calibrate….
The Enter Correct
Position dialog
box opens.
11
12
Enter the true peak
position.
Click OK.
The Enter Correct Position dialog box
disappears.
14-7
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Adjusting slit widths
1
Click on an
icon of a
slit.
Note: A CCD is
mounted on the
side (lateral)
exit, while a
PMT is mounted
on the front exit.
The Slits window
opens. All slits in the TRIAX are adjustable, no matter which slit you click.
2
Enter the desired
slit width.
Note: The exit slit-width parameter is valid only for PMT
users. For CCD detectors,
there is no slit at the exit of
the TRIAX, so any displayed
settings are superfluous.
In bandpass units (e.g.,
nanometers), all slits have the
same setting.
3
Click Move All.
4
Click Close.
This implements the change.
The Slits dialog box closes.
14-8
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
CCD Detector
The CCD detector has a
special symbol in the
Visual Instrument Setup:
1
Click on
the CCD
icon.
The Acquisition
Channel window
opens.
2
3
4
Choose Data
Units.
Choose Gain
Level.
Choose analogdigital conversion.
Note: ADC is available only on the CCD-3500.
5
Click Details.
9
Click OK.
10
Click OK.
The Acquisition Controller –
Details window opens. Details of
the CCD controller are displayed.
The Acquisition Controller –
Details window closes.
The Acquisition Channel window closes.
14-9
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Run Experiment
Within the DataMax main window, CCD experiments using a TRIAX may be performed. To run a CCD acquisition,
1
Choose Collect.
2
Choose Experiment….
A drop-down menu appears.
The CCD Acquisition dialog box opens:
14-10
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
1
3
4
2
6
5
7
In this dialog box—as with other Experiment Acquisition dialog boxes—the
user defines the CCD experiment fully. Several functions specific to the CCDacquisition type are available, such as:
1 CCD Areas
2 CCD Regions
3 Image and Scan radio buttons
4 Integration Time
5 Accumulations
6 Mirrors…
7 Fast Kinetics Mode
These are discussed on the following pages.
3
4
Complete all required parameters.
Click Run.
The CCD Acquisition box disappears; the experiment begins.
14-11
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
CCD Areas
A CCD detector can be divided into several data-collection areas, each acting independently. CCD Areas controls how to divide up the CCD chip into multiple detectors.
1
Click CCD Areas.
The Areas List window appears:
Chip Height and
Chip Width
This shows the
size of the
active area,
using a choice
of units.
Areas
Definitions and
Areas Views
These indexcard tabs give
details about
which pixels
are active,
mathematically
(Areas
Definitions)
and graphically
(Areas
Views).
Comments:
Unlimited amount of information may be entered, but only the first 80 characters are displayed.
Linearization
Normally, data are taken as pixel number versus intensity. The Linearization
checkbox lets the user convert to desired units versus intensity, applying linearization corrections from a correction file created by Jobin Yvon Inc.
Normalize
14-12
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Visible only with two or more active areas, Normalize lets the user choose an
area as the normalization standard. DataMax performs a Dark Subtract on all
data, then divides all areas by the normalization standard.
Reformat
Reformat
divides the
entire CCD
chip equally
(to within 1
pixel) into the
value shown in
the Number
of Areas
slider. If
Number of
Areas is 1,
then the entire
CCD is used
as one area. In
Image mode,
only one area
is allowed.
Changes are
shown in the
Areas
Definitions.
Load and Save
Load and Save custom CCD-area descriptions for later use, with an .arl
extension.
2
When finished, click OK.
The Areas List window closes.
14-13
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
CCD Regions
In Scan mode, a CCD detector can take
“snapshots” of various spectral regions,
which can be attached together to create a
single complete spectrum. CCD Regions
controls how to take and present these
“snapshots”.
1
Click CCD Regions.
Warning: Based on the characteristics of multichannel detectors, discontinuities appear
when various spectral regions
are attached together in Scan
mode.
The Acquisition Setup dialog box opens:
2
Enter the number of regions to scan.
Multiple CCD areas may be defined independently. The Nb region field shows
which area is being set. Extra data-entry fields appear, based on the desired
number of regions.
14-14
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Two methods specify how to acquire data: the Position index card, setting the
central wavelength, and the Range index card, setting the region on the CCD
detector.
Position method
3
Click on the Position index-card tab.
4
Enter the Center wavelength, in nm, for each
region.
The range shown, in nm, depends on the chip size, the TRIAX model, and the
grating’s groove density.
5
6
7
Enter the desired integration time, in s, next to
the Int. Time field for each region.
Enter a comment.
Only the first 80 characters are displayed.
Load and Save
Load and Save custom CCD-area descriptions for later use, with an .arl
extension.
When finished, click OK to close the Acquisition
Setup window.
14-15
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Range method
3
4
5
6
Click on the Range index-card tab.
Enter the starting wavelength in From.
Enter the ending wavelength in To.
Enter the integration time in Intg. time.
7
For piecing together several “snapshots” with
automatic baseline matching, click Auto Adjust.
The Nb Cover field changes depending on the grating, TRIAX, and CCD chip.
8
9
Enter a comment.
Only the first 80 characters are displayed.
Load and Save
Load and Save custom CCD-area descriptions for later use, with an .arl
extension.
When finished, click OK to close the Acquisition
Setup window.
14-16
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Image and Scan
These radio buttons choose between two detection formats:
Using the CCD chip as a two-dimensional detector (Image), or
columns
rows
Binning columns of pixels together to create a one-dimensional detector (Scan) for recording standard spectra.
columns binned
rows
For 2-D detection,
1
Click Image.
For 1-D detection,
1
Click Scan.
When one of these radio buttons is chosen, the CCD Acquisition window may adjust
some of the data-entry fields available.
14-17
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Integration Time
This is the amount of time that the CCD chip is exposed to the sample’s luminescence.
1
2
Enter the Integration Time, in s, in the data-entry
field.
Click on another data-entry field to finalize the
value.
14-18
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Accumulations
An “accumulation” is one exposure to the luminescence for a particular active CCD
area. Accumulations are added together if more than one accumulation is specified.
1
2
Enter the desired number of accumulations in
the data-entry field.
Click on another data-entry field to finalize the
value.
14-19
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Mirrors…
At present, this button is inactive in DataMax.
14-20
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Correction
The Correction button is useful only when working with a PMT.
Warning: Correction factors pertain only to the photomultiplier tube or
other single-point detector, and NOT to a 2-dimensional CCD detector.
Therefore, corrections to raw data cannot be made in CCD mode.
14-21
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Fast Kinetics Mode
Fast Kinetics Mode is a special high-speed data-acquisition mode that uses only a portion of the CCD (top or bottom) as the active area. The Fast Kinetics Mode checkbox
contains hidden data-entry fields.
1
Check the Fast Kinetics Mode checkbox.
2
Choose the Burst or Continuous radio buttons.
When checked, new fields appear, while others disappear:
•
•
Burst means one rapid exposure. With Burst, the CCD Acquisition window
appears as shown above. Burst mode exposes the top edge of the CCD to
the luminescence, while the remainder of the chip serves as a temporary
storage area until the data are read.
Continuous means continue to retake an exposure, spaced by a time increment. This is a slower method of acquisition, using the bottom edge of the
chip. With Continuous, the CCD Acquisition window appears as shown below:
14-22
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
In Continuous mode,
a
b
c
d
3
4
e
Choose the number of Accumulations.
Set the Integration Time.
Choose the Time Increment from the start of one exposure to the start
of the next exposure.
Set the Enable Int. Shutter Move checkbox. If checked, the CCD shutter opens and closes at each accumulation. If unchecked, the CCD shutter opens at the start of the experiment, and closes at the end.
Decide upon a Last Read Out Time.
Set all appropriate parameters.
Click Run.
The experiment begins.
14-23
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Flush
The Flush option can remove all residual charges from the CCD chip before data acquisition. The drop-down menu contains three choices:
Clear out all charges before an accumulation.
• Flush Before Each Acquisition
•
No Flush
Charges are not removed before an accumulation. This is faster, but background noise
may increase between acquisitions. For multiple scans, the first acquisition may have a
higher background-noise level than later
ones.
•
Flush Before First Scan
The charges are removed only before the first
scan.
14-24
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Linearization
Use this function to re-align the TRIAX if the peaks’ wavelengths are correctly assigned near the center of the CCD, but are incorrect near the edges.
1
Choose Collect.
2
Choose
Linearization.
A drop-down menu appears.
Note: Your parameters may
differ from those shown.
The Linearization Parameters
window opens.
3
Enter the desired parameters, or have DataMax
calculate
them for
you.
If you choose to
enter them,
14-25
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
a
b
Do so.
Click OK.
If you choose to have DataMax calculate them,
a
Click OK.
The Linearization Parameters window closes, and the Linearization Procedure
dialog box appears.
4
Choose the
desired
checkbox:
Multi Scans/One Peak, for a single wavelength from a source, such as the
547.074-nm line from a Hg lamp. This takes 5 spectra of the line displaced
across the chip, and requires a motorized spectrometer.
a
Click Define Experiment.
The CCD Acquisition dialog box appears.
b
Click CCD
Areas.
The Area List window
opens.
c
d
Click Reformat,
to use the entire
chip.
De-activate the
Linearization
checkbox.
e
Click OK.
The Area List window
closes.
14-26
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
f
Click CCD Regions.
The Acquisition
Setup window opens.
g
Choose the
Position
index-card
tab.
Five different spectra
are required. The following example uses
a CCD that is 2000
pixels wide × 800
pixels high, with a
Hg line at 546.074
nm.
h
i
j
k
l
m
n
o
Set Nb
Region to 1.
Set the Center to 546.074 nm, and press Enter.
To − From
i=
5
Calculate the increment
, to give 5 positions using the
maximum amount of the chip in linearization.
Set Nb Regions to 5.
Set the first region’s From = 1.05 × i.
Set the second region’s Center2 = Center of region 1 + i.
Repeat steps 12 and 13 until all 5 regions are assigned.
Click OK.
The Acquisition Setup window closes. In the CCD Acquisition dialog box,
p
Enter a file name.
14-27
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
q
r
s
Enter the integration time.
Enter other required parameters, e.g., slit widths.
Click Run.
One Scan/Multi Peaks, for one source with at least five known lines
within the CCD’s area.
This is done with fixedgrating spectrographs and
movable grating systems
not attached to the system.
a
Click Define Experiment.
The CCD Acquisition box appears.
b
Click CCD
Areas.
The Area List window
opens.
c
d
Click Reformat,
to use the entire
chip.
De-activate the
Linearization
checkbox.
e
Click OK.
The Area List dialog box
closes.
f
Click CCD
Regions.
The Acquisition Setup dialog box appears.
14-28
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
g
Choose the
Position index-card tab.
The spectrum must
contain five known
peaks on different areas of the CCD chip.
h
Set Nb
Region to 1.
i
Click OK.
The Acquisition
Setup window closes.
In the CCD
Acquisition dialog
box,
j
k
l
m
Enter a file name.
Enter the integration time.
Enter other required parameters, e.g., slit widths.
Click Run.
If defined correctly for either technique, DataMax collects the spectrum. The Select
Calibration Peak window appears.
A dialog box appears requesting the
calibration wavelengths (for Multi
Scans) or verification (for One
Scan). When enough peaks have
been selected for calibration, the Calc button appears:
5
Select Calc
if correct;
select
Correct to adjust the values.
DataMax calculates new parameters.
6
7
Close and exit all DataMax programs.
Restart DataMax to cause the changes to take
effect.
14-29
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Autoscaling the display
AutoScale is always on, and cannot be switched off.
14-30
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Real Time Display
Unlike typical systems, Real Time Display with a TRIAX and CCD must be used with
the Run Experiment window open simultaneously. The Real Time Display is used to
optimize the CCD detector and TRIAX settings for the best experimental results.
To do a CCD acquisition in the Real Time Display,
1
In Instrument
Control Center,
click the Run Experiment button.
The DataMax main window appears.
2
3
Choose Collect.
A drop-down menu appears.
4
Choose RTD-CCD.
The Real Time Display opens.
14-31
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Note: The Counts window does not appear in Real Time Display with a
CCD detector, because the system is collecting data over many pixels, not reporting a single number.
The Toolbar
When a single-point detector (e.g., PMT, InGaAs, IR, etc.) is attached to a TRIAX and
activated in the correct layout, an HV button appears in the Toolbar.
The Intensity window
The Intensity window is used
only for a single-point detector
(e.g., PMT, InGaAs, IR, etc.).
The Slits window
Note the following terms used in
the Slits window:
• Front = axial slit
• Side = lateral slit
Note: The exit slitwidth parameter is
valid only for PMT
users. For CCD detectors, there is no slit
at the exit of the
TRIAX, so any displayed settings are
superfluous.
Note: Some slits cannot be set in Real
Time Display.
14-32
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
CCD button
The CCD button provides
information about the CCD chip.
1
Click CCD.
2
The Area List
window opens.
Chip Height and Chip
Width
This
shows the
size of the
active
area,
using a
choice of
units.
Areas Definitions and
Areas
Views
These index-card
tabs give
details
about
which
pixels are
active,
mathe-
14-33
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
matically (Areas Definitions) and graphically (Areas Views).
Comments:
Unlimited amount of information may be entered, but only the first 80 characters are displayed.
Linearization
Normally, data
are taken as
pixel number
versus
intensity. The
Linearization
checkbox lets
the user
convert to
desired units
versus intensity, applying
linearization
corrections.
Normalize
Visible only
with two or
more active
areas,
Normalize lets
the user choose
an area as the
normalization standard. DataMax performs a Dark Subtract on all data, then
divides all areas by the normalization standard.
Reformat
Reformat divides the entire CCD chip equally (to within 1 pixel) into the value
shown in the Number of Areas slider. If Number of Areas is 1, then the entire CCD is used as one area. In Image mode, only one area is allowed.
Changes are shown in the Areas Definitions.
Load and Save
Load and Save custom CCD-area descriptions for later use, with an .arl
extension.
3
4
Adjust all parameters as desired.
Click OK.
The Areas List closes.
14-34
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Correcting data with the TRIAX
Introduction
General comments about data correction are found in Chapter 4: Optimizing Data Acquisition. This section deals with the procedures that are different when using the
TRIAX.
Correction-factor file names for multiple gratings
When the Fluorolog®-3 uses a TRIAX (which
contains multiple gratings) or multiple detectors,
multiple correction-factor files are required. Each
of these has a unique file name, which needs
deciphering. Following are rules to guide the
correct choice of correction-factor files.
1
Note: Upper- or lowercase characters are
not important.
The first two characters show the type of correction-factor file.
MC = emission correction-factor file
XC = excitation correction-factor file
2
After MC, the next several
characters determine the
appropriate detector:
2658 = near-IR detector
CD = CCD array
3
Note: Ignore this rule
for XC… files.
928 = R928P photomultiplier tube
DSS = InGaAs detector
The next one or two characters give the grating’s groove-density:
15 = 150 grooves/mm
12 = 1200 grooves/mm
6 = 600 grooves/mm
3 = 300 grooves/mm
4
5
Then comes a separator, x.
Finally, the last characters show the blaze of the
grating:
300 = 300 nm
330 = 330 nm
500 = 500 nm
750 = 750 nm
etc.
14-35
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Here are some examples of deciphering the emission correction-factor file:
Mcorrect.spc
The default emission correction-factor file for an instrument, with no TRIAX, with only one emission grating and
detector.
MC9286x300.spc
An emission correction-factor file for an R928P PMT, with
a grating of 600 grooves/mm and a blaze at 300 mm.
MCCD12x500.spc
An emission correction-factor file for a CCD array, with a
grating of 1200 grooves/mm and a blaze at 500 nm.
MC26586x300.spc
An emission correction-factor file for an IR detector, with a
grating of 600 grooves/mm and a blaze at 300 nm.
Xcorrect.spc
The default excitation correction-factor file for an instrument, with no TRIAX with only one excitation grating.
XC12x330.spc
An excitation correction-factor file for a grating with 1200
grooves/mm and a blaze at 330 nm.
Warning: For CCD arrays, these correction-factor files are only valid under the same conditions, i.e., when used at the same central wavelength as performed in the factory. The files may give invalid corrections
when used at other central wavelengths on the CCD array.
To determine the center wavelength for a particular correction-factor file when used
with a CCD array, see the Performance Test Report that accompanies the instrument.
Warning: DataMax only uses and displays the first 8 characters of a file
name. Therefore, the rest of a correction-factor file name may be truncated, indicated by a tilde (~).
During acquisition
To acquire corrected emission data automatically, enter the appropriate emission correction-factor file, mc…, in the Correction factor file field in the Run Experiment dialog
box. To acquire corrected excitation data, enter the appropriate excitation correctionfactor file, xc….
Note: Before applying correction factors, Jobin Yvon Inc. recommends subtracting the dark counts, and the spectrum of the blank,
from the data. Refer to the software manual for details.
14-36
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
After acquisition
To apply the correction factors after the data have been acquired, multiply the data file
by the appropriate correction factor file (mc… or xc…).
1
Make sure the trace to be corrected is active in
the Run Experiment window.
2
3
Choose Arithmetic from the toolbar.
Choose Functions.
4
Next to Function, select
Multiply.
Next to Operand, choose
Term File*K.
5
6
The Math Functions dialog box opens:
Click Select.
This opens the Select New Term File:
dialog box:
7
Choose the
appropriate
file (mc… or
xc…).
8
Click OK.
9
Click Apply.
The Select New Term
File: dialog box closes.
The file name appears
in the Term File area.
The trace that appears on the
screen is a result of the mathematical operation, giving a corrected spectrum.
14-37
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
TRIAX 320 Specifications
Focal length
Entrance-aperture ratio
0.32 m
f/4.1
Grating size
Image magnification at exit
68 mm × 68 mm
1.00
Scanning range
0–1500 nm (with 1200-grooves/mm grating)
Multichannel coverage
79.2 nm over 30-mm array width (with 1200grooves/mm grating)
30 mm wide × 12 mm high
2.64 nm/mm nominal (with 1200-grooves/mm grating)
Focal plane
Spectral dispersion
Spectral resolution
Wavelength accuracy
0.06 nm nominal (with 1200-grooves/mm grating),
when scanning as a monochromator
±0.3 nm
Wavelength repeatability
Step size
±0.06 nm
0.06 nm (with 1200-grooves/mm grating)
Dimensions
16.25" long × 15.25" wide × 7.5" high
41.3 cm long × 38.7 cm wide × 19.1 cm high
40.7 lb (18.5 kg)
Weight
14-38
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Troubleshooting
Should there be a problem with the TRIAX spectrograph, check the following chart for
possible problems. Try the remedies listed on these pages before contacting Spex®
Fluorescence Service.
Problem
Spectrograph does not respond to any commands
Spectrograph responds to
some commands, but not all
Possible Cause
Spectrometer is off.
Remedy
Check AC outlet (mains), power switch, and
fuse.
Cables are improperly connected.
Check external cable connections.
Internal cables are not connected.
Software is inappropriate.
Check internal ribbon cables, and jumpers on
circuit boards.
Check that system software matches the hardware.
Check that the failing command is valid, and
that parameters are within correct limits for that
function.
Improper parameters used with
correct command.
Software is inappropriate.
Slit and wavelength drives
and accessories do not move
Bad electrical connection.
Background signal is too high
Light leak.
Exit DataMax. Check that the software matches
the hardware, especially device configuration.
Test turret as follows:
1 Exit DataMax.
2 Disconnect power.
3 Remove top cover of TRIAX.
4 Gently move turret partway through its
range of movement.
5 Reconnect power.
6 Turret should automatically return to home
position.
7 Check control-cable connections and interface.
Make sure all covers are in place.
Be sure that the are between the sample and the
entrance slit is enclosed and light-tight. Test by
blocking the entrance slit.
Note: To prevent damage
to the knifeedges, the
slits never
close completely. Therefore some light
always enters.
Signal is too noisy
Signal is too weak.
14-39
Check detector mounting and housing for light
leaks.
Test for stray light:
1 Start at detector, close exit and entrance
slits and shutters in order.
2 Be sure all openings and screw holes are
plugged.
3 Check that the cover, side, and baseplate fit
tightly.
4 In a dark room, shine small flashlight at a
suspicious part or joint, and observe detected signal levels.
Try to increase signal strength at the detector.
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Light leak.
Improper grounding.
Note: Ground
loops often are
difficult to diagnose and
cure. Experiment patiently.
Keep ground
wires short,
make tight
connections,
and avoid
coated surfaces.
“Warning! Connect Failed
(7)” error message appears
Follow directions above for detecting light
leaks.
1 Exit DataMax.
2 Turn off spectrometer.
3 Rearrange power connections so that spectrograph, source, and detector are connected
to the same ground (earth), and if possible,
the same power circuits.
4 Add redundant grounds (earths) to various
points in the system.
Consider a “star” ground (earth) of wires radiating from a single central location, e.g., a
grounded metal table surface under the system.
Electromagnetic interference
from lasers or other high-energy
apparatus
Construct a faraday cage or shield.
CCD-3000 or 3500 controller is
off.
Exit DataMax, turn on CCD controller, and
restart DataMax.
14-40
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
.
.arl ............... 14-13, 14-15, 14-16, 14-34
A
Accumulations.............. 14-11, 14-19, 14-23
Acquisition Channel.................................14-9
Acquisition Controller – Details ..............14-9
Acquisition Setup....14-14, 14-15, 14-16, 1427, 14-28, 14-29
analog-digital conversion.........................14-9
Apply......................................................14-37
Area List ........................ 14-26, 14-28, 14-33
Areas Definitions.... 14-12, 14-13, 14-33,
14-34
Areas List .................... 14-12, 14-13, 14-34
Areas Views .............. 14-12, 14-33, 14-34
Arithmetic ..............................................14-37
Auto Adjust............................................14-16
AutoScale ............................................14-30
axial............................................. 14-1, 14-32
Axial........................................................14-5
CCD Areas...... 14-11, 14-12, 14-26, 14-28
CCD chip 14-4, 14-12, 14-13, 14-16, 14-17,
14-18, 14-33, 14-34
CCD controller .....................14-4, 14-9, 14-40
CCD detector.... 14-9, 14-12, 14-14, 14-15,
14-31
CCD icon................................................. 14-9
CCD Regions.14-11, 14-14, 14-27, 14-28
CCD shutter.................................14-4, 14-23
Center ........................................14-15, 14-27
center wavelength
CCD................................................... 14-36
Change..................................................... 14-5
Change…................................................. 14-6
Chip Height............................14-12, 14-33
Chip Width.............................14-12, 14-33
Close...............................................14-5, 14-8
Collect ...........................14-10, 14-25, 14-31
Comments: ............................14-12, 14-34
Continuous..................................14-22, 14-23
Correct ................................................... 14-29
Correction.................................14-21, 14-36
Correction-factor files
multiple detectors .............................. 14-35
multiple gratings................................ 14-35
B
bandpass...................................................14-8
baseplate adapter.......................... 14-3, 14-39
binning ...................................................14-17
blaze ............................... 14-2, 14-35, 14-36
Burst.......................................................14-22
C
cables ..................................... 14-3, 14-4, 14-39
Calc.......................................................14-29
Calibrate…...............................................14-7
CCD .....14-1, 14-3, 14-4, 14-9, 14-10, 14-11,
14-12, 14-13, 14-14, 14-15, 14-16, 1417, 14-18, 14-19, 14-22, 14-23, 14-24,
14-25, 14-26, 14-27, 14-28, 14-29, 1431, 14-33, 14-34, 14-35, 14-36, 14-40
CCD acquisition........................ 14-10, 14-31
CCD Acquisition ..14-10, 14-11, 14-17, 14-22,
14-26, 14-27, 14-28, 14-29
D
Dark Subtract..........................14-13, 14-34
Data Units................................................ 14-9
DataMax .....14-1, 14-5, 14-10, 14-13, 14-20,
14-25, 14-26, 14-29, 14-31, 14-34, 14-39,
14-40
Define Experiment .................14-26, 14-28
Details.............................................14-3, 14-9
detector .. 14-1, 14-3, 14-4, 14-12, 14-17, 1432, 14-35, 14-36, 14-39, 14-40
E
Enable Int. Shutter Move .................. 14-23
Enter Correct Position ............................. 14-7
Entrance Mirror........................................ 14-5
entrance port ......................... 14-1, 14-3, 14-5
Exit Mirror................................................ 14-5
exit port...........................................14-1, 14-5
14-42
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
Experiment….........................................14-10
F
faraday cage................................................ 14-40
Fast Kinetics Mode ................ 14-11, 14-22
flip mirror....................................... 14-1, 14-5
Flush.....................................................14-24
Flush Before Each Acquisition .........14-24
Flush Before First Scan.....................14-24
From.......................................... 14-16, 14-27
Front .....................................................14-32
Functions................................................14-37
fuse ........................................................... 14-39
G
Gain Level................................................14-9
grating . 14-1, 14-2, 14-6, 14-7, 14-15, 14-16,
14-28, 14-35, 14-36, 14-38
grating icon ..............................................14-6
grating turret.............................................14-1
Grating/Turret...........................................14-6
groove density.....................14-2, 14-6, 14-15
groove-density........................................14-35
H
Hg lamp..................................................14-26
host computer...........................................14-4
HV .........................................................14-32
I
Image ...............14-11, 14-13, 14-17, 14-34
InGaAs ...................................................14-35
InGaAs detector .....................................14-32
Int. Time.................................................14-15
integration time ..14-15, 14-16, 14-28, 14-29
Integration Time........... 14-11, 14-18, 14-23
Intensity .................................................14-32
Intg. time ................................................14-16
IR detector................................14-32, 14-36
L
lamp scan .................................................14-7
laser ....................................................... 14-40
Last Read Out Time .......................... 14-23
lateral ................................. 14-1, 14-3, 14-32
Lateral.................................................... 14-5
layout ............................................14-5, 14-32
Linearization 14-12, 14-25, 14-26, 14-28,
14-34
Linearization Parameters...........14-25, 14-26
Linearization Procedure ........................ 14-26
Load ................. 14-13, 14-15, 14-16, 14-34
M
Math Functions ...................................... 14-37
mcorrect......................................... 14-36
mirror icon............................................... 14-5
Mirrors…...................................14-11, 14-20
monochromator .................. 14-1, 14-5, 14-38
Move All.................................................. 14-8
Multi Scans/One Peak ...................... 14-26
Multiply ................................................. 14-37
N
Nb Cover ............................................. 14-16
Nb region............................................. 14-14
Nb Region ...............................14-27, 14-29
Nb Regions ......................................... 14-27
near-IR detector..................................... 14-35
New Value.......................................14-6, 14-7
No Flush .............................................. 14-24
Normalize ....................14-12, 14-13, 14-34
Number of Areas....................14-13, 14-34
O
OK ..14-6, 14-7, 14-9, 14-13, 14-15, 14-16,
14-26, 14-27, 14-28, 14-29, 14-34
One Scan/Multi Peaks ...................... 14-28
Operand Type ........................................ 14-37
P
Performance Test Report....................... 14-36
photomultiplier tube ........................ See PMT
PMT...........................................14-21, 14-32
Position.........................14-15, 14-27, 14-29
14-2
TRIAX operation with the Fluorolog®-3
Fluorolog-3 v. 2.2 (9 Aug 2002)
power supply............................................14-2
R
R928P.......................................14-35, 14-36
Raman ......................................................14-7
Range....................................... 14-15, 14-16
Real Time Display ...................... 14-7, 14-31
Reformat ..........14-13, 14-26, 14-28, 14-34
ribbon cables ............................................. 14-39
RTD-CCD ..............................................14-31
Run....................14-11, 14-23, 14-28, 14-29
Run Experiment...14-10, 14-31, 14-36, 14-37
S
sample compartment ...................... 14-1, 14-3
sample mount ...........................................14-1
Save .................14-13, 14-15, 14-16, 14-34
Scan ........................... 14-11, 14-14, 14-17
Select......................................................14-37
Select Calibration Peak .........................14-29
Select New Term File ............................14-37
shutter.....................................................14-39
Side.......................................................14-32
slits ...14-3, 14-8, 14-28, 14-29, 14-32, 14-39
Slits .............................................. 14-8, 14-32
14-3
specifications, TRIAX........................... 14-38
SpectrAcq ................................................ 14-4
Spex® Fluorescence Service.................. 14-39
T
Term File ............................................. 14-37
Term File*K .......................................... 14-37
Time Increment .................................. 14-23
To........................................................... 14-16
Toolbar ............................................... 14-32
TRIAX..... 14-1, 14-2, 14-3, 14-4, 14-5, 14-8,
14-10, 14-15, 14-16, 14-25, 14-31, 1432, 14-35, 14-36, 14-39
troubleshooting, TRIAX........................ 14-39
turret .....................................14-2, 14-6, 14-39
V
Visual Instrument Setup....... 14-2, 14-5, 14-9
voltage converter ..................................... 14-2
X
xcorrect......................................... 14-36
xenon lamp .............................................. 14-7
Fluorolog-3 v. 2.2 (12 Jul 2002)
Technical Specifications
15: Technical Specifications
Each Fluorolog®-3 system consists of:
• An excitation source
• An excitation spectrometer
• A sampling module with reference detector
• At least one emission spectrometer
• At least one emission detector.
Each system is controlled by an IBM-PC-compatible computer, and may include a
printer for hard-copy documentation.
Important
The Fluorolog®-3 spectrofluorometer system is designed to comply with the requirements of the Low Voltage Directive 73/23/EEC and the EMC Directive 89/336/EEC
and, as of 1 January 1997, carries the CE marking accordingly. The system was tested
using standard (Jobin-Yvon-Inc.-authorized) components, cables, etc.
The details and specifications for each component of the Fluorolog®-3 series of spectrometers follow.
15-1
Fluorolog-3 v. 2.2 (12 Jul 2002)
Technical Specifications
Spectrofluorometer system
The Fluorolog®-3 spectrofluorometer consists of modules and components controlled
by the specialized software. Although the system can be configured in various ways for
a variety of applications, the basic (standard) Fluorolog®-3 spectrofluorometer system
consists of the following components:
Excitation Source
450-W xenon short arc, mounted vertically in an air-cooled
housing. Light collection and focusing by an off-axis mirror for
maximum efficiency at all wavelengths.
Optional pulsed lamp or laser port interface available.
Spectrometers
Single-grating excitation and emission spectrometers (standard).
Spectrometers are Czerny-Turner design with kinematic classically-ruled gratings and all-reflective optics.
The following specifications are based on 1200-grooves/mm
gratings:
Resolution
Accuracy
Speed
Range
Gratings*
Excitation
Emission
0.2 nm
±0.5 nm
150 nm/s
0–1300 nm
330-nm blaze (200–700 nm range)
500-nm blaze (300–1000 nm range)
*Other gratings available for ranges above 1 µm.
Optional double-grating units available for highest stray-light rejection and sensitivity.
Sample Module
T-format sample module with excitation reference detector. The
T-box design allows a second emission-detection channel to be
incorporated. The sample module also has a removable gap-bed
assembly for sampling accessory replacement.
Optional front-face collection assembly available.
Detectors
Calibrated photodiode for excitation correction from 240–1000
nm. Emission detector is an R928P for high sensitivity in photon-counting mode (240–850 nm).
Other PMTs to 1100 nm, with thermoelectrically cooled option.
Solid-state detectors for higher wavelength emissions. CCD multichannel detector for instant emission spectra and sample spatial
information.
15-2
Fluorolog-3 v. 2.2 (12 Jul 2002)
Technical Specifications
Lifetime Option
Lifetime range: 10 picoseconds to 10 microseconds
Frequency range: 0.2 to 310 MHz.
Sensitivity
Double-distilled deionized ICP-grade water Raman scan 4000:1
S/N at 397 nm, 5-nm bandpass, 1 s integration time, background
noise first standard deviation at 450 nm.
15-3
Fluorolog-3 v. 2.2 (12 Jul 2002)
Technical Specifications
Minimum computer requirements
•
•
•
•
•
•
•
•
•
•
Pentium III 90 MHz
64 megabytes memory (more is recommended)
Minimum 4-gigabyte hard drive
SVGA display card and SVGA monitor to match display card
At least one 3½" high-density floppy
drive
CD-ROM drive
Note: Additional COM ports
101-key enhanced keyboard
may be required to control
Mouse is strongly recommended
accessories such as the
2 COM ports minimum (1 for
MicroMax, temperature
communication, 1 for plotter)
bath, etc.
Windows™
Software
DataMax software for data acquisition and manipulation through the Windows™ environment.
15-4
Fluorolog-3 v. 2.2 (12 Jul 2002)
Glossary
16: Glossary
3D excitation/emission
display
This maps a specified emission-scan wavelength range using various
excitation wavelengths.
3D synchronous/offset
scan
A scan that maps a specified synchronous scan using various offset
wavelengths between the spectrometers.
Absorption
Transition, when a photon enters a molecule, from the ground state
to the excited singlet state. This process typically occurs in ~10–15 s.
Absorbance
The extent of absorption by a substance. Absorbance, A, is –log T,
where T is the transmittance of the sample. Absorbance is also synonymous with optical density (OD). Absorbance can be calculated
using the Beer-Lambert Law:
OD = A = εcl = –log T
ε = the extinction coefficient (M–1 cm–1)
c = sample concentration (M)
l = path length (cm)
Bandpass
The wavelength range of light passing through the excitation and
emission spectrometers. The wider the bandpass, the higher the signal intensity.
Bandpass filter
Optical element that selectively transmits a narrow range of optical
wavelengths.
Bioluminescence
Emission of light originating from a chemical reaction in a living
organism.
Blaze wavelength
Wavelength at which a grating is optimized for efficiency. Generally, the gratings are efficient to 2/3 before the blaze wavelength to
twice the blaze wavelength. The excitation and emission gratings are
blazed in the UV and visible respectively.
Chemiluminescence
Emission of light originating from a chemical reaction.
Color effect for pulse
technique
Time-dependent wavelength distribution of the lamp pulse.
Color effect for phasemodulation technique
Phase of the excitation and the degree to which it is modulated.
Corrected emission scan
An emission scan corrected for the wavelength characteristics of the
emission spectrometer and the response of the signal detector. To
obtain a corrected emission scan, an emission spectrum is multiplied
by the emission correction factors. A set of emission correction factors is supplied with the instrument and stored as mcorrect.spt
on the software disks.
Corrected excitation scan
An excitation scan corrected for the wavelength characteristics of the
xenon lamp, the aging of the xenon lamp, and the gratings in the excitation spectrometer. To obtain a corrected excitation scan, the de-
16-1
Fluorolog-3 v. 2.2 (12 Jul 2002)
Glossary
tector signal is ratioed to the reference signal, which provides 90%
of the corrected excitation scan. To obtain a completely correct scan,
the excitation scan acquired in the manner described above is multiplied by correction factors. A set of excitation correction factors is
included on the software disks.
Correction factors
Compensates for the wavelength-dependent components of the system, like the xenon lamp, gratings, and signal detector. Emission and
excitation correction factors are included with the software and are
titled xcorrect.spt and mcorrect.spt.
Cut-on filter
Optical component that passes light of a higher wavelength.
Cut-off filter
Optical component that passes light of a lower wavelength.
Dark counts
Inherent background signal of the photomultiplier when high voltage
is applied. Cooling the detector decreases the dark counts.
Demodulation
(less modulation) Usually refers to the demodulated emission relative to the excitation.
Demodulation factor
Can be calculated using the following equation:
m=
AC f / DC f
AC x / DC x
where ACf is the amplitude of the emission voltage, DCf is the excitation offset voltage, ACx is the amplitude of the excitation voltage,
and DCx is the offset of the emission voltage.
Emission scan
Shows the spectral distribution of light emitted by the sample. During an emission scan, the excitation spectrometer remains at a fixed
wavelength while the emission spectrometer scans a selected region.
Energy transfer
The transfer of the excited energy from a donor to an acceptor. The
transfer occurs without the appearance of a photon and is primarily a
result of dipole-dipole interactions between the donor and acceptor.
Excitation scan
Shows the spectral distribution of light absorbed by the sample. To
acquire an excitation scan, the excitation spectrum scans a selected
spectral region while the emission spectrum remains at a fixed wavelength.
Extrinsic fluorescence
Inherent fluorescence of probes used to study non-fluorescent molecules.
Flash lamp
A lamp that provides pulsed-light output used to excite a sample.
Can be either “free running” or “gated.”
Fluorescence
The emission of light or other electromagnetic radiation during the
transition of electrons from the excited singlet state to the ground
state. Fluorescence typically occurs within about ~10–9 s.
Fluorescence lifetime
The average length of time that a molecule remains in the excited
state before returning to the ground state.
16-2
Fluorolog-3 v. 2.2 (12 Jul 2002)
Glossary
Front-face detection
A mode of detection in which fluorescence is collected off the front
surface of the sample. Front-face detection usually is selected for
samples such as powders, thin films, pellets, cells on a cover-slip,
and solids.
Grating
Optical element in the spectrometer, consisting of finely scribed
grooves that disperse white light into a spectrum.
Intrinsic fluorescence
The natural fluorescent properties of molecules.
Laser
A monochromatic light source that provides high excitation intensity.
Mercury lamp
Light source that emits discrete, narrow lines as opposed to a continuum. A mercury lamp can be used to check the spectrometer calibration.
Mirror image rule
When the emission profile appears to be the mirror image of the absorption spectrum.
Modulated light
Light whose intensity varies in a sinusoidal manner, with a specified
frequency.
Optical density
A synonym of Absorbance. See Absorbance.
Optical-density effects
Fluorescence intensities are proportional to the concentration over a
limited range of optical densities. High optical densities can distort
the emission spectra as well as the apparent intensities.
Phase angle
The delayed relationship between the modulated emission relative to
the excitation.
Phase-modulation method
A technique for measuring fluorescence lifetimes, where a sample is
excited with light whose intensity is modulated sinusoidally. The
emission is a forced response to the excitation.
Phosphorescence
The emission of light or other electromagnetic radiation during the
transition of electrons from the triplet state to the ground state. Phosphorescence is generally red-shifted relative to fluorescence and occurs within ~10–6 s to several seconds. To enhance phosphorescence
detection, samples are often chilled to liquid-nitrogen temperature
(77 K).
Pockels cell
A light modulator that transmits ultraviolet and visible light, and can
be operated at variable frequencies. Highly collimated excitation
light is required.
Pulse-sampling method
A technique for measuring fluorescence lifetimes, in which an initial
population of fluorophores is excited by infinitely short pulses of
light. An advantage of this technique is the direct recording of timeresolved emission spectra.
Raman scattering
Scattering caused by vibrational and rotational transitions. Raman
bands generally appear red-shifted relative to the incident electromagnetic radiation. The primary characteristic of Raman scatter is
that the difference in energy between the Raman peak and the inci-
16-3
Fluorolog-3 v. 2.2 (12 Jul 2002)
Glossary
–1
dent radiation is constant in energy units (cm ).
Rayleigh scattering
Light scattering from particles whose dimensions are much smaller
than the wavelength of incident light. The scattered light is of the
same energy as the incident light. Rayleigh scatter shows scatter radiation intensity inversely proportional to the 4th power of the wavelength of incident radiation.
Rayleigh-Tyndall
scattering
Combination of Rayleigh and Tyndall scatter. These two scattering
phenomena cannot be separated. If the molecule’s Stokes shift is
small, Rayleigh-Tyndall scatter will limit the ultimate resolution.
Red-sensitive
photomultiplier
A detector that extends fluorescence detection to 850 nm.
Reference photodiode
Detector used to monitor the output of the xenon lamp.
Resolution
The ability to separate two closely spaced peaks. Resolution can be
improved by decreasing the bandpass and the increment (step size).
Right-angle detection
Collection of fluorescence at 90° to the incident radiation. Rightangle detection typically is selected for dilute and clear solutions.
Scatter
A combination of Raman, Rayleigh, and Rayleigh-Tyndall scattering, which can distort fluorescence spectra with respect to intensities
and wavelengths.
Signal photomultiplier
Detector used to measure excitation and fluorescence from the sample. It is operated in the photon-counting mode of detection to provide high sensitivity. Different detectors cover different wavelength
regions.
Singlet state
The spin-paired ground or excited state. The process of absorption
generally produces the first excited singlet state that emits fluorescence, or undergoes intersystem crossing to form a triplet state.
Spectrometer
The component in a fluorometer system that is scanned to provide
the excitation and emission spectra. The spectrometer is chosen for
stray-light rejection, resolution, and throughput.
Stokes shift
Generally, the energy-difference between the absorption peak of
lowest energy and the fluorescence peak of maximum energy.
Synchronous scan
Scan that characterizes the overlap between the excitation and emission. The excitation and emission spectrometers are scanned at the
same time, with a constant offset specified as either nanometers
(wavelength units) or in cm–1 (energy units).
Time-base scan
Scan in which the sample signal is monitored while both the excitation and the emission spectrometers remain at fixed wavelengths.
Time-base data are used to monitor enzyme kinetics, dual wavelength measurements, and determine the reaction rate constant.
Time-resolved emission
scan
Scan in which the emission spectra are acquired at various times after the excitation pulse. Provides insight into excited state reactions,
charge-transfer-complex formation, solvent dipolar relaxation and
16-4
Fluorolog-3 v. 2.2 (12 Jul 2002)
Glossary
other experiments.
Triplet state (T1)
The spin-paired ground or excited state formed from the excited
singlet state when paired electrons become unpaired. The triplet state
emits phosphorescence.
Tyndall scattering
Scatter that occurs from small particles in colloidal suspensions.
Xenon lamp
Lamp that produces a continuum of light from the ultraviolet to the
near-infrared for sample excitation.
Xenon-lamp scan
A profile of the lamp output as a function of wavelength. The lamp
scan is acquired with the reference detector while scanning the excitation spectrometer. The maximum xenon-lamp peak at 467 nm can
be used to determine proper calibration of the excitation spectrometer.
16-5
Fluorolog-3 v. 2.2 (12 Jul 2002)
Bibliography
17: Bibliography
P.M. Bayley and R.E. Dale, Spectroscopy and the Dynamics of Molecular Biological
Systems, Academic Press, London, 1985.
R. Becker, Theory and Interpretation of Fluorescence and Phosphorescence,
Wiley-Interscience, 1969.
B. Berlman, Handbook of Fluorescence Spectra in Aromatic Molecules, Vols. I & II,
Academic Press, New York, 1965 & 1971.
C.R. Cantor and P.R. Schimmel, Biophysical Chemistry, Freeman, New York, 1980.
M. Chalfie, Green Fluorescent Protein: Properties, Applications, and Protocols,
Wiley-Interscience, New York, 1998.
R.F. Chen, et al., Biochemical Fluorescence: Concepts, Vol. I & II, 1964 & 1970.
J.N. Demas, Excited State Lifetime Measurements, Academic Press, New York, 1983.
Enrico Gratton, David M. Jameson, and Robert D. Hall, “Multifrequency Phase and
Modulation Fluorometry,” Ann. Rev. Biophys. Bioeng. 13, 105–124 (1984).
G.G. Guilbault, Ed., Fluorescence—Theory, Instrumentation and Practice, Marcel
Dekker, New York, 1976.
_______, Practical Fluorescence: Theory, Methods and Techniques, 2nd ed., Marcel
Dekker, 1990.
_______, “Molecular Fluorescence Spectroscopy,” Anal. Chem. 8, 71–205 (1977).
D.M. Hercules, Ed., Fluorescence and Phosphorescence Analysis, Wiley-Interscience,
New York, 1965.
J. Ingle and S. Courch, Spectrochemical Analysis, Prentice-Hall, Englewood Cliffs, NJ,
1988.
F.H. Johnson, The Luminescence of Biological Systems, Amer. Assoc. Adv. Sci.,
Washington, D.C., 1955.
S.U. Koney, Fluorescence and Phosphorescence of Proteins and Nucleic Acids,
Plenum Press, New York, 1967.
M.A. Konstantinova-Schlezinger, Ed. Fluorometric Analysis, Davis Publishing Co.,
New York, 1965.
17-1
Fluorolog-3 v. 2.2 (12 Jul 2002)
Bibliography
nd
Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy, 2 ed., Plenum Press,
New York, 1999.
_______, Ed., Topics in Fluorescence Spectroscopy, Vols. 1–5, Plenum Press, New
York, 1991–1998.
_______, Badri P. Melinal, Enrico Gratton, “Recent Developments in FrequencyDomain Fluorometry,” Anal. Instr., 14 (314), 193–223 (1985).
_______, S. Soper, and R. Thompson, Advances in Fluorescence Sensing Technology
IV, SPIE Proc. Series, Vol. 3602 (1999).
W.T. Mason, Ed., Fluorescent and Luminescent Probes for Biological Activity: A
Practical Guide to Technology for Quantitative Real-Time Analysis, 2nd ed., Academic
Press–Harcourt Brace & Co., 1999.
W.H. Melhuish and M. Zander, “Nomenclature, Symbols, Units and Their Usage in
Spectrochemical Analysis VI: Molecular Luminescence Spectroscopy,” Pure App.
Chem., 53, 1953 (1981).
J.N. Miller, Ed., Standardization & Fluorescence Spectrometry: Techniques in Visible
and Ultraviolet Spectrometry, Vol. 2, Chapman and Hall, 1981.
W.G. Richards and P.R. Scott, Structure and Spectra of Molecules, John Wiley & Sons,
1985.
A. Schillen, et al., Luminescence of Organic Substances, Hellwege Verlag, Berlin,
1967.
S. Schulman, Ed., Molecular Luminescence Spectroscopy: Methods and Applications,
Vols. 1–3, Wiley–Interscience, New York, 1985–1993.
A. Sharma and S. Schulman, Introduction to Fluorescence Spectroscopy, Wiley
Interscience, New York, 1999.
D. Skoog, Principles of Instrumental Analysis, 5th ed., Saunders College/Holt, New
York, 1998.
N.J. Turro, Modern Molecular Photochemistry, Benjamin/Cummings, New York, 1978.
K. Van Dyke, Bioluminescence and Chemiluminescence: Instruments and Applications,
Vol. 1, CRC Press, Boca Raton, FL, 1985.
T. Vo-Dinh, Room Temperature Phosphorimetry for Chemical Analysis, John Wiley &
Sons, 1984.
I.M. Warner and L.B. McGowan, Ed., Advances in Multidimensional Luminescence, Jai
Press, Greenwich, CT, 1991.
17-2
Fluorolog-3 v. 2.2 (12 Jul 2002)
Bibliography
E.L. Wehry, Ed., Modern Fluorescence Spectroscopy, Vol. 1–4, Plenum Press, New
York, 1981.
C.E. White and R.J. Argauer, Fluorescence Analysis: A Practical Approach, Marcel
Dekker, 1970.
J.D. Winefordner, et al., Luminescence Spectrometry in Analytical Chemistry,
Wiley-Interscience, New York, 1972.
In addition, the following journals may prove useful:
Analytical Chemistry
Biophysics and Biochemistry
Fluorescence
17-3
Fluorolog-3 v. 2.2 (11 Jul 2002)
Requirements & Installation
18: Index
Key to the entries:
Times New Roman font.........subject or
keyword
Arial font................................command,
menu choice,
or data-entry
field
Arial Condensed Bold font .....dialog box
Courier New font .........file name or
extension
1
1,2-propanediol ..................................... 11-15
1908MOD Scatter Block Assembly 6-7, 11-3
3
320M........................................................2-11
9
9-CA.........................................................3-20
A
About Instrument Control Center..........7-8
accessory
450-W xenon lamp...................................6-28
absorption/transmission accessory.............6-3
autotitration injector .................................6-26
CCD detector ............................................6-10
cut-on filter................................................6-15
cut-on filter holder ....................................6-18
dual-position thermostatted cell holder ...6-21
external trigger accessory...............6-33, 6-35
fiber optic mount ......................................6-14
four-position thermostatted cell holder....6-19
front-face viewing option........................ 6-16
grating....................................................... 6-17
HPLC flow cell.......................................... 6-8
injector port.............................................. 6-33
lamp housing, dual................................... 6-25
lamp housing, universal........................... 6-25
liquid nitrogen dewar................................. 6-6
liquid nitrogen dewar assembly ................ 6-6
micro cell (with Model 1923A adapter) ... 6-8
micro cell (with Model 1924A adapter) ... 6-8
microscope interface................................ 6-27
microwell plate reader ............................. 6-31
Peltier thermocouple drive ...................... 6-29
phosphorimeter ...............................6-30, 10-6
photodiode reference detector................... 6-9
polarizer.................................................... 6-32
quartz cuvette............................................. 6-8
reduced volume sample cell...................... 6-8
room temperature signal detector............ 6-11
sample cell.................................................. 6-8
scatter assembly
11-2
scatter block assembly............................... 6-7
solid sample holder.................................. 6-23
standard lamp assembly ...................6-7, 11-2
temperature bath ...................................... 6-34
thermoelectrically cooled near-IR signal
detector ................................................ 6-13
thermoelectrically cooled signal detector6-12
Accumulations .............. 14-11, 14-19, 14-23
Acquisition Channel ................................ 14-9
Acquisition Controller – Details.............. 14-9
Acquisition Setup ...........14-14–16, 14-27–29
adjustment
lamp procedure .......................................... 5-8
Alconox® ................................................... 4-1
Alternate Lamp...................................... 6-30
amplitude ................................................... 8-2
analog-digital conversion ........................ 14-9
anisotropy-decay ....................................... 8-4
anthracene.........................................3-20, 4-9
Apply ..................................................... 14-37
Area List ......................... 14-26, 14-28, 14-33
Areas Definitions..........14-12–13, 14-33–34
18-1
Fluorolog-3 v. 2.2 (11 Jul 2002)
Area List ............................... 14-12–13, 14-34
Areas Views ....................... 14-12, 14-33–34
Arithmetic4-17, 4-20, 11-6, 11-11, 11-13,
11-18, 14-37
.arl .........................14-13, 14-15–16, 14-34
Auto Adjust ...........................................14-16
Auto Zero ............................................... 11-1
AutoScale .............................................14-30
axial.............................................. 14-1, 14-32
Axial........................................................ 14-5
azulene .................................................... 10-2
B
bandpass 3-14, 4-4, 4-13–14, 4-16, 11-5, 14-8
baseplate adapter.......................... 14-3, 14-39
binning ...................................................14-17
Binomial ................................................. 4-17
biological samples..................................... 4-2
blank..................................................... 11-6
Blank.....................................4-18, 11-1, 11-7
blaze ....................................... 14-2, 14-35–36
BNC connector.........................................13-5
boot disk.................................................... 7-2
Burst ......................................................14-22
C
cables. 5-1, 5-6, 5-14, 5-1, 7-2, 14-3–4, 14-39
Calc........................................................14-29
Calibrate... ...........................3-10, 3-17, 14-7
calibration
excitation spectrometer ..............................3-9
capillary cell.............................................. 4-2
CAS Number........................................... 3-20
CCD2-8, 2-12, 14-1, 14-3–4, 14-9–19, 1422–29, 14-31, 14-33–36, 14-40, 15-3
CCD acquisition......................... 14-10, 14-31
CCD Acquisition14-10–11, 14-17, 14-22, 1426–29
CCD Areas ..............14-11–12, 14-26, 14-28
CCD chip14-4, 14-12–13, 14-16–18, 14-33–
34
CCD controller....................14-4, 14-9, 14-40
CCD detector ...14-9, 14-12, 14-14–15, 14-31
CCD icon .................................................14-9
CCD Regions ..........14-11, 14-14, 14-27–28
Index
CCD shutter ..................................14-4, 14-23
CE marking..............................................15-1
Center ........................................14-15, 14-27
center wavelength
CCD ....................................................... 14-36
Change ................................................... 14-5
Change… ............................................... 14-6
Chip Height ...............................14-12, 14-33
Chip Width.................................14-12, 14-33
chopper ....................................................10-5
circular frequency......................................8-2
Close.................................... 3-10, 14-5, 14-8
Collect.................11-16, 14-10, 14-25, 14-31
colloidal silica..........................................3-20
COM port...................................................7-2
Comments:................................14-12, 14-34
configurations
custom .........................................................2-9
standard .......................................................2-3
connections, cable....................................13-1
Constant Wavelength Analysis.........3-2, 4-10
contaminated water................................7-5–6
Continuous............................12-4, 14-22–23
controller....................................................2-1
corrected data..................................4-18, 11-1
Correct .................................................. 14-29
Correction........................ 4-19, 14-21, 14-36
CORRECTION ....................................11-19
Correction-factor files
multiple detectors................................... 14-35
multiple gratings .................................... 14-35
correction factors ............................4-18, 11-1
CRE..................................................11-10–11
cuvette3-12, 4-1–3, 5-8, 6-4, 6-20, 6-22, 7-5–
7, 11-15
Czerny-Turner .........................................15-2
D
damage.......................................................0-4
danger ........................................................0-4
dark counts............................ 12-1, 12-4, 12-6
Dark Offset.............................................4-18
Dark Subtract............................14-13, 14-34
data
corrected..........................................4-18, 11-1
Data Acquisition Parameters ................11-19
data collection methods .............................4-3
18-2
Fluorolog-3 v. 2.2 (11 Jul 2002)
Data Units...............................................14-9
DataMax0-1, 4-18, 7-8, 8-1, 10-1, 12-3, 14-1,
14-5, 14-10, 14-13, 14-20, 14-25–26, 1429, 14-31, 14-34, 14-39–40, 15-4
D-connector .............................................13-2
Define Experiment .................. 14-26, 14-28
demodulation .............................................8-2
Details .....................................................14-9
detector2-4–7, 2-10, 3-3, 3-6, 3-13, 4-4, 4-10,
4-14, 4-16, 4-18, 5-9, 5-14–15, 6-1, 6-4–5,
6-7, 6-11–13, 7-1, 7-6, 10-1, 10-4–5, 11-5–
7, 11-14–15, 14-1, 14-3–4, 14-12, 14-17,
14-32, 14-35–36, 14-39–40
dispersion .................................................4-16
dissolved solids ..........................................4-2
DM302 Photon Counting Module ...........6-13
dynamic depolarization..............................8-4
E
Edit Table .............................................. 11-11
Electrical requirements ..............................1-3
Emission Acquisition3-4, 3-12–13, 4-5, 4-8,
4-11, 11-6, 11-8–9, 11-16
emission scan ...........................................10-4
Enable Int. Shutter Move ...................14-23
Enter Correct Position...........3-10, 3-17, 14-7
Entrance Mirror........................................14-5
entrance port ......................... 14-1, 14-3, 14-5
environmental requirements ......................1-2
europium(III) chloride hexahydrate.........3-20
Excitation Acquisition.3-4–5, 4-8, 11-16–17
Excitation Acquisition ................................3-5
excitation spectrometer
calibration ...................................................3-9
Exit Mirror ................................................14-5
exit port .......................................... 14-1, 14-5
Exp Type… .3-4, 3-12, 4-5, 4-8, 11-8, 11-16
Experiment .............11-6, 11-8, 11-16, 14-10
exposing
lamp, the......................................................5-3
Index
F-3004 Sample Heater/Cooler Peltier
Thermocouple Drive ........................... 6-29
F-3005/6 Autotitration Injector ............... 6-26
factors, correction...........................4-18, 11-1
faraday cage........................................... 14-40
fast-Fourier transform........................See FFT
Fast Kinetics Mode ..................14-11, 14-22
FF ............................................................ 6-24
FFT.......................................................... 4-17
filter ................................................6-15, 6-18
FL-1001 Front-Face Viewing Option...... 6-16
FL-1010 Filter Holder ....................6-15, 6-18
FL-1011 Four-Position Thermostatted Cell
Holder.................................................. 6-19
FL-1012 Dual-Position Thermostatted Cell
Holder.................................................. 6-21
FL-1013 Liquid Nitrogen Dewar Assembly66
FL-1015 Injector Port.............................. 6-33
FL-1039 Xenon Lamp Housing .............. 6-28
FL-1040 Dual Lamp Housing ........6-25, 6-28
FL-1044 L-Format Polarizer ................... 6-32
flash lamp ............................... 2-8, 2-12, 6-30
flip mirror .......................................14-1, 14-5
fluorescein ..................................3-20, 10-2–3
fluorescence polarization......................... 10-7
Fluorescence Service Department ..5-16, 5-20
fluoroimmunoassay ................................. 10-7
fluorophore ................................................ 8-4
Flush ..................................................... 14-24
Flush Before Each Acquisition.......... 14-24
Flush Before First Scan ..................... 14-24
FM-2003 Sample Compartment Accessory 619, 6-21
From...........................................14-16, 14-27
Front...................................................... 14-32
front-face data collection..................4-3, 10-6
Functions.....................................4-20, 14-37
fuse ........................................................ 14-39
fused silica.................................. 4-2, 6-6, 6-8
G
F
F-1000/1 Temperature Bath.....................6-34
F-3000 Fiber Optic Mount.......................6-14
G factor.............................................. 4-10–12
G Factor ................................................. 4-11
Gain Level.............................................. 14-9
glycogen .................................................. 3-20
18-3
Fluorolog-3 v. 2.2 (11 Jul 2002)
grating4-16, 4-18, 5-8, 5-16–19, 6-7, 6-15, 617–18, 10-1, 14-1–2, 14-6–7, 14-15–16,
14-28, 14-35–36, 14-38
grating factor............................... See G factor
grating icon ..............................................14-6
grating turret.............................................14-1
Grating/Turret........................3-10, 3-17, 14-6
groove density 4-16, 14-2, 14-6, 14-15, 14-35
H
hazardous condition .................................. 0-4
Help .......................................................... 7-8
Hg lamp..................................................14-26
High Voltage ........... 11-10, 11-17, 12-3, 12-5
highly scattering samples........................ 10-4
host computer0-1, 1-3, 2-1, 3-2, 6-31–32, 123, 13-1–3, 13-6, 14-4, 15-1
humidity level ........................................... 1-2
HV ...................................11-10, 11-17, 14-32
I
Image ..................14-11, 14-13, 14-17, 14-34
imaging spectrograph....................... 2-9, 2-11
infrared.................................................... 10-5
InGaAs ....................................... 14-32, 14-35
inserting
lamp, the......................................................5-5
installation................................................. 1-4
Instrument Control Center ..........3-2, 7-2, 7-8
Int. Time ................................................14-15
integration time4-13–14, 5-8, 14-15–16, 1428–29
determining optimum...............................4-14
Integration Time3-5, 3-13, 11-6, 12-3, 1411, 14-18, 14-23
Intensity .................................................14-32
Intg. time ...............................................14-16
IR detector............................ 14-32, 14-35–36
IRR ................................................. 11-11–13
irradiance............. 11-2, 11-5, 11-8, 11-10–11
isamain.ini ........................................ 7-2
isascan.set ........................................ 7-2
isascan.vw........................................... 7-2
Index
L
lamp
adjustment...................................................5-8
exposing ......................................................5-3
inserting.......................................................5-5
removing .....................................................5-4
lamp scan3-5, 3-7, 3-11–12, 5-1, 7-3–4, 7-8,
11-7, 14-7
lamp.exp.......................................3-5, 3-19
lamp.spc................................................3-5
laser....................................... 2-8, 2-12, 14-40
Last Read Out Time ........................... 14-23
lateral .................................. 14-1, 14-3, 14-32
Lateral..................................................... 14-5
layout .................................. 4-11, 14-5, 14-32
Layout Selection........................................3-2
LDS 750...................................................3-20
lifetime.................................. 3-20, 8-1–2, 8-4
lifetime measurements...............................8-2
lifetime resolved ........................................8-4
limit of detection......................................10-3
Linearization 14-12, 14-25–26, 14-28, 14-34
Linearization Parameters...........14-25, 14-26
Linearization Procedure ........................ 14-26
liquid nitrogen ...........................................6-6
Load ......................... 14-13, 14-15–16, 14-34
lock-in amplifier ......................................10-5
low-temperature scans .............................10-6
LUDOX® .................................................3-20
M
magnetic stirrer................... 4-2, 6-8, 6-19–22
maintenance...............................................5-1
detector, reference.................................... 5-15
detector, signal ......................................... 5-14
gratings..................................................... 5-16
lamp.............................................................5-1
housing................................................. 5-12
replacement.....................................5-1, 5-2
mirrors...................................................... 5-20
mask.........................................................11-2
Math Functions .............................4-20, 14-37
mcorrect....4-18–20, 11-1–2, 11-13, 14-36
(Me)2POPOP ...........................................3-20
meniscus ....................................................7-6
MHV connector ....................................... 13-5
18-4
Fluorolog-3 v. 2.2 (11 Jul 2002)
MicroMax .................................3-1, 6-2, 6-31
Microscope Interface ...............................6-27
mirror icon ...............................................14-5
Mirrors… ................................... 14-11, 14-20
mm..................................................... 3-6, 6-8
Model 1630 Field Lens Adapter ..............6-12
Model 1692M Selection Mirror...............5-20
Model 1914F Thermoelectrically Cooled
Signal Detector ....................................6-12
Model 1923 Micro Cell..............................6-8
Model 1924 Micro Cell..............................6-8
Model 1933 Solid Sample Holder 4-2–3, 6-23
Model 1938 Cut-On Filter ............. 6-15, 6-18
Model 1939 Cut-On Filter ............. 6-15, 6-18
Model 1908 Standard Lamp Assembly .. 11-2
Model 1908MOD Scatter Assembly....... 11-2
Model 1940 Absorption/Transmission Model
Accessory...............................................6-3
Model 1976 Accessory Controller.............7-2
Model FL3-11 ............................................2-4
Model FL3-12 ............................................2-5
Model FL3-21 ............................................2-6
Model FL3-22 ............................................2-7
Model FL3-XXX .....................................2-10
Model TRIG-15/25 External Trigger
Accessory.............................................6-35
models
Fluorolog-3 .................................................2-3
modulation ......................................... 8-2, 8-4
modulation lifetime....................................8-2
monitor.......................................................1-3
monochromator2-3, 3-2–4, 3-7, 3-9–12, 316–18, 4-16, 5-17, 7-1–2, 10-5, 11-15, 141, 14-5, 14-38
monolayer ...............................4-2, 6-16, 10-6
Move All ..................................................14-8
Multi Scans/One Peak........................14-26
Multiply.............................4-20, 11-11, 14-37
N
NADH......................................................3-20
Nb Cover ..............................................14-16
Nb region ..............................................14-14
Nb Region ................................ 14-27, 14-29
Nb Regions ..........................................14-27
near-IR detector .................................... 14-35
neutral-density filter................................ 11-2
Index
New Polarization Sample........................ 4-10
New Value ........................................... 14-6–7
Nikon ................................................6-2, 6-27
nitric acid................................................... 4-1
nm ........................................................... 3-14
No Flush ............................................... 14-24
no trace view object................................ 7-2
Normalize ............................14-12–13, 14-34
Number of Areas......................14-13, 14-34
Number of Scans .........................4-15, 11-6
O
offset.................................................8-2, 10-4
OK14-6–7, 14-9, 14-13, 14-15–16, 14-26–
29, 14-34
Olympus ...........................................6-2, 6-27
One Scan/Multi Peaks ....................... 14-28
Operand ......................................4-20, 14-37
optics ....................................................... 4-10
optimum
integration time ........................................ 4-14
optimum wavelengths
determining ................................................ 4-3
options ..............................................2-8, 2-12
custom ...................................................... 2-12
standard ...................................................... 2-8
Options ............................................3-6, 3-14
outliers ..................................................... 4-17
P
Peaks/Settings......................................... 4-17
performance test report...................3-3, 14-36
phase angle ................................................ 8-2
phase lifetime ............................................ 8-2
phase shift.................................................. 8-4
phosphorescence................. 2-2, 3-20, 10-5–6
photobleach ............................................. 10-7
photodiode 2-2, 3-4, 5-15, 6-2, 6-9, 11-14–15
photomultiplier detector .................. See PMT
photomultiplier tube ........................ See PMT
plateau voltage............................12-1, 12-5–6
PMT2-3, 2-8, 2-12, 3-3, 5-14, 6-2, 6-13, 1421, 14-32
Polar Scan… ......................................... 4-12
polarization............................. 2-2, 2-10, 4-10
18-5
Fluorolog-3 v. 2.2 (11 Jul 2002)
Polarization............................................ 4-11
polarizer .................. 4-10–12, 6-32, 7-1, 10-7
Polarizer Settings ................................... 4-10
poor resolution .......................................... 7-4
POPOP .................................................... 3-20
Position ..........................14-15, 14-27, 14-29
potassium bromide .................................... 4-2
power supply5-2, 5-7, 6-1, 6-7, 6-12, 6-25, 71, 11-2, 11-5, 14-2
PPD ......................................................... 3-20
PPO ......................................................... 3-20
printer..........................................1-3, 2-1, 3-2
Proceed to Acquisitions....................... 4-10
p-Terphenyl............................................. 3-20
Q
quantum yield.............. 4-13, 4-18, 10-4, 11-1
quartz.................. 3-12, 6-2, 6-6–8, 6-23, 11-2
Quick Polarization................................... 4-12
R
R ..............................................3-6, 4-18, 6-5
R928P2-4–7, 3-3, 5-14, 6-11–12,11-7, 11-14,
15-2, 14-35–36
RA ...........................................3-14, 6-3, 12-3
radiometric correction.................... 11-1, 11-8
radiometric correction factors ........ 4-18, 11-1
Raman3-3, 3-12–13, 3-15, 3-18–20, 5-1, 5-8–
10, 7-5–8, 9-1, 14-7
Range..............................................14-15–16
raw polarization ...................................... 4-10
Rayleigh scatter....................................... 3-15
re-assembly ...................................... 1-4, 13-1
Real Time Display3-2, 3-9, 3-16, 4-4, 4-14,
5-9, 11-3, 11-15–17, 12-3, 14-7, 14-31
red-sensitive photomultiplier .................. 10-5
reduced-volume samples......................... 10-3
reference detector........ 2-2, 4-18, 8-1, 10-1–2
reflectance plate ...................................... 11-2
Reformat .............14-13, 14-26, 14-28, 14-34
remove
lamp.............................................................5-4
replacement procedure
lamp.............................................................5-2
resorufin .................................................. 10-3
Index
rhodamine-B ..........................................11-15
ribbon cables.......................................... 14-39
right-angle data collection .........................4-3
rose Bengal ..............................................3-20
RTD-CCD............................................. 14-31
Run. 4-7, 4-9, 11-18, 14-11, 14-23, 14-28–29
Run Experiment3-2, 3-4, 3-11–12, 3-18, 4-5,
4-8, 4-11, 4-14–15, 4-17, 4-19–20, 5-10,
11-6, 11-8, 11-13, 11-16, 11-18, 14-10, 1431, 14-36–37
S
S .............................3-13, 5-8–9, 6-5, 12-3–4
S/N ratio .......................... 4-13–15, 6-12, 15-3
S/R .................4-9, 4-18, 6-4–5, 11-15, 11-17
sample changer .................................8-1, 11-3
sample compartment2-1, 4-4, 11-3–4, 11-15,
14-1, 14-3
sample mount........................................... 14-1
sample preparation
biological samples ......................................4-2
dissolved solids...........................................4-2
small-volume sample..................................4-2
solid samples...............................................4-2
Save ......................... 14-13, 14-15–16, 14-34
Savitsky-Golay ......................................4-17
Scan ............................... 14-11, 14-14, 14-17
scanning, multiple times..........................4-15
scatter block.............................................11-2
scatter plate..............................................11-3
scattering..................................................3-20
screwdriver .......................................5-1, 5-16
Select ...........................................4-20, 14-37
Select Calibration Peak......................... 14-29
Select Experiment Type3-4, 3-12, 4-5, 4-8,
11-8–9, 11-16
Select New Term File ...................4-20, 14-37
Selected Signal ...................................11-17
serial number .............................................7-9
Service Department1-4, 7-1–2, 7-8–9, 11-2,
14-39
shutter ......... 5-9, 7-1, 8-1, 11-1, 11-15, 14-39
Side ....................................................... 14-32
Signals.................................. 3-6, 3-13, 4-8–9
Signals....... 3-6, 3-13, 4-6, 4-8, 11-10, 11-17
signal-to-noise ratio ....................See S/N ratio
singlet ......................................................10-5
18-6
Fluorolog-3 v. 2.2 (11 Jul 2002)
slits2-4–7, 3-6, 3-14, 4-4, 4-16, 5-8, 5-17, 61, 6-4–5, 7-1–2, 7-4, 7-6–7, 8-1, 11-3, 115, 11-15, 14-3, 14-8, 14-28–29, 14-32, 1439
Slits ...........3-6, 3-14, 4-6, 11-17, 14-8, 14-32
Slits... ............................3-6, 3-14, 4-6, 11-17
small-sample volume .................................4-2
smoothing.................................................4-17
solid samples..............................................4-2
specifications
computer ...................................................15-4
software.....................................................15-4
spectrofluorometer system .......................15-2
TRIAX ................................................... 14-38
SpectrAcq......................................... 3-1, 14-4
standards ..................................................3-20
stdlamp................................................ 11-6
stdlamp2 ............................ 11-6, 11-12–13
Styryl 7.....................................................3-20
support
technical ......................................................7-8
surface requirements ..................................1-1
symbols ......................................................0-4
synchronous scan ............................. 6-5, 10-4
system configuration..................................1-1
system controller..................... 1-3, 13-1, 13-3
Index
troubleshooting, TRIAX ....................... 14-39
turret ................................... 14-2, 14-6, 14-39
U
Units.................................................3-6, 3-14
Use Polarization Modes....................... 4-10
V
ventilation.................................................. 1-2
View ...................................................... 11-10
Visual Instrument Setup3-2, 3-6, 3-9–10, 314, 3-16–18, 4-14, 7-1, 14-2, 14-5, 14-9
voltage converter ..................................... 14-2
W
water ........................................................ 3-20
water.exp ..................................3-13, 3-19
water.spc ........................................... 3-13
wavelengths
determining optimal................................... 4-3
web site...................................................... 7-8
Windows™.......................................0-1, 15-4
X
T
T-box2-2–3, 6-14–15, 6-18, 6-32, 10-6, 1115
Table View........................................... 11-10
technical support ........................................7-8
temperature ................................................1-2
temperature bath .... 3-1, 6-2, 6-19, 6-21, 6-34
Term File...............................................14-37
Term File *K ............................... 4-20, 14-37
thin film...................................... 4-2, 6-23–24
time-based fluorescence...........................10-6
Time Increment....................................14-23
time-resolved .............................................8-4
time-resolved data....................................10-5
To...........................................................14-16
Toolbar..................................................14-32
TRIAX2-9, 2-11, 14-1–5, 14-8, 14-10, 1415–16, 14-25, 14-31–32, 14-35–36, 14-39
triplet........................................................10-5
troubleshooting ..........................................7-1
Xe ......................................... See xenon lamp.
xenon lamp1-3, 2-2, 3-1, 3-3–5, 3-8, 3-10, 312, 3-19, 4-18, 5-1, 5-2, 5-4–5, 6-1, 6-9, 625, 6-28, 7-1, 7-3–4, 9-1, 12-3–4, 14-7
xcorrect.. 4-18–20, 11-1, 11-18–19, 14-36
XFER .................................................... 11-17
Z
Zap .......................................................... 4-17
Zeiss..................................................6-2, 6-27
18-7
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