LS M 5 1 0 M ETA L aser S cann in g M ic roscope

LS M 5 1 0 M ETA L aser S cann in g M ic roscope
M i c r o sc o p y f r o m Ca r l Z e i ss
LS M 5 1 0 M ETA
L a s e r S c a n n in g M ic r o s c o p e
F lu o r e s c e n c e S ig n a ls R e lia b ly S e p a r a t e d
H ig h lig h t s o f
L a s e r S c a n n in g M ic r o s c o p y
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The first Laser Scanning Microscope from Carl Zeiss.
The prototype of the LSM 44 series is now on display in the
Deutsches Museum in Munich.
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The LSM 10 – a confocal system
with two fluorescence channels.
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The LSM 310 combines confocal laser
scanning microscopy with
state-of-the-art computer technology.
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The LSM 410 is the first inverted microscope of the LSM family.
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The LSM 510 – the first system of the
LSM 5 family and a major breakthrough
in confocal imaging and analysis.
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The LSM 510 NLO is ready for multiphoton microscopy.
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The LSM 5 PASCAL –
the personal confocal microscope.
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The LSM is combined with
the ConfoCor 2 Fluorescence
Correlation Spectroscope.
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The LSM 510 META –
featuring multispectral analysis.
LS M
510
M ETA –
t h e d if f e r e n c e b e t w e e n
“ s e e in g a lo t ” a n d “ d e t e c t in g c le a r ly ”
Conventional multifluorescence microscopy alw ays
reaches its limits w hen the emission signals of the
dyes overlap. The LSM 510 M ETA solves this problem. You w ill obtain brilliant images w ith an information content unachievable until now.
The Greek prefix “M ETA” stands for
“going beyond” the currently available.
The LSM 510 M ETA is the new generation of laser
scanning microscopes w hich leaves the old standard
far behind – to allow you to see a lot, and to detect
things clearly.
Clear separation by Emission
Fingerprinting: Section through
the eye of the fruit fly
(Drosophila melanogaster) .
Actin filaments marked
with Alexa Fluor 532 (green),
Na+/K+ ATPase with Cy3 (red),
autofluorescence (blue).
Specimen: Dr. Otto Baumann,
University of Potsdam, Germany
C ont ent s
Confocal Principle
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System Components
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Emission Fingerprinting
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Online Fingerprinting
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Dynamic Spectral Analysis
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Softw are
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FRET and FRAP
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Physiology Softw are
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Quantitative Colocalization
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Tools
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Specification
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System Overview
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Services
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Glossary
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LS M
5 1 0
M ETA
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T h e C o n f o c a l P r in c ip le :
M a x im u m R e s o lu t io n a n d E f f ic ie n c y
The advantage of confocal light microscopy is that
A pinhole conjugated to the focal plane obstructs
it can collect the light reflected or emitted by a
the light coming from objects outside that plane,
single plane of the specimen.
so that only light from in-focus objects can reach
the detector. A laser beam scans the specimen
pixel by pixel and line by line. The pixel data are
then assembled into an image that is an optical
section through the specimen, distinguished by
high contrast and high resolution in x, y and z.
A number of images generated with the focal
plane shifted in small steps can be combined into
a 3-dimensional image stack which is available for
digital processing.
Beam path
in the confocal laser scanning microscope
Detector
Confocal Pinhole
Laser source
Main Dichroic Beamsplitter
Collimator
Scanning Mirrors
Objective
Specimen
Focal Plane
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Energy
T h e O r ig in o f F lu o r e s c e n c e
Energy diagram of fluorescence excitation,
single photon excitation (left), multiphoton excitation (right).
Under irradiation with light of a wavelength λex,
Intensity (I)
certain electrons of a fluorochrome are raised to a
higher energy level. During a very short dwell
time, they lose some of their energy and drop back
Excitation
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Emission
to their original level while emitting light of a
longer wavelength λem > λex. The difference in
wavelengths is known as the Stokes shift. In multi-
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photon excitation, the energies of several photons
with n times the excitation wavelength add up to
raise the electrons to the higher energy level.
0
Wavelength (λ)
T h e P r o p e r t ie s
o f F lu o r e s c e n c e S p e c t r a
Excitation efficiency and emission intensity as a function of excitation wavelength.
A fluorescence molecule can be irradiated with
different wavelengths within its excitation spectrum and, accordingly, will emit light with a char-
I
acteristic emission spectrum. The amplitude of the
Detector
emission spectrum is determined by the intensity
of radiation and the excitation efficiency, which is
a function of the excitation wavelength.
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S e p a r a t io n o f E m is s io n S p e c t r a
I
One way to separate fluorescence emissions is by
high-quality dichroic beamsplitters with a threshold wavelength λ τ. Thus, the beamsplitter reflects
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all wavelengths shorter, and transmits all wavelengths longer, than the threshold. The META
detector with Emission Fingerprinting provides an
alternative, much more flexible way of separating
even strongly overlapping emission spectra.
Dichroic
Beamsplitter
Detector
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Separation of fluorescence emissions by means of dichroic filters.
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S ys t em C om ponent s :
A P er fect M at ch
In the way it implements the confocal principle, the
design of the LSM 510 META system is unsurpassed.
It allows multifluorescence images to be collected
without compromising resolution and efficiency.
M ic r o s c o p e s
Every LSM 510 META is based on one of the high-performance research microscopes from Zeiss. Depending on your specific applications, you can choose
between the following instruments:
Axioplan 2 imaging M OT, Axiovert 200M and
Axioskop 2 FS MOT. All of them are equipped with
ICS optics, which are unsurpassed for image quality,
f lexibilit y and opt ical perf ect ion. The mot orized
microscope models are interchangeable and fully
supported by the LSM software. The software automatically identifies the microscope settings and the
objectives used, and controls all movements and
measurements carried out by the system with high
precision.
The unique scanning module is the core of the LSM
510 M ETA. It cont ains mot or-driven collimators,
scanning mirrors, individually adjustable and positionable pinholes, and highly sensitive detectors
including the META detector. All these components
are arranged to ensure optimum specimen illumination and efficient collection of reflected or emitted
light. A highly efficient optical grating provides an
innovative way of separating the fluorescent emissions in the META detector. The grating projects the
entire fluorescence spectrum onto the 32 channels
of the META detector. Thus, the spectral signature is
acquired for each pixel of the scanned image and
can then be used for the digital separation into
channels reflecting dye distributions.
C ont r ol C om put er and S oft w ar e
Carl Zeiss objectives are highly regarded for their performance excellence. For the wide range of types
and specifications, users can select those providing
the optimum combination of resolving power, aperture, working distance and correction for their specific applications.
The LSM 510 META comes with an IBM-compatible
PC equipped with a powerful processor. The easy-touse LSM software enables you to control all system
components. The Windows operating system provides multitasking capability and easy linking to
existing computer networks. All components have
been carefully selected and tested. The high-performance graphics card with OpenGL capability ensures
fast presentation of 2D and 3D graphics and animations.
L a s e r M o d u le
E le c t r o n ic s M o d u le
For excitation of fluorescent dyes and fluorescent
proteins, the LSM 510 META is provided with different lasers emitting a number of lines in the UV and
visible spectral ranges. The laser light is guided into
the scanning module safely and efficiently via optical
fibers. It is also possible to use direct or fiber-coupled
tunable short-pulse lasers for multiphoton excitation.
By means of an AOTF or an AOM, the excitation light
is precisely cont rolled and can be blanked or
unblanked down to one pixel. This provides the best
possible specimen preservation and enables targeted
photobleaching, e.g. for FRAP experiments.
The LSM 510 META is controlled by digital signal
processors (DSP). This brings about fast, flexible synchronization of the scanners, the AOTF and the
detectors, and enables such sophisticated functions
as Multitracking, Spot Scan, fast Step Scan, rROI
Scan, Spline Scan, or ROI Bleaching f or FRAP,
Uncaging and Photoactivation. Moreover, this technology permits the implementation of new scanning
functions through simple software upgrades.
O b je c t iv e s
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S c a n n in g M o d u le
Beam path inside the
Scanning Module (schematic)
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1 Optical fibers
2 Motorized
collimators
3 Beam combiner
4 Main dichroic
beamsplitter
5 Scanning mirrors
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Scanning lens
Objective lens
Specimen
Secondary dichroic
beamsplitters
10 Confocal pinhole
11 Emission filters
12 Photomultiplier
13 META detector
14 Neutral density
filters
15 Monitor diode
16 Fiber out
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E m is s io n F in g e r p r in t in g :
C le a r C o lo r S e p a r a t io n
in M u lt if lu o r e s c e n c e
So far, the quality and information content in laser
A c q u is it io n
scanning microscopy has been determined by the
of a Lam bda S t ack
spectral properties of the dyes used. As soon as
several dyes with overlapping fluorescence emis-
The Lambda Stack, an image stack containing
sion spectra were used, a clear separation was
information on the dimensions x, y, z, t and λ,
possible only to a limited extent. The innovative
records the spectral signature of your specimen.
LSM 510 META overcomes these restrictions in a
The simultaneous, and therefore fast recording of
sophisticated, yet easy way: through Emission
spectrally resolved images guarantees optimum
Fingerprinting.
protection of your delicate specimens. Furthermore, Lambda Stacks allow you to capture even
Emission Fingerprinting enables you to precisely
fast dynamic processes reliably and with a high
separate the emission spectra of different dyes
information content.
and lets you see things in an entirely new way. This
technique for the recording, analysis and separation of emission signals (patent pending) generat es an unmist akable and separat e “ emission
fingerprint” of each dye used. Many scientific
analyses which could not be performed so far can
now be implemented.
The separat ion of t he emission signals is performed as follows:
• Acquisition of a Lambda Stack
• Determination or selection of reference spectra
• Separation of mixed color spectra.
Four-population mix of single-labeled polystyrene beads:
Lambda Stack with spectral distribution of fluorescence emissions
“The solution that Zeiss has developed is very
much targeted towards the problem we have –
which is being able to follow multiple dyes within the
preparation at the same time.
I should say I'm very impressed
with the data I have seen.”
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Prof. Scott E. Fraser,
Biological Imaging Center, Caltech, Pasadena, USA
D e t e r m in a t io n o r s e le c t io n
S e p a r a t io n
of r efer ence s pect r a
o f m ix e d c o lo r s p e c t r a
Depending on your requirements, you determine
The Linear Unmixing function separates the mixed
the reference spectra of the various dyes used
signals pixel by pixel, using the entire emission
either automatically or interactively, using the
spectrum of each dye in the examined specimen.
Mean-of-ROI function. You can store these refer-
As a result , even w idely overlapping emission
ence spectra in the spectra database of the LSM
spectra, e.g. those of GFP and FITC, are separated
510 META and recall them for further experiments.
precisely. Broadband aut of luorescence can be
eliminated reliably.
Four populations of single-labeled polystyrene beads:
Lambda-Coded representation with Regions Of Interest (ROI)
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Intensity
1
500
550
600
650
Wavelength
Spectral signatures of the fluorescence emission in the Regions Of
Interest shown above: each dye can be clearly determined
(1) Separation of emissions
with bandpass filters:
unsatisfactorily
differentiated images
(2) Separation by Linear
Unmixing: clear delimitation
.
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O n lin e F in g e r p r in t in g :
E f f ic ie n c y M e e t s H ig h S p e e d
Our close cooperation with scientists in universit ies and research inst it ut es has enabled us t o
consistently continue developing the Emission
Fingerprinting technique.
In the Online Fingerprinting dialogue, reference
spectra are selected prior to scanning. The spectrum is unmixed during the scanning procedure,
and the result is displayed immediately. You no
longer have to wait for the end of the scanning
procedure to assess dynamic processes in living
cells. Thus, the appropriate time to induce a reaction or apply a stimulus is easy to determine. You
don’t need to focus on the technique of your
application, but can devote all your attention to
CFP, CGFP, GFP and YFP in cultivated cells
after Emission Fingerprinting
(Specimen: Dr. A. Miyawaki, RIKEN, Japan)
the analysis of your work.
“The new scan modes of the system offer
a completely new quality of analysis.
The interpretation of the data is far more
reliable than with any conventional system
based on filter sets and bandpass acquisition.”
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Online Fingerprinting.
All the required settings from
excitation to emission are
made in a single menu.
Dr. Frank-D. Böhmer,
Research Unit M olecular Cell Biology, Friedrich Schiller University of Jena, Germany
D y n a m ic S p e c t r a l A n a ly s is :
T im e a n d C o lo r S e p a r a t io n
Almost every specimen conceals inf ormat ion
which the scientist can only obtain and use by
specifically searching for it through special analysis
functions.
Dynamic processes in the emission
spectrum can only be “ visualized” if the microscope system can appropriately analyze the time
dimension. The LSM 510 META meets this requirement by creating Lambda-t data series.
Concealed emission spect ra in t he
specimen are of major importance for research
results. To be able to detect them, the LSM 510
META features a special analysis function: Automatic Component Extraction (ACE). This statistical
Lambda-t data series visualize dynamic processes.
technique extracts the dye spectra contained in
the Lambda Stack, complementing the interactive
detection of reference spectra.
The quality and “intelligence”of the analysis functions determine the results of the
microscopic examinations to a large extent. The LSM 510 META detects concealed
emission spectra via the Automatic Component Extraction (ACE) function.
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T he S oft w ar e :
U s e r - f r ie n d ly O p e r a t io n
1
S w it c h o n t h e L a s e r
w it h L a s e r C o n t r o l
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S e t t h e S p e c im e n
w it h M ic r o s c o p e C o n t r o l
In the development of the LSM 510 META soft-
Since the system is entirely motorized and coded,
ware, great attention has been paid to high oper-
all system configuration parameters can be stored
ating convenience and mastering the combination
and recalled with a single mouse click. This ReUse
of ease of use with high functionality.
approach guarantees high reproducibility of your
The effort has been a success. This is confirmed by
results.
our cust omers, w hose suggest ions have con-
At the push of a button, the Find function will
tributed materially to design improvements.
search for the ideal detector settings and automatically control each detector. This and many
other functions support you in your work so that
you can concentrate on what is really important.
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1
3
2
3
4
S e t D e t e c t io n
w it h C o n f ig u r a t io n
C ont r ol or R eU se
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S can
w it h S c a n C o n t r o l
o r F in d
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FR ET and FR A P :
T r a c k in g d o w n B io lo g ic a l F u n c t io n s
CaM: Calmodulin
M13: Calmodulin binding domaine
Convent ional imaging t echniques depend on
No FRET
FRET
acquiring closely limited emission bands in order
to minimize crosstalk. This applies, for example, to
Stimulation:
+ Ca2+
the examination of protein-protein interactions
using the FRET technique, and to experiments with
ion-sensitive dyes such as Indo-1 or SNARF. Compared to such conventional methods, the Emission
Fingerprinting technique of the LSM 510 META
offers substantial advantages because the entire
Calcium imaging using the FRET indicator Yellow Cameleon 2.
signal is used.
After separation by Emission Fingerprinting:
The image shows a clear separation of the YFP and
CFP fluorescence of Yellow Cameleon 2.
The Region Of Interest (ROI) is marked with a white square.
First, you can follow spectral signatures of the
fluorescence and their changes in your specimen
by means of acquiring a series of Lambda Stacks.
Intensity
After acquisition of the Lambda Stacks, separate
the emission signals and gain direct information
YFP
about the FRET partners or of the binding statuses
of the ion sensors.
FRET
Intensities of the YFP
and CFP signals within
the ROI shown above.
0s
0
100 s
FRET analysis providing brilliant results.
The ratio between YFP and CFP fluorescence of Yellow Cameleon 2 was analyzed.
The calcium concentration markedly increases in the course of time.
(Dr. A. Miyawaki, RIKEN, Wako, Japan; Prof. Y. Hiraoka, KARC, Kobe, Japan)
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100
200
200 s
CFP
300
400
300 s
500
Time (s)
400 s
Prof. Yasushi Hiraoka,
Kansai Advanced Research Center, Kobe, Japan
“This system provides a very easy way
to do FRAP experiments.
I regret not having used this system earlier.”
With the bleach function the acceptor can be
FRET
sw it ched off f or checking FRET event s. The
precise int eract ion of AOTF and DSP in t he
LSM 510 META guarantees the pixel-precise con-
CFP YFP
trol of the laser intensity, which is also the major
requirement for FRAP and Uncaging experiments.
before Acceptor Bleaching
before Acceptor Bleaching
FRET between CFP and YFP in
cultivated cells, detected by pixelprecise bleaching of the acceptor
(YFP) and an increased donor
signal (CFP)
after Acceptor Bleaching
after Acceptor Bleaching
YFP
YFP
CFP
CFP
before Acceptor Bleaching
Bleaching
after Acceptor Bleaching
“The new META unit makes FRET imaging
really easy because you get a spectral readout
of both proteins.”
M ary Dickinson, PhD,
Biological Imaging Center, Caltech, Pasadena, USA
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P h y s io lo g y S o f t w a r e :
C o m p le t e R e c o r d in g a n d A n a ly s is T o o l
In physiological examinat ions, t he superb
D is p la y a n d A n a ly s is
advantages of the LSM 510 META are particularly
o f I o n C o n c e n t r a t io n s :
obvious. This is mainly due to the extremely fast
and efficient scanning modes of the system. Plus,
online ratio calculations permit direct data display
Online and offline ratio for ratiometric dyes
Online and offline F/F0 for single-wavelength dyes
Various modes are available for the calibration of
Calibration for single-wavelength and ratiometric dyes
• in situ and in vitro
• including background correction
• after titration with various curve fittings
• according to Grynkiewicz
dyes for concentration analyses. This technological
Interactive scaling of image data series
even while the recording is still running. To make
t his possible, t he syst em uses preset analysis
formulas with user-defined parameter settings.
configuration makes the LSM 510 META suitable
Interactive graphic display of the measured data from ROIs
f or every dye and it s specif ic f luorescence
properties.
Hormone-induced calcium changes
in the salivary gland of an insect,
visualized with Fluo-4.
(Dr. B. Zimmermann, Dr. B. Walz,
University of Potsdam, Germany)
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Software dialog for the
interactive calibration
of ion-sensitive dyes
Q u a n t it a t iv e C o lo c a liz a t io n :
F in d in g t h e N e e d le in t h e H a y s t a c k
The LSM 510 META enables you to easily perform
D is p la y a n d A n a ly s is o f
quantitative colocalization analyses with a reliabil-
C o lo c a liz a t io n E x p e r im e n t s :
ity and precision never achieved before. Image display, scattergram and data table are interactively
linked to the ROI and tresholding tools. To give
you an example: you select an area in the scattergram, and the existence of colocalization will be
shown immediately in the unmixed image. Data
table, histogram and image are interlinked in the
same way. Data analysis can hardly be any more
intuitive and precise.
Interactively linked image display, scattergram and data table
Interactive or automated determination of thresholds
Overlay of image channels with results of the colocalization analysis
Quantitative colocalization analysis for up to 99 ROIs with:
• area and average gray level intensity
• colocalization degree
• colocalization coefficient
• Pearsons’ correlation coefficient
• Overlap coefficient according to Manders
Export of analysis results
Qualitative (color-coded) colocalization analysis is
often misleading – only quantitative tools (left) make
things clear: cerebral cortex of the rat, double-stained
mitochondria (Mn-SOD) marked green and red, and
microtubuli (MAP2) marked yellow.
(Dr. J. Lindenau, University of Medical Neurobiology,
University of Magdeburg, Germany)
Use first-class tools correctly:
image display, scattergram, and data table are interactively
linked to the ROI and tresholding tools
“My people were thrilled.
We have been working on this for a year and now we have
conclusive evidence that the proteins really interact.”
Colin C. Collins, PhD, Cancer Research Institute,
University of California, San Francisco, USA
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S e le c t io n o f T o o ls :
R esear ch M ade E asy
M u l t it r a c k i n g –
L in e a r U n m ix in g
M e t a t r a c k in g
The fast, line-by-line change between excitation
The Linear Unmixing function separates the mixed
laser lines, known as Multitracking, is an appreciated
signals pixel by pixel by using the entire emission
procedure for the separation of overlapping fluo-
spect rum of each f luorescence marker in t he
rescence spectra. Of course, you can also perform
specimen. As a result, even greatly overlapping
this technique with the LSM 510 META. Using
emission spectra, e.g. those of GFP and FITC, are
t he new M et at racking t echnique, you can
separat ed reliably, and broadband aut of luo-
now optimize the bandwidth of your detection
rescence is eliminated. This provides solutions to
channels according to the emission characteristics
problems unsolvable so far and enables completely
of each dye and switch between different, yet
new experimental approaches.
overlapping bandpass characteristics line by line.
This ensures optimum signal detection without
any disturbing crosstalk in the case of critical dye
combinations.
Multifluorescence in-situ hybridization (MFISH)
of human metaphase chromosomes
(Dr. T. Liehr and Dr. V. Beensen, University of Jena, Germany)
Of course, all the image recording, image
analysis and image display functions of
t he LSM 510 have been int egrat ed in
the LSM 510 M ETA, and have even been
systematically improved and extended.
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ECFP-RanGAP (blue), GFP-emerin (green)
and YFP-SUMO1 (red) expression in cultured cells
(Prof. Y. Hiraoka, KARC, Kobe, Japan)
CFP (blue), GFP (green) and YFP (red) expression in the
nuclei of NIH3T3 cells (Dr. M. Dickinson, Dr. R. Lansford,
Prof. S. Fraser, Caltech, Pasadena, USA)
William C. Hyun, Comprehensive Cancer Center,
University of California, San Francisco, USA
“The power of the Zeiss system is not only
in its sensitivity, its software and its
user-friendliness, but also in the technical
enhancements for spectral selection.”
3 D V is u a liz a t io n
M u lt if lu o r e s c e n c e
The extensive 3D visualization modes of the LSM 5
To optimize multifluorescence analyses, the LSM
Image VisArt sof t w are package provide new,
510 META provides the unique possibility of com-
undreamed-of insights into the spatial structures
bining the META detector with other single detec-
of your specimen. Fast 3D and 4D reconstruction
tors. This enables you to configure the spectral
and various project ion and animat ion opt ions
range of the META detector as required, and to
afford an entirely new understanding of interrela-
achieve maximum signal yield via t he single
tions for research and training.
detector at the same time. In fact, individually
For higher resolution demands, deconvolution
adjust able and posit ionable pinholes of each
functions have been implemented on the basis of
det ect or off er you an easy w ay t o make your
calculated Point Spread Functions (Nearest Neigh-
experiments perfect.
bor, Maximum Likelihood, Constraint Iterative).
Capillary network
of a rat after
injection of
rhodamine
GPI-GFP (green) and FM4-64 (red) fluorescence in
wing buds of the fruit fly Drosophila melanogaster
(V. Greco and Dr. S. Eaton, Max Planck Institute
of Cell Biology and Genetics, Dresden, Germany)
Zebrafish embryo, eye and part of the brain;
cell adhesion molecule Tag-1 (Alexa Fluor 488, green),
tubulin (Cy3, red), sugar epitope PSA (Cy5, purple),
cell nuclei (DAPI, blue). (Dr. M. Marx and Prof. M. Bastmeyer,
Constance University, Germany)
Actin (Alexa 488-phalloidin, green) and paxillin
(Texas Red, red in cultured fibroblasts)
(Dr. M. A. Woodrow, University of California,
San Francisco, USA)
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S p e c if ic a t io n
LS M 5 1 0 M ETA
S ys t em C om ponent s
M ic r o s c o p e s
Models
Upright: Axioplan 2 imaging MOT, Axioskop 2 FS MOT; Inverted: Axiovert 200 M BP (Base Port) or SP (Side Port)
Z drive
DC motor with optoelectronic coding, smallest increment 25 or 50 nm; fast piezo objective focus attachment
HRZ 200 (option)
High-precision galvanometric fine focusing stage, total lift 200 µm, smallest increment 10 nm
XY stage (option)
Motorized XY scanning stage, with Mark & Find (xyz) and Tile Scan (mosaic scan) functions, smallest increment 1 µm
Accessories
Digital microscope camera AxioCam, integration of incubation chambers, micromanipulators, etc.
S c a n n in g m o d u le
Models
META scanning module with two single-channel detectors and a polychromatic multichannel detector
(each genuinely confocal with selected, high-sensitivity PMTs) prepared for lasers from UV to NIR
Scanner
Two independent galvanometric scanning mirrors, DSP-controlled, providing ultrashort line and frame flyback times
Scanning resolution
4x1 to 2048x2048 pixels, also for several channels, continuous adjustment
Scanning speed
13x2 speed stages; up to 5 frames/s with 512x512 pixels (max. 77 frames/s with 512x32 pixels);
min. 0.38 ms for a line of 512 pixels
Scanning zoom
0.7x to 40x, digital, variable in steps of 0.1
Scanning rotation
Free 360° rotation, variable in steps of 1 degree, free xy offset
Scanning field
18 mm diagonal field (max.) in the intermediate image plane,
homogeneous illumination
Pinholes
Pinholes for each epi-illumination channel (single-channel detector or META multichannel detector),
individual adjustments of size and position, preadjusted
Detection
Standard: three confocal epi-illumination channels simultaneously (META detector + 2 single-channel detectors),
each with a high-sensitivity PMT detector. Options: transmitted-light channel with PMT;
monitor diode for measuring the excitation intensity. New: Simultaneous acquisition of up to 8 channels;
META: fast acquisition of Lambda Stacks, also in combination with time series
META detector
Polychromatic 32-channel detector for fast acquisition of Lambda Stacks and Metatracking
Data depth
Selectable between 8 bit and 12 bit, individual 12-bit A/D converter for each of 8 channels
L a s e r m o d u le s
VIS laser module
Polarization-preserving single-mode fiber, temperature-stabilized VIS-AOTF for simultaneous intensity
control of up to 6 visible-light laser lines, switching time < 5 µs; AOTF reprogramming via the LSM software;
Diode laser (405 nm) 25 mW; Ar laser (458, 477, 488, 514 nm) 30 mW; ArKr laser (488, 568 nm) 30 mW;
HeNe laser (543 nm) 1 mW; HeNe laser (633 nm) 5 mW (end-of-lifetime specification)
UV laser module
Polarization-preserving single-mode fiber, temperature-stabilized UV-AOTF for simultaneous
intensity control of two ultraviolet laser lines, switching time < 5 µs; Ar laser (351, 364 nm) 80 mW;
optional Kr laser (413 nm) 40 mW (end-of-lifetime specification)
Multiphoton option
Direct or fiber coupling of pulsed NIR lasers into the scanning module; various makes are supported.
Grating Dispersion Compensator (GDC) and Post Fiber Compressor (PFC) for optimum pulse shaping.
Fast change of laser intensity by means of AOM. Up to 4 external detectors for Non-Descanned Detection (NDD).
Objectives optimized for use in the NIR range
E le c t r o n ic s m o d u le
22
LSM 510 Control
Control of the microscope, the VIS and UV laser modules, the scanning module and further accessories.
Monitoring of data acquisition and synchronization by a Digital Signal Processor (DSP).
Data exchange between DSP and computer via ultra-wide SCSI
Computer
High-end PC with ample RAM and hard disk storage capacity, ergonomic high-resolution monitor or
TFT flat-panel display, many accessories; Windows 2000/NT 4.0 operating system with multi-user capability
S t andar d S oft w ar e
System configuration
Convenient control and configuration of all motorized microscope functions, of the laser and scanning modules.
Saving and restoration of application-specific configurations
ReUse function
Restoration of acquisition parameters per mouse click
Acquisition modes
Spot, Line/Spline, Frame, Z Stack, Lambda Stack, Time Series and combinations: xy, xyz, xyt, xyzt, xz, xt, xzt, Spot-t, xλ, xyλ, xyzλ, xytλ,
xyztλ, xzλ, xtλ, xztλ, On-line computation and presentation of ratio images.
Averaging and summation (linewise or framewise, configurable). Step Scan (for higher frame rates, configurable)
Auto-Z function
On-line adaptation of Z Stack acquisition parameters for uniform brightness distribution
Crop function
Convenient selection of scanning ranges (zoom, offset, rotation simultaneously)
RealROI scan
Scanning of up to 99 ROIs (regions of interest) of any shape, with pixel-accurate laser blanking
ROI bleach
Localized photobleaching of up to 99 bleaching ROIs for applications such as FRAP (Fluorescence Recovery After Photobleaching)
or uncaging
Spline scan
Scanning along a freehand defined line
Multitracking
Acquisition of multiple fluorescences; fast change of excitation lines minimizes signal crosstalk
Metatracking
Extension of Multitracking by fast electronic change of detection channels, even with overlapping bandpasses,
ensures optimum signal detection (only with META detection module)
Lambda Stack scan
Fast simultaneous acquisition of image stacks with spectral information for every pixel
(only with META detection module)
Emission
Fingerprinting
Technique for generating crosstalk-free multiple-fluorescence images with fast simultaneous excitation,
unmixing possible online or offline, automatic or interactive
Presentation
Orthogonal view (xy, xz, yz in a single presentation),
cut view (3D section made under a freely definable spatial angle),
2.5D view for time series of line scans,
projections (stereo, maximum, transparent) for single frames and series (animations),
depth coding (pseudo-color presentation of height information). Brightness and contrast adjustments;
off-line interpolation for Z Stacks, selection and modification of color lookup tables (LUT),
drawing functions for documentation
Analysis
Advanced tools for colocalization and histogram analysis with individual parameters and options,
profile measurement of straight lines and curves of any shape, measurement of lengths, angles, areas, intensities, etc.
Image operations
Addition, subtraction, multiplication, division, ratio, shift, filters (low-pass, median, high-pass, etc.; user-definable)
Data archiving,
export, import
LSM image database with convenient functions for managing images together with their acquisition parameters;
Multiprint function for creating assembled image and data views; more than 20 file formats (TIF, BMP, JPG, PSD, PCX,
GIF, AVI, Quicktime …) for compatibility with all common image processing programs
S o f t w a r e O p t io n s
LSM Image VisArt
Fast 3D and 4D reconstruction and animation
(various modes: shadow projection, transparency projection, surface rendering)
3D Deconvolution
Image restoration based on computed point spread functions (modes: nearest neighbor, maximum likelihood, constraint iterative)
Multiple Time Series
Complex time series with change of application-specific configurations, autofocus and bleaching functions
3D for LSM
3D presentation and measurement of volume data records
Physiology
Extensive Software for the analysis of time series, graphical mean-of-ROI analysis,
online and offline display and calibration of ion concentrations
Topography package
Visualization of 3D surfaces (fast rendering modes) plus many measurement functions (roughness, surface areas, volumes)
VBA Macro Editor
Acquisition and editing of routines for the automation of scanning and analysis functions
Im a g e B r o w s e r
Free Software Package for display, editing, archiving, print and export/import of LSM 5 images
23
S y s t e m O v e r v ie w L S M
510
M ETA
VIS Scan module LSM 510
VIS/ UV Scan module LSM 510
VIS/ NLO-Scan module LSM 510
VIS/ 405 Scan module LSM 510
NLO kit for fiber coupling
l
1
Ar-laser, 458, 477, 488,
514 nm, 30 mW
NLO kit for direct coupling
Diode laser, 405 nm, 25 mW
Upgrade kits LSM 510 to
LSM 510 M ETA
HeNe laser, 543 nm, 1mW
n
Laser Enterprise II 653 (80 mW, 351, 364 nm)
Laser module kit
HeNe laser, 633 nm, 5 mW
VIS Scan module LSM 510 M ETA
VIS/ UV Scan module LSM 510 M ETA
Laser module VIS
VIS AOTF (6 channels)
VIS/ NLO Scan module LSM 510 M ETA
VIS/ 405 Scan module LSM 510 M ETA
Fiber decoupling, channel 4
Options:
-Fluorescence Lifetime Imaging
M icroscopy (FLIM )
-Fluorescence Correlation
Spectroscopy (FCS)
Argon Krypton laser, 488, 568 nm, 30 mW
LCD TFT-Flatscreen 18"
M onitor 21" (50 cm)
Actively vibration-absorbed system table
for LSM 5
Table surface 30" x 30"
Host computer
Large LSM 5 system table
w idth 1500 mm, height 780 mm, depth 800 mm
w ith vibration absorption
Granite slab
24
Small LSM 5 system table
w idth 650 mm, height 780 mm, depth 800 mm
w ith vibration absorption
Granite slab
System table NLO w ith active absorption
w idth 1800 mm, height 750 mm, depth 1400 mm
System table NLO w ith active absorption
w idth 1200 mm, height 750 mm, depth 1400 mm
Illuminator N HBO 103
VIS Scan module LSM 510 M ETA
VIS/ UV Scan module LSM 510 M ETA
Piezo objective focus
Pow er supply unit N HBO 103
VIS/ NLO Scan module LSM 510 M ETA
VIS/ 405 Scan module LSM 510 M ETA
Non Descanned Detection kit
Epifluorescence
motorized NDD module
w ith shutter
AxioCam HRm
AxioCam HRc
HRZ 200 fine focusing stage
Detection module
external PM
for Non Descanned
Detection
10
9
8
7
6
5
4
3
2
1
I
32
se
ar
Co ne
Fi
Scanning stage DC100x100 (65x50 mm)
00
O
K
Axioplan 2 imaging M OT
100 HAL lamp housing
w ith collector, lamp mount
Halogen lamp 12 V 100 W
HRZ 200 fine focusing stage
Sw itching mirror mot
Axiovert 200 M SP
1:1
Axiovert 200 M BP
Transmitted-light
channel for LSM 5
Several solutions for
incubation w ill be offered.
100 HAL lamp housing
w ith collector,
lamp mount
Non Descanned
Halogen lamp Detection kit
12 V 100 W
motorized NDD module
w ith shutter
Detection module
external PM T for Non
Descanned Detection
Scanning stage DC 120x100
w ith mounting frame
VIS Scan module LSM 510 M ETA
VIS/ UV Scan module LSM 510 M ETA
VIS/ NLO Scan module LSM 510 M ETA
VIS Scan module LSM 510 M ETA
VIS/ UV Scan module LSM 510 M ETA
VIS/ 405 Scan module LSM 510 M ETA
VIS/ NLO Scan module LSM 510 M ETA
VIS/ 405 Scan module LSM 510 M ETA
AxioCam HRm
AxioCam HRc
Detection module
external PM T for Non
Descanned Detection
M otor control M CU 28
AxioCam HRm
AxioCam HRc
100 HAL lamp housing
w ith collector, lamp mount
2-axes control panel
Halogen lamp 12 V 100 W
Pow er supply 12 V DC 100 W,
stabilized
04
08
01
07
02
06
03
05
90
100
Sw itching mirror mot
Axioskop 2 FS M OT
ECU for LSM 510 / LSM 510 M ETA
w ith scanner drive
Transmitted-light
channel for LSM 5
25
O u r S e r v ic e s
Thanks to many years of experience in the devel-
P r o f e s s io n a l S u p p o r t
opment of laser scanning microscopes, we are
able to offer you a system the components of
The laser scanning system you purchase should be
which are perfectly matched to each other and
configured to suit the range of your applications.
which can be combined and extended. Here we
Especially in a multi-user environment, making the
profit from the application-oriented design of the
right decision is a complex task, with many differ-
fifth generation of laser scanning microscopes
ent requirements to be matched.
from Carl Zeiss.
Our LSM team specialists, familiar with the market
The new detection module permits LSM 510
and components from other manufacturers, will
systems already installed to be easily upgraded
guide you in selecting the right system.
into the LSM 510 M ETA at the customer’s site.
We are commit t ed t o support ing you in your
Existing optical, mechanical and electronic inter-
efforts w ith specific advice on applications
faces enable step-by-step upgrading for further
and technology for your examination methods.
techniques, for example the measurement of molecule interactions via FCS (Fluorescence Correlation Spectroscopy), multiphoton microscopy or
FLIM (Fluorescence Lifetime Imaging).
New scanning and analysis techniques are
made available quickly and easily via softw are upgrades.
Our experts are continuously developing new software and hardware modules to meet your challenging application requirements. Over the past
two decades, your applications expertise, combined with our know-how in scientific instrument
design, have helped us to transform the laser
scanning microscope from a 3D imaging device
int o a very versat ile and f lexible imaging and
analysis center.
This makes the LSM 510 M ETA a rew arding
long term investment.
26
F u n c t io n s o f
L a s e r S c a n n in g M ic r o s c o p e s
f r o m C a r l Z e is s
R e lia b le S e r v ic e
To ensure smoot h operat ion of your LSM 510
META, we offer you the following services:
Our regional consultants and technicians provide reliable services and technical support to
assist you in your research.
After every system installation, a comprehensive
introduction to LSM applications is offered to
the users.
Furthermore, Carl Zeiss offers training courses
and w orkshops, which provide in-depth knowhow about practical topics and applications in
laser scanning microscopy.
Automatic Component Extraction
Statistical procedure for the detection of single dye spectra
in a Lambda Stack.
Emission Fingerprinting (patent pending)
Method available with the LSM 510 META for the recording,
analysis and separation of emission signals in multifluorescence
images; also suitable for widely overlapping spectra.
Lambda Stack
Image stack with information in x, y and λ; combinable
with z and/or time series; for the determination of spectral
signatures at any specimen location.
Linear Unmixing
Mathematical procedure for the spectral deconvolution
of multiple emission signals.
M etatracking
Scanning mode available with the LSM 510 META,
similar to Multitracking, but with additional fast switching
between detection settings.
M ultitracking
Scanning mode available with the LSM 5, generates multifluorescence images without crosstalk of emission signals, by means of
fast switching between excitations, and quasi-simultaneous detection.
RealROI (rROI) Scan
Scanning mode in which freely definable specimen areas
are excited and imaged; guarantees maximum specimen
protection thanks to exact blanking of the laser lines outside
the selected specimen areas.
ROI Bleaching
Defined photobleaching of several, freely defined specimen areas,
e.g. for FRAP, Uncaging, or Photoactivation experiments.
Spline Scan
Scanning along a freehand-defined line for recording fast
(physiological) processes, e.g. along neurons.
Spot Scan
Scanning mode in which the signal intensity at a confocal point
can be tracked with extremely high temporal resolution.
Step Scan
Fast overview scan in which intermediate lines are added by
interpolation.
Tile Scan
Records an overview image consisting of a number of tiled partial
images for the recording of larger objects with improved resolution.
27
G lo s s a r y
ACE
Automatic Component Extraction
ADC
Analog-to-Digital Converter
AOM
Acousto Optical Modulator
AOTF
Acousto Optical Tunable Filter
CFP
Cyan Fluorescent Protein
DIC
Differential Interference Contrast
(Nomarski)
DSP
Digital Signal Processor
FCS
Fluorescence Correlation Spectroscopy
FLIM
Fluorescence Lifetime Imaging Microscopy
FRAP
Fluorescence Recovery After
Photobleaching
FRET
Fluorescence Resonance Energy Transfer
GFP
Green Fluorescent Protein
NLO
Non-Linear Optics
(multiphoton imaging)
ROI
Region Of Interest
YFP
Yellow Fluorescent Protein
2002: The LSM 510 M ETA w ins the renow ned
R&D 100 aw ard for technical developments.
Carl Zeiss
Advanced Imaging M icroscopy
07740 Jena
GERMANY
Phone: ++49-36 41 64 34 00
Telefax: ++49-36 41 64 31 44
E-Mail: [email protected]
w w w.zeiss.de/ lsm
Subject to change.
Printed on environment-friendly paper,
bleached without the use of chlorine.
For further information, please contact :
45-0008 e/10.02
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