Zeiss LSM 510 Multiphoton Confocal Microscope

Zeiss LSM 510 Multiphoton Confocal Microscope
Zeiss LSM 510 Multiphoton Confocal
Microscope
User Guide
LSU Health Sciences Center-Shreveport
Research Core Facility
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
Table of Contents
1
2
3
4
5
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7
8
9
10
11
12
13
14
15
16
Safety……………………………………………………………………………………………………………………....
Turn On the System ………………………………………………………………………………….................
Start Up the ZEN Software ……………………………………………………………………………………….
Turn On the Lasers .………………………………………………………………………………………………….
Select and Mount Objectives……..………………………………………………………………………………
Change Stage and Mount Samples ……………………………….……………………………………………
View Samples Through Eyepiece………………………………………………………………………………..
Configure the Beam Path ………………………………………………………………………………………….
Set the Parameters for Scanning……………………………………………………………………..……….
Optimize Image Acquisition ……………………………………………………………………………………..
Z Stack Acquisition …………………………………………………………………………………………………….
Using the NDD Detectors ………………………………………………………………………………………….
Save ………………………………………………………………………………………………………………………….
Shutdown..………………………………………………………………………………………………………………..
Specifications for Publication..…………………………………………………………………………………..
Additional Information on Software Options ……………………………………………………………
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1. SAFETY
Page 2 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0

The system contains components with dangerous voltage. The system must not be opened by
anybody else other than authorized Carl Zeiss Service staff.

Maintenance, repair, modification, removal or exchange of components, or other interference
with the equipment beyond the operations described in this manual may only be carried out by
the manufacturer Carl Zeiss or by persons expressly authorized by Carl Zeiss to do so.
This applies especially to the microscope system, the laser scanning module, lasers, the PC
system, the power supply units, cable connections and other system components.

The openings for ventilation must not be covered.

There are hot surfaces on the HBO and HAL lamp.

The LSM 510 and LSM 510 META are laser hazard class 3 B instruments. If equipped with a
Ti:Sa Laser, the LSM 510 and LSM 510 META are devices that belong to laser hazard class 4.
These moderate and high-risk classes embrace medium-power and high power lasers. You must
take care not to expose yourself to the radiation of such lasers. In particular, never look into the
laser beam! Only personnel who have been instructed on laser safety are allowed to operate the
system.

If used properly, the LSM 510 and LSM 510 META will not pose any laser radiation risks for operating
staff. Nevertheless, you should observe the following warnings:
•
Do not place any reflecting objects into the beam path.
•
Never open any covers or panels.
•
Never look into the laser beam, not even to simply view the specimen, whether with the aid
of optical instruments or without. Otherwise you risk going blind!
•
Do not leave any empty objective positions of the nosepiece uncovered.

If a class 4 laser is attached to the system, already stray light can impose danger to the
operator.

With class 4 lasers take special care of fire protection requirements. Do not use or store
flammable or explosive solids, fluids or gases in the vicinity of the system.

Class 4 lasers can ignite flammable materials like cloth or paper. Do not put such
materials into the beam path.

For NLO systems equipped with a specific push and click filter for NDD imaging be aware that
the NDD reflector cube in the reflector turret leads to a strong back reflection of HBO light into
the specimen plane and the ocular lens. When observing the specimen through the ocular lens
the use of the NDD reflector cube should be avoided. The light flash is not harmful but
unpleasant. The reflex of closing the eyelid is sufficiently protective. To completely avoid this
situation an additional filter (#1261-345) can be mounted into the NDD reflector cube which
prevents the back reflection of the HBO light in the ocular plane.
Page 3 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
2. TURN ON THE SYSTEM
Always turn on in the order of 1->4. Numbers are labeled on the
machines
1) Turn on the FluoArc mercury arc power supply (No. 1).
2) Wait two minutes until ignition is complete on the FluoArc
timer/controller. Then you may select the percent lamp
intensity by pressing F1 or F2. The 50% intensity level will
cause less photobleaching while you are observing your
sample. (Record the bulb time in the sign-up sheet)
3) Turn on the main power switch on the laser cart, turning 90
degrees clockwise, to vertical (No. 2).
4) Press the Components and System/PC switches to ON, one at
a time (No. 3).
5) Press the On button to turn on the computer. Log in with
your LSUHSC ID and password. Make sure the domain is set
to LSUMC-MASTER.
Page 4 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
6) If you are going to use the Chameleon multiphoton laser,
turn the key on the front of the control panel from Standby
to On.
3. START UP THE ZEN SOFTWARE
Double-click on the ZEN 2009 desktop icon to start the program.
The Login “Zen 2009” window appears. Select
Start System to initial microscope system for
acquisition. If you want to only analyze your
images, select Image Processing.
Wait for the Initialization to complete.
The main menu opens. This guide will only cover
features from the File, Acquire, and Maintain
submenus. There are many other very useful
features which are explained in the Zeiss
Operating manual, located in the Help menu.
Page 5 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
4. TURN ON THE LASERS
From the Setup Manager submenu, click on the
Laser menu.
In the Laser window, click on the name of the
laser to activate it.
For the argon laser, click Standby. When the
status goes from warming up to ready, click On
and then use the slider to set output to 50%. If
you exceed 50%, you will get a warning. Above 50
% is only used for intentional photobleaching.
For the HeNe 543 and 633, select On.
If you log out of the system, the argon laser will
go to standby.
Page 6 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
When you turn the Chameleon laser on, all you
are doing is opening a shutter and activating the
software control. The laser is actually turned on
with the key on the front of the Chameleon
control panel. The status will read Key Locked, if
you have not turned the key, and Heating if only
a few minutes have passed since you turned the
key. It is ready to use when it says Modelocked.
Note: Please inform staff when the chameleon
laser has problem going into the Modelock
status.
You can tune the wavelength of the Chameleon
laser from 705nm to 980nm by clicking on the
laser icon in the Light Path tab or in the
Channels window by entering the wavelength of
choice. The status will read Busy while the
mirrors are adjusted, and then it will read
Modelocked. (This is indicated with a red box
over the laser wavelength for Busy and
disappears when in Modelocked.) Please modify
the wavelength in multiple steps if you are
moving it through a large portion of the range,
i.e. three 50nm steps to go from 800nm to
950nm.
For quantitative work, the lasers should be on for one hour before collecting data.
Note: The tuning process should only take a few seconds to complete, when there is a delay or takes longer time
please inform staff.
Page 7 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
5. SELECT AND MOUNT THE OBJECTIVES
The microscope holds only two objectives, and the ones you plan to use need to be activated in the software
because many of the optimal settings for imaging are related to the objective chosen and the software needs to
be told what you are using.
From the main menu, select the Maintain Tab, then select Objectives.
In the Objectives window, two objectives will be
displayed. They may/may not be the actual
objectives that are screwed into the turret mount.
To change the objective, in the software, click on
the objective icon. See if the desired objective is
already in the software by clicking one of the
change objective options: Favorite, User-Defined,
or Potential. Find the correct objective and then
click on Select Objective.
Page 8 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
Before changing objectives, use the ↑ focus
button to move the objective carrier to the highest
up position, to prevent the objectives hitting the
stage while operating.
To mount the objective(s), turn the front silver
lever clockwise and raise the objectives. Push or
pull the lever gently to move the objectives out of
the load position. Then unscrew the mounted
one(s) clockwise. Screw in the one(s) you want
counterclockwise and tighten slightly.
To use a selected objective, Turn the front silver
lever clockwise to raise the objectives, then push
or pull the lever gently to put the objective of
choice in the load position.
Note: Lower the objective slowly by turning the
lever counterclockwise, but do not let it drop down
into the complete down position. Since the stage
can be (and often is) manually raised or lowered to
different positions, you may not have enough
working distance to fully lower the objectives.
Available objectives are in a box on the shelf
labeled: Zeiss LSM Objectives. All objectives are
listed on the RCF ->microscopy ->Zeiss Confocal
webpage.
Page 9 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
6. CHANGE STAGE AND MOUNT SAMPLES
Slide-samples and live animals use different stages. You need to change the stage according to your need. If you
are not experienced with stage changing, ask for assistance.
animal stage
slide stage
To change stage, screw or unscrew the stage holder
as shown in the image. Both slide-stage and animalstage have magnatic legs to fix on the stage.
For the slide stage, you need to fit the groove on the
back of the stage to the microscope, and make sure
the groove sits on the knob of the microscope.
stage holder
Knob on the microscope
Place the slide, coverslip facing up, in the slide carrier on the stage.
If you are using an immersion objective, place a VERY small drop of the appropriate immersion fluid on the
coverslip. Be sure to only use the immersion fluids located next to the microscope. If you accidentally use the
wrong fluid, please contact staff right away so they can clean the objective.
Page 10 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
7. VIEW SAMPLES THROUGH EYEPIECE
To observe your sample through the eyepieces, click
the Online button on the Ocular submenu or press
the Vis button on the left side of the microscope.
Manually turn the turret to the desired filter cube
with its number in front and center.
1 = scanning
2 = DAPI (EX 365,EM LP 420)
3 = FITC (EX BP 470/40, EM BP 525/50)
4 = TRITC (EX BP 546/12, EM LP 590)
5 = Brightfield or NDD (for 2-photon)
HBO lamp
For fluorescence excitation, use position 2,3, or 4 on
the turret, and make sure that the manual shutter to
the HBO lamp is pulled out, and the manual slider on
the right side of the microscope is pulled out to the
first stop (open position).
When performing a scan, the manual slider on the
the microscope needs to be pushed in all the way
(close position), and the filter turret turned to
position 1.
Note: The HBO lamp (mercury bulb) only provides
fluorescence light for eyepiece observation. For confocal
scanning, the laser are involved.
Page 11 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
For Bright field observation, make sure that the Bright
field filter cube is loaded (position 5). The #5 position
is also used with another filter cube in place for
observation with the NDD detectors, and you must
confirm that a previous user has not left the NDD
cube in place. You will be shown how to do this
during training.
Turn on the HAL illumination power supply, with
bulb intensity adjusted. (HAL is the light source for
bright field transmitted light)
When you are doing fluorescence scanning, you will need
to have the HAL bulb intensity turned to zero, or it may
add background signal to your images.
To focus on your sample, use the focus control
module.
Slow = fine focus
Speed = fast focus
Turn wheel = ultra-fine focus (CW ↑, CCW ↓)
↑ = moves the objective carrier up
↓ = moves the objective carrier down
You can also focus using the software by selecting
Focus menu from the Online Acquisition submenu.
Page 12 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
8. CONFIGURE THE BEAM PATH
Click on the folder icon under to add or change an
experiment Configuration. This allows you to load
predetermined configurations for the light path
and for tracks used during acquisition.
From the opened pull down menu, select the
cofiguration that meets your needs. A custom
configuration can also be written and saved to the
list through setting the Light Path. Any method
that has NDD in the name uses the NDD
detectors, and should only be selected if you are
using them.
Examples of Light Path configurations using the standard confocal detectors, LSM, and the Non Descanned
Detectors, NDD.
The light path is initially difficult to understand, and you should not write your own methods until you do
understand it. Please ask staff for assistance in writing a custom method, and they will explain the positions of
dichroic mirrors and emission filters.
Page 13 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
9. SET THE PARAMETERS FOR SCANNING



Before any scanning, turn the filter cube turret to Scan (1) and push in the manual slider on the right
side of the microscope (see picture page 12).
In the Ocular tab, select Offline to switch from eyepiece to software.
In the Acquisition Tab, click on Acquisition
Mode from the Online Acquisition submenu.
 Choose your objective from the pull down menu.
 In the Scan Mode menu icon, select Frame.
 Select the frame size by clicking on one of the
predefined selections, define your own, or click on
Optimal to select the appropriate frame size for
your objective N.A. and λ. Pixel size is very
important for successful imaging. Pixels smaller
than optimal result in oversampling, more
photobleaching, and longer scan times. Pixels
larger than optimal produce images with reduced
spatial resolution. High resolution may not be
important to you – you decide. Remember that
more pixels will result in a larger image file.
 Use the slider to set Scan Speed. Use a fast speed
to focus on your sample, center it, and do other
basic adjustments. Use a slower speed for
scanning. Fast scanning produces a lot of
undesirable noise in the image.
 Select the Data Depth as 8 bit or 12 bit. 12 bit is
recommended for publication and quantitative
imaging.
 Set Scan Direction as unidirectional or
Scan Area: The default zoom position is 1. With Zoom set at 0.7,
you are using the entire scanning field. With zoom >0.7, the scan
area is decreased, but the frame size (number of pixels in x,y)
remains the same. Thus the pixel size is smaller. Click Optimal
under Frame Size to readjust the pixel size. The red-blue box
representing the scan field can be moved by dragging it with the
mouse or clicking the Offset arrows. Pressing Reset re-centers the
scan field.
Page 14 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
bidirectional. Bidirectional is twice as fast, and is
mostly used for live sample scan.
 Set Scan Average, which will improve the signal
to noise ratio. You can avg. line by line (smoother
image) or frame by frame (less photobleaching).
You can either average the number of scans you
choose, or sum them for weak signals.
 Select Channels from the Online Acquisition
submenu.
 Set the Pinhole size to 1 (Airy Unit) by clicking on
the 1. Pinhole adjustment changes the optical slice
thickness, the z thickness from which signal is
collected. When collecting multichannel images,
adjust the pinholes so that each channel has the
same optical slice thickness. This is important for
colocalization studies.
 Set the Detector Gain, for each channel, between
600 and 800.
 Make sure a laser is checked for each channel
(clicking on each channel name will display the
Excitation pane for each channel. Select a usual
Transmission % to start (488nm – 5%, 543nm 50%, 633nm – 20%, and the Chameleon laser –
2%).
 Select Snap to acquire a single image.
 Select Continuous for continuous scanning with
the selected scan speed.
 Select Stop for stopping the current scan
procedure.
 The best settings for gain, offset, and laser power
are determined while scanning in continuous
mode at a medium speed, and adjusting the sliders
to optimal positions while using the range
indicator from the Color Palette window.
Page 15 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
10.OPTIMIZE IMAGE ACQUISITION
If you do not already have an image window open, do a fast
scan to get one, or click on New Acquisition Document.
From the bottom panel of the image window, select
Dimensions.
Then click on the display indicator below the channel
indicator. This will display a list of possible choices, choose
Range Indicator. This false-color LUT colors saturated pixels
red (255 intensity value for 8 bit and 4095 for 12 bit images)
and black pixels (0 intensity value) are colored blue. This
will help you set laser power, gain, and offset correctly so
that there are almost no oversaturated red pixels and only a
few blue pixels.
Set the Scan Speed to a medium speed, around 6-7.
To reduce noise in the image, you can reduce scan
speed, and/or use average successive scans (Mean) by
Line or Frame. To increase signal you can Sum
successive scans or increase laser power or detector
gain.
Click the Continuous button, which scans continuously. Set
the Detector Gain (Master) to 800, and then adjust the
laser power slider until you do not have any saturated
pixels. Next, set the Digital Offset with the slider until you
have a few blue pixels. When you are finished with your
adjustments, stop the continuous scan by clicking on Stop,
and then collect an image by clicking on Snap.
Page 16 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
11. Z -STACK ACQUISITION
Select Z Stack in the Scan Control Window. This
will insert a menu in the Acquisition Tab in the
Multidimensional Acquisition submenu, which is
circled.
To set the Z scan region, Click on Continuous or
Live and focus to a lower position of the sample
where the Z Stack is to start. Click Stop, then Set
First. Repeat this procedure, focusing to the top
of the sample and Set Last.
The Interval is the distance the motorized
objectives will move with each step. On this
microscope, the motorized objectives will only
move in the up direction during a z stack. To
determine the ideal interval size that will collect
the most information without oversampling, click
on Optimal, below the Interval number which is
circled.
Click on X:Y:Z = 1:1:1 to set the Z interval in such a
way that the voxel has identical dimensions in x, y
and z. This is sometimes important in 3D image
reconstruction.
Refractive Index Correction is important if there
is a difference in the refractive index of the
objective immersion medium (n) and the
mounting medium of the specimen (n’). Use Ratio
= n/n’ to determine the proper setting with the
slider.
Auto Z Brightness Correction allows you to set
different gains, offsets, and laser power at
different positions of the stack and linearly
interpolate between them.
Click Start Experiment, in the Acquisition Tab, to
begin the Z Stack.
Page 17 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
12.USING THE NDD DETECTORS-For Two photon
These instructions come from a Zeiss Applications Specialist. They cover the process from turning on the
Chameleon laser to scanning your sample. Some pieces of these instructions have been covered in this guide
already.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Normal startup of the system, plus turn the Chameleon from “Standby” to “On” via the key on the
Chameleon power supply
Select the “Laser” button and click on Chameleon.
Click “On”. This opens the shutter on the Chameleon, and turns the AOM on for attenuation control of
the laser.
If you need to change the wavelength, click “Modify” in the laser window, then type in the wavelength
desired and click “Store”.
After locating your sample as you normally would with the “Vis” button and proper filters, go back to the
LSM mode, and open the Configuration window. Here, about half of the way down, you will see two
tabs, “LSM 510”, and “Nondescanned”. Click “Nondescanned”
All of the configurations that use the NDDs have ndd in their descriptions. You have the choice of single
or one multitrack. You can choose one of those, or make your own.
 To make your own, click on the 4 position drop-down near the middle of the diagram, which
includes 3 dichroic filter choices, and “HBO Mirror”
 Choose one of the dichroics, and the corresponding filters for the 2 channels will be listed.
Choose the channel that you want to use with a check mark.
 Make sure that the primary dichroic in the upper left hand corner is on “KP 650”.
Click your laser to on in the laser window in the diagram. It should be at relatively low power when
using a high NA objective when in the 750 to 800nm range, say 1 to 5%, and incrementally higher power
away from this center of the range. Lower NA objectives pass less power, so will also require higher
power settings.
Make sure the NDD dichroic is in the 5 position on the reflector turret at the front of the microscope.
Select the 5 position to send the emission light to the NDDs.
If the HBO lamp is on, push the black slider that sticks out to the back of the tube that connects the lamp
house to the closed position.
Make sure that the black slider on the right side of the microscope is in the middle or outermost position
Turn the lights off, and keep light minimally in the room. Cover the sample stage with the black vinyl
cloth with Velcro strip. Consider turning the monitors off or covering them.
Go to scan control and use it as you would for normal imaging, keeping in mind that there is no pinhole.
Page 18 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
13. SAVE
Click on the Save or Save As button in the Image
window or in the File toolbar of the Main menu.
In the opened Save As window, select a file
format. LSM 5 is the current software file format
and LSM 4 should be openable by AIM or LSM
software programs. All files should be saved to
the E drive. Afterwards, enter a name for the file,
type in any description and notes. Make sure the
appropriate file is highlighted and click OK.
To save an image in a different data format, open
the .lsm image, choose File from the main menu,
then select Export. Then select the image Format.
Page 19 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
Raw data: single plane or series (single image or
time series/z stack with no LUT or overlays)
Contents of image window: single plane or series
(single image or time series/z stack as viewed on
the monitor – easy way to save the panel image)
Full resolution image window: single plane or
series (single image or time series/z stack with the
pixel resolution as collected, 512 x 512, 1024 x
1024, etc.)
Be sure to save all images to the C Drive in a folder with your first and last name. This is only temporary
storage. Transfer your files to external storage as soon as possible.
14. SHUTDOWN
If someone is signed up after you within two hours, do not turn off the system, but do turn the Multiphoton
laser to standby with the key, then turn it off in the laser panel. Turn the argon laser to standby in the laser
panel. Close the software, and log out. If no one else is signed up to use the system for 2 hours, shut the
system down in the following order:










Save your images to your database
Click on the file button in the Main Menu of the ZEN software and then click Exit.
In the pop up window, shut off all lasers that are running.
Log off and shut down the computer.
Wait until the argon laser fan has switched off – about 5 minutes. The fan is on top of the laser cart,
and you can hear it running and feel the air flow. It is important for the life of the laser that it not be
turned off prematurely. If you turn off the System/PC switch prematurely, it will shut the fan off.
If the Chameleon multiphoton laser was used, turn the key on its front panel to standby.
Turn off the Components and System/PC switches, one at a time (No. 3).
Turn off the main power switch on the laser cart, turning 90 degrees counter-clockwise, to horizontal
(No. 4).
Record the Bulb time and time of day in 24 hour format in the sign-up sheet.
Switch off the HBO 100 mercury lamp switch (No. 1).
Page 20 of 21
RCF Microscopy Manual
Authors Chaowei Shang, C. Nook and K. Lorens
7/1/2017
Version 3.0
15. SPECIFICATIONS FOR PUBLICATION
The system is a Zeiss LSM 510 NLO with a Coherent XR multiphoton laser. It has three standard
Photomultiplier Tube (PMT) detectors, two Non-Descanned Detectors (NDD), and a transmitted light detector.
The attached microscope is a Zeiss Axioscop 2 FS MOT with an upright configuration.
It has the following lasers and objectives:
LASERS
1.
2.
3.
4.
Argon (458,477,488,514nm)
Green HeNe (543nm)
Red HeNe (633nm)
Multiphoton Coherent
Chameleon XR tunable pulsed
laser (705– 980nm)
OBJECTIVES
10x /.3NA Fluar WD 12.5mm
25x/.8NA DIC LCI Plan Neofluar (for use with/without coverslips, water or glycerol
immersion)
20x/0.5NA Acroplan WD 1.9mm, water dipping
40x/0.8NA Acroplan WD 3.6mm, IR corrected, water dipping
63x/0.9NA Acroplan WD 2.0mm, IR corrected, water dipping
63x/1.4NA DIC Plan Apochromat WD .18mm, oil immersion
16. ADDITIONAL INFORMATION ON SOFTWARE OPTIONS
This manual is only a quick start guide and does not cover many additional and useful features in the software.
You can also access information on software features through the Help section, accessed from the Main
Menu.
Page 21 of 21
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