ConfoCor 3
Microscopy from Carl Zeiss
ConfoCor 3
Laser Scanning Detection Module
Quantitative Image and Fluctuation Analysis –
Every Photon Counts
Unraveling Cellular Processes
Unraveling cellular processes requires describing
them in quantitative terms – a task requiring
supreme­ly sensitive detection methods capable of
detecting single molecules.
This is the task where the combination of the
LSM 710 and the ConfoCor 3 displays its strengths:
Imaging with the very best signal-to-noise ratio for
every requirement.
Sensitivity however is not the only feature that
counts. Another essential contribution to optimum
results is intelligent data analysis that can compute
dynamic information from intensity images.
With its special modules for fluctuation analysis,
the ZEN software fulfills your needs.
ConfoCor 3 on the LSM 710 –
your key to quantifiable success.
Contents
ConfoCor 3 on the LSM 710
4
APD Imaging
6
FCS
8
RICS
10
Beam Path
12
ZEN Software
14
ZEN for APD Imaging
15
ZEN for FCS
16
ZEN for RICS
17
System Properties
20
Specification
21
System Overview
22
2
ConfoCor 3
on the LSM 710
3
ConfoCor 3 on the LSM 710
A Clever Combination that Taps Synergies
An expert duo of supreme sensitivity and dynamics.
The very combination that makes this microscope
for the most exacting requirements.
The ultimate in sensitive detection – simply perfect.
Flexibility in image acquisition
Continuous or single photon counting provides the broad
basis for sensitive detection. The most efficient acquisi­tion
mode is always available, whether expression is high or
low.
Images with quantifiable data
The ZEN software offers powerful algorithms to display the
dynamics derived from the fluctuations in the image.
The ConfoCor 3 is coupled to the external channel output
of the LSM 710 via a free parallel beam.
Best optical quality and stability are guaranteed.
4
The inner workings of the ConfoCor 3
shows the high degree of automation.
All optical elements can be controlled
through the software, to ensure high
reproducibility and precision.
5
APD Imaging
Sensitive Imaging and Photon Counting
Seeing where there is hardly anything to be seen: an assignment that calls for
the power of an avalanche photodiode (APD). Counting every single photon,
APDs provide the ultimate in photon counting sensitivity. The dark noise of these
detectors is extreme­ly low, resulting in an exceptionally high signal-to-noise ratio.
Specimen-preserving procedures
The use of APDs allows laser outputs to be substantially
reduced where delicate specimens are involved. With less
unnecessary stress on the specimens, cells can be observed
over a much longer period.
Particularly strong with weak expression
Thanks to their low dark noise, APDs have an excellent
signal-to-noise ratio. So you can safely sum up the interesting signals from several images, whilst the background
remains nicely dark.
Particularly strong in the red wavelength range
The APDs used in the ConfoCor 3 are highly efficient especially in the red wavelength range, just where fluorochromes can typically be very weak. Combination with the
Quasar detector easily allows the balancing of different
intensity fluorochromes.
Detect subtle differences
The extremely sensitive APDs detect the tiniest and fastest
changes in the fluorescent signal.
C-Apochromat 40x / 1.2 W Korr UV-VIS-IR from Carl Zeiss.
Excellent color correction plus high transmittance –
from IR to VIS to UV.
6
Avalanche photodiode from Perkin Elmer.
Extreme sensitivity in the red range, low dark noise.
HeLa cell expressing DsRed1.
Despite laser output reduced approximately four times, the increased sensitivity
of the APD is clearly visible. Specimen: Takako Kogure and Atushi Miyawaki,
RIKEN Brain Institute, Wako, Japan
HepG2 cells expressing EGFP-CENP I.
A time series of 500 images acquired with laser power reduced five times clearly
displays less bleaching due to the use of APDs. Specimen: Stefanie WeidtkampPeters and Peter Hemmerich, Fritz Lipmann Institute, Jena, Germany
Distribution of the proteins Keima-PKC (red channel) and EGFP-MARCKS
(green channel) in HeLa cells at rest (left), and after stimulation
with PMA (right).
Subtlest changes can be followed extremely well with APD detectors.
Specimen: Takako Kogure and Atushi Miyawaki, RIKEN Brain Institute,
Wako, Japan
7
FCS
Fluorescence Correlation Spectroscopy
Determining the diffusion, concentration, localization and interaction of molecules
with single-pixel accuracy is the function of FCS. All this information is directly
obtained and analyzed from the fluctuation of the fluorescent signal, with no need
to calibrate. Performing an analysis is as easy as this:
Define the site of measurement
Define the measuring site within an LSM image
and simply mark it. Start the measurement, and
the LSM scanners will precisely target the marked
site. This way you can examine several sites of
interest in automatic succession and with high
resolution.
Determine correlation
From the fluctuation, the system determines time
correlation in real time during the measurement.
The signal is compared either with itself (autocorrelation) or with another signal (cross-correlation), to show similarity to itself or another
signal, respectively.
Start fluctuation analysis
A mouse click starts the recording of the fluctuations caused by molecule movement against
time. The APD detectors will collect almost all of
the photons captured by the objective.
Adapting to the model
Now you can ascertain parameters of interest by
adapting the correlation to a particular model.
The correlation amplitude provides information
on the number of particles, while the correlation
decay time indicates their speed.
2.2
Assessing conformity
How well the end predictions describe the system examined can be assessed from the deviations between correlation and the theoretical
model derived. The smaller the deviations, the
greater the probability that the theory and reality match.
2.0
1.8
G []
In the temporal correlation diagram the correlation G() is plotted versus the correlation
or lag time D . The number of particles and
the their diffusion time can be computed from
the non-normalized amplitude G(0) and the
characteristic decay time, respectively. Please
note that the diagram displays normalized
correlation functions top emphasize time the
different decay times.
1.6
1.4
1.2
1.0
1E-7 1E-6 1E-5 1E-4 1E-3 1E-2 1E-1 1E+0 1E+1
 [s]
0–40 µs
20 µs
10 µs
0 µs
30 µs
0 µs
40 µs
10 µs
20 µs
30 µs
40 µs
8
Snapshots of diffusion of particles of different
speeds during a point measurement.
The faster molecule (red) will have left the
stationary observation volume after a smaller
time delay, as it covers a greater distance per
unit of time. Therefore, its time correlation
decays faster.
In the examples, the measuring positions
in the LSM image are marked by crosses;
the resulting cross-correlation functions
are superimposed on the images.
HepG2 cell expressing EGFP-HP1. The protein binds
chromatin. Correlation analysis shows two molecule
classes having diffusion coefficients of 5.8 µm²/s
(unbound) and 0.2 µm²/s (transiently bound to
chromatin).
Specimen: Karolin Klement and Peter Hemmerich,
Fritz-Lipmann-Institute, Jena, Germany
tsEoS-Paxillin expressed in an HFF-1 cell. Paxillin is
involved in the formation of foval adhesion sites in
the cell. Due to transient binding to the structures, ­­
the motility compared to the free molecules decreases
from 2.5 to 0.18 µm²/s.
GFP-Bim1 expressed in baker‘s yeast.
In proliferation, the protein accumulates in spindle
pole bodies. In the cytosol, it remains motile, with
a diffusion coefficient of 1.4 µm²/s.
Topaz A1 adenosine receptor expressed on the
membranes of CHO cells. In the membrane, the
receptor has a diffusion coefficient of 1.3 µm²/s.
Specimen: Harald Hess, Howard Hughes Medical
Institute, Janelia Farms, USA
Specimen: Susanne Trautman, Eidgenössische
Technische Hochschule, Zurich, Switzerland
Specimen: Steve Briddon, Queens Medical Institute,
University of Nottingham, UK
9
RICS
Raster Image Correlation Spectroscopy
If an image is generated by scanning, it contains not only spatial but also temporal
information. From such an image you can determine information on molecule speeds
and concentrations without upsetting its cellular equilibrium.
Select the measuring window
Use a suitable zoom to select the area of interest
from an overview image.
Start the measurement
Record a time series stack of intensity images at
a suitable scanning speed.
Filter the data
Disturbing structures in the image, such as stationary objects or slow drifts, are eliminated by
20 µs
subtraction of a moving average.
0–40 µs
10 µs
0 µs
Determine correlation
The system computes spatial correlation from
the pixel fluctuations, comparing the image either with itself (auto-correlation) or with another
image (cross-correlation).
Adapting to the model
Allowing for the settings used for image acquisition, you can adapt the correlation to a particular
model, and thus ascertain parameters of interest,
such as the relative number of particles and their
speed.
30 µs
0 µs
Assessing conformity
How well the model employed describes the
measurement data can be seen from the deviations between correlation and fit. The better the
model fits, the smaller the deviations.
40 µs
10 µs
20 µs
0.40
G [ ,0]
30 µsis
In the spatial correlation diagram (depicted
0.35
a cut through the x-axis) the correlation G( ,0)
is plotted versus the pixel shift . The relative 40 µs
0.30
molecule number can be computed from the
0.25
amplitude G(0,0). The decay shape contains
the information on the diffusion time D of
0.20
the molecule.
0.15
0.05
0
0 µs
10 µs
20 µs
0
30 µs
10
20

30
40
40 µs
20 µs
10 µs
0 µs
30 µs
0 µs
40 µs
10 µs
20 µs
30 µs
40 µs
10
Snapshots of diffusion of particles of different
speeds with moving beam. The faster molecule
(red) will have left the moving observation
volume faster with smaller spatial distances,
but may re-enter the volume with long distances, as it covers greater distances per unit
of time. Therefore, its spatial correlation first
diminishes faster but remains to exist longer.
Intensity image of spherules sized 20 nm in solution (top).
The resulting correlation diagram (bottom) contains information
on the number (N=0.8) and speed (D=8.9 µm²/s) of the spherules.
Specimen: InVitrogen – Molecular Probes
Time series of an HFF-1 cell expressing tdEoS-Paxillin
(the first of 100 pictures). Correlation analysis shows a mean
diffusion coefficient of 0.78 µm²/s (middle). Mapping reveals
the local difference in the dynamics of Paxillin (bottom).
The protein is distinctly slower in the region of the focal
adhesion structures.
Specimen: Harald Hess, Howard Hughes Medical Institute
(HHMI), Janelia Farms, USA
11
1
3
1
2
4
9
7
5
4
6
8
10
11
14
13
12
12
The beam path of the LSM 710 / ConfoCor 3
setup combines spectral detection
and single photon counting.
19
18
17
20
15
18
16
1 V PCT laser coupling ports
2 IR PCT laser coupling port
(IR laser not usable in combination with ConfoCor 3)
3 VIS PCT laser coupling ports with VIS AOTF
4 Monitor diode
5 InVis TwinGate color beamsplitter (retrofit)
6 Vis TwinGate color beamsplitter (user-exchangeable)
7 Scanning mirror (SF 20, 6k x 6k)
8 Central pinhole
9 Beamsplitter for external channel
10 Spectral beamsplitter and recycling loop
11 12 13 14 15 16 17 18 19
20
Spectral beam guide
QUASAR PMT, spectral channel # 1
QUASAR PMT, spectral channels # 2-33 (or # 2)
QUASAR PMT, spectral channel # 34 (or # 3)
External channel
Blocking filter wheel
Secondary beamsplitter wheel
Emission filter wheels
APD detector (# 1)
APD detector (# 2)
13
ZEN Software
Efficient Setting, Recording, Analysis and Cataloguing
The LSM 710 and the ConfoCor 3 are controlled via the ZEISS ZEN software.
The easy-to-understand user interface is an ideal environment for microscopy.
Perfect integration
The ConfoCor 3 is operated through special controls that
perfectly integrate with the existing software structure.
­Interplay with the LSM takes place in the background, unnoticed by the user.
Everything important available at a click
Checking whether the experiment is really worth while,
starting the measurement, and a lot more: everything is
available at a click in the software. That way you save time
and can concentrate on the essentials.
Control window,
with ConfoCor 3 group of controls
Count rate window opened by one
of the ConfoCor 3 action buttons
14
ZEN for APD Imaging
Ultimate Sensitivity in Imaging
Like photomultiplier tubes (PMTs), avalanche photodiodes (APDs) are point detectors, but of
higher quantum efficiency. The detectors used in the ConfoCor 3 are twice as sensitive in the
green spectral range, and even three to four times as sensitive in the red one. Thanks to their
low dark noise, APDs have an excellent signal-to-noise ratio.
Photon counting at its best
Avalanche photodiodes count individual photons – a process that is far more sensitive than analog detection. This
also means less specimen stress with the same image intensity.
Optimum combinability and flexibility
APD detectors can be freely combined with the Quasar detector, so that weak and strong fluorescence can be simultaneously captured and balanced. A large number of filters
in the ConfoCor 3 module permits optimization for almost
every fluorescent dye.
Group of beam path
controls
Emission filter group
in the ConfoCor 3
15
ZEN for FCS
Measuring Dynamics with
Pinpoint Accuracy
The ZEN software renders the best possible support
to the precise, high-resolution measurement of
molecule concentrations, speeds and brightnesses.
Selecting the measuring position
in the intensity image
FCS window displaying the count rate track (top),
the correlation function (centre left), the photon count
histogram (center right), and the result table with
the adapted parameters (bottom).
16
ZEN for RICS
Discovering Temporal Information Hidden in the Image
In the RCS module you determine the number and speed of moving molecules
from images. The ZEN software offers many setting options facilitating
the interpretation and display of the data.
Determining correlation
Select the RICS register tab, and ZEN automatically computes correlation and adapts it to easy-to-assemble models.
Moreover, the ZEN software helps you answer the question
how well the model fits the recorded data.
Fast detection of heterogeneities
ZEN automatically computes the number and speeds of
molecules from overlapping regions. Display by molecule
number and diffusion maps permits differences to be assigned to cell regions and to be realized at a glance.
Menu for setting the model function
that describes the scanning pattern
of the laser beam.
Example of a molecule number map. This is
composed of the displacement of a 64 x 64
pixel region by 32 pixels each per X and Y
increment in an image sized 512 x 512 pixels.
The number per pixel is color-coded according
to the scale on the left.
(specimen description see pages 18/19)
Menu for setting and filtering the
molecule number and diffusion
maps.
17
ZEN for RICS
See everything at a glance:
fluctuation,
correlation,
mapping and
parameters –
for efficient data analysis and
the resulting scientific findings.
RICS image window with display
of correlation in 2.5 D (left),
the intensity image (top right),
the diffusion map (bottom right),
and the results table with adapted
data.
Specimen: EGFP-MS2 protein
expressed in U2OS cells. The cells
produce an HIV reporter transcript containing MS2 binding
sites.
Ute Schmidt and Edouard
Bertrand, IGMM-CNRS,
Montpellier, France
18
19
System Properties
The ConfoCor 3 used on the LSM 710 makes up a system with a wide range of
dynamics and sensitivity, adapted to widely varied expression rates and kinetic
behaviors. Correlation algorithms of the ZEN software permit molecule concentration and speed to be quantified.
Integration
ConfoCor functions are integrated in the existing
ZEN software for easy system handling
Optics
Objective specially adapted to FCS and corrected
for UV-VIS-IR, ideal for cross-correlation
Ergonomics
Automated changing between configurations
Sensitivity
The objective is just overfilled to create a diffraction-limited excitation volume for the maximum
signal-to-noise ratio
Stability
The ConfoCor3 connects to a free parallel beam
of the LSM 710
Positioning modes
Positioning by means of the scanning mirrors or
the scanning stage
Positioning accuracy
Positioning by the scanning mirrors with nm
precision
Imaging
APDs can be freely combined with the Quasar
detector for simultaneous use
Spatial correlation
Linear scanning is excellently suitable for raster
image correlation spectroscopy (RICS)
Statistics
Average correlation computed from several
measurements
Reproducibility
Call up previous settings for re-use
20
Laser suppression
TwinGate beamsplitter arranged at 10 degrees
relative to the incident beam, with excellent
laser suppression over optical density OD 4.
An optional filter wheel blocks unwanted secondary lines of the argon laser
Flexibility
Optional iris diaphragm in the excitation beam
permits controlled variation of the confocal
volume
Pinhole setting
Automatic adjustment of the pinhole relative to
the beam path in X and Y
Automation
Selection of multiple measurement positions,
which are targeted in succession
Specification
Microscope
Stand
Inverted: Axio Observer.Z1 with sideport
Z drive
Smallest increment: <25 nm (Axio Observer.Z1).
Options: fast piezo objective focus or stage focus; Definite Focus for stands
XY stage
Motorized XY scanning stage; smallest increment 1 µm (Axio Observer.Z1)
Accessories
Digital microscope camera AxioCam, integration of incubation chambers, micromanipulators, etc.
LSM scan module
Detectors
2, 3 or 34 spectral detection channels, high QE, low dark noise, setting of up to 10 digital gains;
prepared for violet light lasers (405, 440 nm)
ConfoCor 3 detection module
Detectors
2 fiber-coupled, actively suppressed avalanche photodiodes (APDs).
Detector sensitivity in the visible range: 40–75 % (depending on wavelength).
Dark count rate <250 Hz, dead time 40 ns, time resolution 50 ns (corresponding to 20 MHz time resolution for photon counting).
Pulse width: 15 ns, afterpulsing (100–500 ns): 0.5 %
Filter
2 filter configurations: Basic and extended filter sets
Pinhole
Central pinhole
Beam path
Changeable TwinGate major beamsplitter with up to 50 combinations of excitation wavelengths, excellent laser line suppression; optional laser notch
filters for fluorescence imaging on reflecting substrates (on request); outcoupling for external ConfoCor 3 detection module
Iris diaphragm
Optional iris diaphragm in the excitation beam path
for adjustable filling of the objective‘s rear aperture (for ConfoCor 3 on request)
Data depth
Selectable: 8, 12 or 16 bit; simultaneous detection in up to 37 channels
Lasers
Laser modules (VIS, V)
Pigtail-coupled lasers with polarization-preserving single-mode fiber; stabilized VIS-AOTF for simultaneous intensity control, switching period <5 µs,
or direct modulation; up to 6 V/VIS lasers directly connectable to the scanning head; 30mW diode laser (405 nm, CW/pulsed), 25mW diode laser
(440 nm, CW+pulsed), 25 or 35mW argon laser (458, 488, 514 nm), 1 mW HeNe laser (543 nm), 20mW DPSS laser (561 nm), 2mW HeNe laser
(594 nm), 5mW HeNe laser (633 nm) (manufacturers‘ pre-fiber specs.)
External lasers (NLO)
Prepared laser ports for the extension of all systems
Electronics module
Real-time electronics
Control of microscope, lasers, scanning module and accessories; monitoring of data acquisition and synchronization by real-time electronics.
Oversampling read-out logic for best sensitivity and excellent SNR; data exchange between real-time electronics and user PC via gigabit Ethernet,
with option of online data analysis right during image acquisition
User PC
Workstation PC with ample RAM and HD memory space; ergonomic, high-resolution 16:10 TFT flat-panel display,
many accessories, Windows VISTA OS (as available), multi-user capability
ZEN software
LSM 710
Basic software, plus options: LSM Image VisArt plus, 3D Deconvolution, Physiology, FRET plus, FRAP,
Visual Macro Editor, VBA Macro Editor, RICS Raster Image Correlation Spectroscopy (PMT & APD)
ConfoCor 3
Basic software, plus options: Extended models, global and interactive fit, and photon count histogram (PCH)
21
System Overview: ConfoCor 3 on LSM 710
22
Milestones of FCS Technology
in Bioscience
1903 M. von Smoluchovski explains the interrelation
between auto-correlation and Brownian movement.
1972 First fluorescence correlation spectrometers
are developed in the labs of Cornell University,
Ithaca, USA and Max Planck Institute, Germany
1988 First confocal microscope set up for FCS
measurements developed at Karolinska Institute
in Stockholm, Sweden
1996 Carl Zeiss launches the world’s first automated fluorescence correlation spectrometer,
the ConfoCor 1.
1999 Carl Zeiss designs the ConfoCor 2 for
fully automated dual-channel cross-correlation
spectrometry.
2000 Carl Zeiss combines the ConfoCor 2 with
the LSM 510 to make up a platform which, for the
first time, makes biophysical methods in cellular
biology available to a wide range of users.
2005 Carl Zeiss introduces the ConfoCor 3,
a module tailor-made for observing live cell processes.
2008 Carl Zeiss combines ConfoCor 3 and
LSM 710 into a platform.
23
Working jointly with the LSM 710, the ConfoCor 3 now can
bring its strengths to bear better than before.
Highest sensitivity without compromising dynamics. Intelligent
algorithms for new interpretations of images. Tried-and-tested
quality, continually redefined as technology advances. Quality
by Carl Zeiss.
LITERATURE
HP-1: Schmiedeberg L, Weisshart K, Diekmann S, Meyer Zu Hoerste G, Hemmerich P. (2004). High- and low-mobility populations
of HP1 in heterochromatin of mammalian cells.
Mol Biol 15(6): 2819-2833.
HIV transkripts: Boireau S, Maiuri P, Basyuk E, de la Mata M,
Knezevich A, Pradet-Balade B, Bäcker V, Kornblihtt A, Marcello A,
Bertrand E. (2007). The transcriptional cycle of HIV-1 in real-time
and live cells. J Cell Biol 179(2): 291-304.
A1-Adenosin rezeptor: Briddon SJ, Middleton RJ, Cordeaux Y,
Flavin FM, Weinstein JA, George MW, Kellam B, Hill SJ. (2004).
Quantitative analysis of the formation and diffusion of A1-ade­
nosine receptor-antagonist complexes in single living cells.
Proc Natl Acad Sci U S A 101(13): 4673-4678.
Paxillin: Digman MA, Brown CM, Horwitz AR, Mantulin WW,
Gratton E. (2008). Paxillin dynamics measured during adhesion
assembly and disassembly by correlation spectroscopy.
Biophys J 94(7): 2819-31.
CENP I: Hemmerich P, Weidtkamp-Peters S, Hoischen C, Schmiedeberg L, Erliandri I, Diekmann S. (2008). Dynamics of inner
kinetochore assembly and maintenance in living cells.
J Cell Biol 180(6):1101-1114.
Keima: Kogure T, Kawano H, Abe Y, Miyawaki A. (2008) Fluorescence imaging using a fluorescent protein with a large Stokes
shift. Methods 45(3): 223-226.
60-1-0012/e – printed 01.09
Carl Zeiss Microscopy GmbH
07745 Jena, Germany
BioSciences
[email protected]
www.zeiss.com/microscopy
Subject to change. Printed on
environmentally friendly paper,
bleached without the use of chlorine.
Bim: Guertin DA, Trautmann S, McCollum D. (2004). Cytokinesis
in eukaryotes. Microbiol Mol Biol Rev 66(2):155-178.
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