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Microscopy from Carl Zeiss
LSM 5 PASCAL Laser Scanning Microscope
Dynamic Tracking of Cellular Processes
We make it visible.
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The LSM 5 PASCAL from Carl Zeiss is a confocal laser scanning microscope for fundamental research in medicine and biology. Equipped with optimized, user-friendly hardware and software, this system delivers excellent confocal images and image stacks, especially in fluorescence applications. It is optimally designed for the acquisition of time series for the analysis of molecule mobilities by bleaching or photoconversion.
Corneal epithelium: Cytokeratin K3 labeled with FITC (green), nuclei with propidium iodide (red). Specimen: Fatima Anees, L.V. Prasad Eye Institute, Hyderabad, India
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Contents
Confocality
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Components
6
Configurations
8
MultiTracking
10
Colocalization
12
3D Image Stacks
14
FRAP
16
Photoconversion
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FRET
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Physiology
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Visualization
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Archiving
23
Specification
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System Overview
26
Glossary
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LSM 5 PASCAL
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Fascinating Contrast Experience Confocality
The LSM 5 PASCAL is a confocal microscope system
Confocal ...
that scans a specimen with laser light, point by point and line by line, in order to acquire an optical sec-
The special advantage of confocal laser scanning
tion. Many optical sections collected from different
microscopy results from the use of a pinhole dia-
Z planes form a 3D image stack. Confocal image
phragm located conjugate to the focal plane. The
stacks acquired with fluorescent light provide infor-
pinhole only admits light coming from the focal
mation on selectively labelled functional regions of
plane, while emissions from planes above or below it
cells, tissues and organisms.
are rejected.
Intensity (I)
... laser ...
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Excitation
A fluorochrome can be excited by different wavelengths within its excitation spectrum. Light is then emitted with a characteristic emission spectra. The emission intensity is a function of the radiation intensity and excitation efficiency of the light.
Emission
50
For the LSM 5 PASCAL, lasers with several lines ranging from 405 to 633 nm are available for excitation. The lasers are coupled to the scanning module reliably and efficiently via separate optical fibers. Laser intensities can be adjusted either with a softwarecontrolled mechano-optical tunable filter (MOTF), or a fast acousto-optical tunable filter (AOTF). ... scanning
0
Wavelength (λ)
Point-by-point and line-by-line scanning of the specimen with a focused laser beam produces twodimensional images free from scattered light.
Detector
Scanning at different levels as the laser focus is shift-
I
ed along the Z axis generates a series of optical sections (slices), which can then be combined into a
three-dimensional image stack.
I
Dichroic beamsplitter
Detector I
In case of a multiple-stained specimen, the various emission signals are separated by highquality dichroic filters, which can be selected and changed to suit the application.
1 2 3 4 5 6 7 8 9 10 11
Fiber (from laser source) Collimator Main dichroic beamsplitter* Objective* Specimen Pinhole Emission filter* Detector Scanning mirror Scanning optics Focal plane *user-exchangeable components
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7
8
3 6
1 2
3
9 10
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3 2
xy
1
9 4 5 11
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A Perfect Team Components Matched for Efficient Operation Carl Zeiss configures every LSM 5 PASCAL to suit the
tioning of the two scanning mirrors, and continuous
user’s scope of applications. For that purpose, a
laser attenuation to permit the user to apply many
great number of well-matched, high-quality system
different scanning strategies. Fluorescence signals
components are available.
can for example, be detected along a straight line or a freely defined curve, or at a diffraction-limited spot. Pixel resolutions of confocal images can be
The heart of the system
freely selected between 4 x 1 and 2,048 x 2,048 pixels. The two independent scanning mirrors allow
The scanning module includes collimators, the scan-
the scanning field to be rotated to any angle
ner, and a freely positionable and adjustable pinhole.
between 0° and 360°. The scanning speed can be
Detection is by highly sensitive photomultipliers.
precisely varied in 26 steps, with line frequencies
Advanced scanning control allows the precise posi-
ranging from 4 to 2,600 Hz.
LSM 5 PASCAL with Axiovert 200 M / Sideport: The 2D and 3D false-color images are rendered through a high-resolution graphics card.
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C-Apochromats: Optimized for confocal microscopy.
The intelligent control center
The electronics module houses the main component of scanner control and image acquisition, the digital signal processor (DSP). A trigger interface allows synchronization of external devices (TriggerOut), e.g., a micromanipulator, as well as “remote-controlled” triggering of confocal image acquisition (TriggerIn) by external devices such as a breath sensor.
EC Plan-Neofluars: High contrast for general use.
The stable basis
Depending on the user’s application, several high-end research microscopes are available as platforms for the LSM 5 PASCAL system: Axio Imager, Axiovert 200 M, and Axioskop 2 FS MOT. All of them are equipped with IC2S optics, which guarantee unrivalled image quality, brilliant contrast, and perfect color correction. The LSM software automatically detects all objectives, reproduces saved microscope settings at the touch of
Plan-Apochromats and Fluars: High-aperture photon collectors.
a button, and accurately controls the system’s image acquisition process. The keen eyes
High-grade Carl Zeiss objectives allow you to set just the right combination of resolving power, speed, working distance, the refractive index of the immersion liquid, etc., as required for your application. C-Apochromat objectives provide the ultimate in confocal microscopy: diffraction-limited resolution
Achroplans: Water-immersed objectives for electrophysiological specimens.
and perfect chromatic correction from the UV to the NIR range. The flare-reduced EC Plan-Neofluar objectives deliver enhanced contrast, and the slender LCI Plan-Neofluars provide extra space for convenient manipulation and temperature control of live cell specimens. LCI Plan-Neofluars: With temperature compensation for living specimens.
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Proper Settings Configuration with just a Mouse Click
The success of a microscopical experiment is a matter of correct settings. All parameters of the fully motordriven LSM 5 PASCAL can be selected quickly and correctly, via its intuitive software. The parameters of the experiment – from laser setting to image acquisition – are automatically saved and can be precisely restored whenever they are needed again. So rather than having to care too much about the microscope, you can fully concentrate on your research.
(2+1) detection channels
In the LSM 5 PASCAL, one or two channels are available for fluorescence and reflection measurements, each featuring a highly sensitive, low-noise photomultiplier. XY adjustment of the pinhole is effected through a software-controlled motor; the pinhole diameter can be controlled continuously, as can the intensity of the laser. In some experiments it is helpful to superimpose transmitted-light and fluorescence images to “get the whole picture”. Especially with differential interference contrast (DIC), the optional transmitted-light channel supplies important information about the topology of your specimen.
Clear and easy to use: The software of the LSM 5 PASCAL.
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Cultured cells. Fluorescence image superimposed with differential interference contrast in the transmitted-light channel. HeLa cells, mitochondria labeled with DsRed. Specimen: Prof. S. Yamamoto, Hamamatsu Medical University, Japan
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Complex functionality under control
Use the Configuration Control dialog window to select the main and secondary dichroic beamsplitters and emission filters in the confocal beam path, and to optimize the detection parameters. Alternatively, the system falls back on settings made in earlier experiments: the tried-and-approved ReUse function not only speeds up procedures in the lab but also exactly reproduces experimental conditions. Use the Scan Control dialog window to define all scanning parameters, such as frame size (up to 2,048 x 2,048 pixels), scanning speed, data resolution, and scanning direction (uni- or bidirectional). Use the Find function to have the system find the optimum contrast and brightness settings, within seconds of clicking the mouse.
The Crop function allows a new scanning area to be selected and rotated with speed and ease. Cultured A 549 cells: Alpha-tubulin labeled with FITC (green), cell nuclei with propidium iodide (red). Specimen: Prof. H. Ayabe, Nakasaki University, Japan
The images can be displayed individually or superimposed. Cell division of HEp-2 cells. Microtubules labeled with rhodamine (red), centromeres with FITC (green), PML nuclear bodies with Alexa 633 (blue). Specimen: S. Weidtkamp-Peters, Fritz-Lipmann-Institute Jena, Germany
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No Crosstalk MultiTracking for Clear Signal Separation Framewise or linewise MultiTracking is an elegant and reliable solution which efficiently prevents interchannel crosstalk between several fluorochromes. Colocalization studies, in particular, benefit from the increase in reliability gained from MultiTracking.
Unique capabilities
The LSM 5 PASCAL allows the user to optimize excitation parameters and detection settings for a single dye signal, and to define and save it as a track. A list of such tracks for independently optimized dye sig-
Isolated salivary gland of a cockroach. Cell nuclei are labeled with DAPI (blue), Na+/K+ ATPase with Cy2 (green), and F-actin with Alexa 568 phalloidin (red). Specimen: Dr. D. Malun, Free University of Berlin, Germany The simultaneous recording of either DAPI and Cy2, or Cy2 and Alexa 568 shows strong bleedthrough of the DAPI signal into the Cy2 channel (middle), or of the Cy2 signal into the Alexa 568 channel (right).
nals can then be run automatically as a MultiTracking experiment. Unblanking and blanking of the laser lines between the tracks is effected by mechano- or acousto-optical devices. Up to eight fluorescent signals can thus be MultiTracked in a single run, section by section (MOTF) or line by line (AOTF). Due to the selective excitation and detection of the dyes, signal crosstalk is prevented reliably.
The same specimen recorded by MultiTracking (with the same laser intensity). The channels are clearly separated.
MultiTracking is as easy as pie: Select the laser lines, main dichroic beamsplitters and emission filters for each channel in the Configuration Control dialog window. Once compiled, a list of tracks can be saved and activated later whenever needed.
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Increased efficiency
MultiTracking is a distinct benefit especially where signals are very faint. Here, you can now use longpass emission filters so as to use the entire emission spectrum of the dyes. The pinhole diameter can be set individually for each track to help balance the signal intensity for the respective dye type or dye concentration. This allows either optimum adjustment for greatly different dye intensities, or to perfectly match the optical slice thicknesses in critical colocalization studies.
Superposition of the images obtained from three channels clearly illustrates the difference: Simultaneous recording results in massive signal crosstalk, which wrongly suggests a colocalization (shown in orange and turquoise). With MultiTracking, the signals from the various channels are clearly separated, and optimum signal yield is guaranteed. The Spectra function displays the selected laser lines and filters in a clear overview. Example of a configuration for detecting Cy2 (green) and Alexa 568 (red) as a MultiTrack with long-pass filters.
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Perfect Coincidence 3D Colocalization Quantified
With the LSM 5 PASCAL, quantitative colocalization
Visualization and analysis of
analyses can be made with unprecedented reliability
colocalization experiments :
and precision. Image presentation, scatter plot and data table are interactively linked to the region-ofinterest (ROI) and thresholding tools.
• Interactive linking of images, scatter plots and data tables • Interactive or automatic threshold determination
The colocalization of labeled cell structures is often considered a first indicator of a potential function-
• Results of the colocalization analysis
al interaction. With MultiTrack configurations of
superimposed on image channels
the LSM 5 PASCAL, genuine colocalization can readily be distinguished from emission channel crosstalk. Therefore, a region of interest can be selected immediately in the scatter plot, whereupon the system immediately indicates the occurrence of this colocalized fluorophores in the image. In the same way, the data table is inter-
• Quantitative colocalization analysis for up to 99 ROIs, including – area and mean gray level intensity – degree of colocalization – coefficient of colocalization – Pearson’s correlation coefficient – Manders overlap coefficient
linked with the scatter plot and the image. There is no more intuitive and precise way of analyzing your data.
Artery of a rat's lung. Endothelin B labeled with Alexa 555 (red), α-actin (SMA) with Alexa 647 (blue), autofluorescent elastic fibers (green). Specimen: L. Villeneuve, Heart Montreal Institute Research Center, Montreal, Quebec, Canada.
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• Export of analysis results
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First-rate tools properly applied: Image presentation, scatter plots and data table are interactively linked with the ROI and thresholding tools. Expression of GFP-hMsrA (green), and mitochondria labeled with Mitotracker (red). Specimen: Prof. S. H. Heinemann, Jena University, Germany
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Governed Space Analyzing 3D Image Stacks to µm Accuracy Confocal microscopy is distinguished from conven-
Slice by slice
tional microscopy mainly by its capability to precisely analyze a specimen in three dimensions. From 3D
Thanks to the use of the confocal pinhole, which
image stacks, the researcher can gain new insights
only admits light from the focal plane while rejecting
into the complex structures and interrelations on the
emission from planes lying above or below it, the
cellular level. The LSM 5 PASCAL can turn such image
system produces a confocal “optical section”, which
stacks into projections of infinite depth of field, even
is free from out-of-focus scattered light. The thick-
from thick tissue sections.
ness of the slice depends on the pinhole diameter and the wavelength of the laser light. Digitally stacked one above the other, many of such sections made at equidistant intervals along the Z axis assemble into a 3D representation of quasi-infinite depth of field. This stack of images is the basis for an analysis of the specimen’s spatial structure.
Guard cells, transfected with AtVAM3-GFP (green), and autofluorescence in chloroplasts (red). The gallery view of the image stack shows the in-focus information in different Z positions. Specimen: Dr. M. Sato, Kyoto University, Japan
Indication of optical slice thicknesses (intervals) for various wavelengths. All relevant parameters are automatically used to calculate the resultant slice thickness, displayed as a graph, and equalized at the click of a button.
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Rendering the section planes
Using the LSM 5 PASCAL, you can generate virtual optical sections of any orientation inside the image stack. These virtual sections show you the spatial arrangements inside the specimen, even with structures that are only faintly fluorescent. Integrated measurement functions furnish geometric measurements such as length, angles, circumference or area, as well as densitometric parameters. The Profile function measures signal intensities along freely defined curves. The gray level data are presented in a clearly arranged table.
Orthogonal presentation of a stack of 15 images. Simultaneous view of XY, XZ and YZ planes.
The Cut function allows virtual section planes of any orientation to be visualized.
Intensity measurement of the various channels along a freely defined curve by means of the Profile function.
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FRAP Dynamic Processes Observed with Single-Pixel Accuracy Using acousto-optical attenuation (AOTF) of the laser
Fast, precise light flashes
beam, and the FRAP tool of the LSM 5 software, you can conveniently study the distribution dynamics of
The AOTF technology allows the laser output to be
biomolecules. The LSM 5 PASCAL delivers quantita-
maximized within microseconds to efficiently bleach
tive results within a few minutes.
a region defined with single-pixel accuracy. Therefore, the LSM 5 PASCAL is capable of running many photobleaching techniques such as FRAP (Fluores-
In addition to the mere imaging of cellular struc-
cence Recovery After Photobleaching) or FLIP
tures, it is also possible to irreversibly quench the
(Fluorescence Loss In Photobleaching), which have
fluorescence of many dyes or fusion proteins from
become established as standard experiments in bio-
defined regions. Non-bleached dye molecules from
medical analyses.
the surrounding regions will then diffuse into the bleached region, which therefore regains signal
The FRAP tool
intensity. The LSM 5 PASCAL readily supports other designs of bleaching experiments as well. Various kinetic models (single- and bi-exponential fits) are provided for the reliable determination of regeneration times and mobile or immobile molecule fractions – all with simple menu prompts.
FRAP In a FRAP experiment, a defined region in a cell expressing, e.g., a GFP fusion protein is bleached by brief but intense laser irradiation. The recovery of fluorescence is documented by time-lapse shots and measured.
FLIP In a FLIP experiment, the same region within a cell is bleached repeatedly, and the loss in fluorescence outside that region is measured.
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Telling Changes The Use of Photoconvertible Fluorescent Proteins The fluorescence of organelles or tissue cells express-
Tracking the green-to-red ratio
ing PA-GFP, KAEDE or DRONPA can be selectively activated, deactivated or spectrally changed by local
The Time Series functionality and the unmixing of
irradiation with violet light. With the proper light,
spectrally overlapping emission signals allow you to
the LSM 5 PASCAL visualizes your photoactivable
observe the changing ratio of photoconverted (red)
proteins by fluorescence.
to non-converted (green) KAEDE. Labeled molecules taking part in transport processes in cells can thus be precisely identified and localized at any time.
Single-molecule activation The blinker protein
Compared to bleaching experiments, photoactivation has the advantage that individual molecules can
The photoswitchable protein DRONPA can be
be activated selectively and their movements ana-
”turned on“ with a dose of 405 nm light, and
lyzed directly. With fast AOTF laser switching and the
”turned off“ again with 488 nm, repeatedly. Using
405 nm laser diode of the LSM 5 PASCAL, you have
the Visual Macro Editor of the LSM 5 software, you
everything you need to directly follow the journey of
can thus repeat experiments with the same cell
a protein inside a living cell.
several times. This facilitates optimization of the ambient conditions (including stimulants), and it increases the certainty of your assessment of the result.
PA-GFP + DRONPA DRONPA is a fluorescent protein which can be optically stimulated to switch between a fluorescent and a non-fluorescent state. DRONPA PA-GFP
KAEDE KAEDE is a fluorescent protein which changes from red to green color when irradiated with ultraviolet light.
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FRET Visualize Molecular Interactions
FRET (Fluorescence Resonance Energy Transfer) is a
FRET-sensitive emission
radiationless energy transfer between two fluorophores located close to each other. Its intensity
Regions of arbitrary shape can be selected precisely
change can be harnessed for the investigation of
thanks to fast, pixel-accurate switching of the laser
intramolecular protein-protein interactions, enzyme
intensity by the AOTF. With the linewise or frame-
activities, ion concentrations, and interactions
wise MultiTracking capability of the LSM 5 PASCAL,
between messenger substances in cells.
donor, acceptor and FRET signal portions and their overlaps can be clearly identified.
Proper excitation
The intensive bleaching program
With its wide range of suitable laser lines, dichroic
Alternatively, the FRET effect can be detected ele-
beamsplitters and emission filters, the LSM 5 PASCAL
gantly and quickly by the bleaching of the acceptor
is optimally suited to excite selected dye combina-
fluorophore. An increase in donor intensity in a
tions – known as FRET fluorophore pairs – such as
Region of Interest (ROI) immediately after bleaching
CFP/YFP, GFP/mRFP or GFP/Rhodamine.
can be unambigeously proved by means of the Time Series function of the LSM 5 PASCAL.
Example beam path configuration for the simultaneous acquisition of CFP and YFP for FRET studies. Continuous variation of laser intensities with an MOTF or AOTF. One or two ratio channels can be defined in addition and the result displayed during the measurement.
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Time series of the CFP and YFP fluorescences of Yellow Cameleon 2 (in hepatocytes) after stimulation with ATP and ionomycin. Specimen: Prof. T. Kawanishi, National Institute of Health Science, Tokyo, Japan
The Visual Macro Editor of the LSM 5 PASCAL allows the convenient and reproducible configuration of complete experiments for the unambiguous detection of the FRET signal portion, optionally in combination with MultiTracking. A number of bio-sensors have been designed recently that permit ion concentrations to be changed via a variation of FRET intensity. One of these bio-sensors used to detect changes in intracellular calcium concentration is Yellow Cameleon 2.
Intensity (I) ATP
The intensities of the CFP and YFP fluorescences and the YFP/CFP signal ratio in the ROI marked above.
Ionomycin
Ratio
YFP CFP
Time (s)
Spectral configuration for FRET on the LSM 5 PASCAL using CFP/YFP
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Fast Action Close Monitoring of Physiological Processes Physiological experiments are another field in which
Release pulses on time
the LSM 5 PASCAL can show its strengths. Its image acquisition and processing functions are ideally suit-
Outgoing trigger signals provide precise control of
ed to the investigation of dynamic processes in live
external equipment, e.g., micro injectors. Incoming
cells and tissues.
trigger signals can be used, e.g., for synchronization with electrophysiological experiments in order to start
Capture processes
confocal image acquisition.
with correct timing Vary experimental setups
Whether you study a fast process or a long-time
from time to time
change: the LSM 5 PASCAL covers a wide range of biologically relevant time intervals between 0.1 ms
The Multiple Time Series option can be used to cre-
and 10 hours. Highly frequent, linear scanner move-
ate complex time series experiments. This allows you
ments ensure reliable image data. All parameters can
to automatically switch between complete configu-
be optimized on-line during image acquisition.
rations, e.g., capture of an XY image in one configu-
Ratio
Intensity (I)
ration (GFP), and recording of a Z stack in another.
Time (min)
Selection of Regions of Interest (ROIs) within a specimen. ROI1: Cytosol ROI2: Cell membrane
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Time (min)
Individual intensities (left), and the ratio of intensities (right) of the two ROIs boxed in the picture (far left). Colors are assigned accordingly.
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Results available at any time
With freely defined regions of interest (ROIs) you can investigate precisely those structures of a specimen you are looking for. During image acquisition, either the series of images or the intensity curves inside the ROIs or both can be displayed. In ratiometric measurements, rather than waiting until the end of the time series, you can keep track of the results in a separate channel in real time. On-line ratio calculations allow the data to be presented live during image acquisition. The system utilises preset calculation formulae with user-defined parameters. Various calibration routines and display modes are available for calibrating fluorophores to be used in concentration analyses. Equipped with these process features, the LSM 5 PASCAL can do Dialog window for the interactive calibration of ion-sensitive dyes.
justice to any fluorescent dye.
Investigation of protein movements Time series experiment in HeLa cells transfected with PKC-GFP. Stimulation of the cells with PMA at the time t =1 min causes redistribution of PKC from the cytosol to the cell membrane. Specimen: Dr. S. Yamamoto, Hamamatsu Medical University, Japan
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Spectacular Views Fast Visualization and Archiving
Lucid presentation
you to present and publish your findings in the most perfect way. Thanks to fast algorithms, such visual-
The optional Image VisArt software package
izations are generated at an amazing speed.
provides a broad range of functions for 3D and 4D
Handling this tool is extremely easy: To create an ani-
visualizations of image stacks and time series, e.g.,
mation, simply specify the desired viewing angles.
as shadow or transparency projection. Image VisArt
The system then automatically computes a virtual
not only delivers added information but also helps
“camera flight” around, into or through the specimen.
3D reconstruction from 108 individual images. Epithelial cells from the lachrymal gland. Actin filaments of myoepithelial cells labeled with BODIPY-FL phallacidin (green), cytoplasm and nuclei of acinal cells with ethidium homodimer-1 (red). Specimen: Prof. Y. Satoh, Iwate Medical University, Japan
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Convenient management
To integrate the documents into other programs, the system offers many export formats. The same applies
Transparency and lucidity were prime concerns in the
to measured data and tables. As a genuine multi-
development of an archiving system for the LSM 5
user system, the LSM 5 PASCAL allows every user to
PASCAL. The database not only saves all settings
save configurations in their preferred way. This way,
(laser lines, pinhole sizes, scanning mode, objective,
the system is optimized to match the special applica-
etc.) but allows them to be retrieved and reproduced
tion of every investigator in its user team.
with a mouse click, thanks to the approved ReUse function.
LSM image database in the clear gallery mode of presentation. Each image represents the entire acquisition method. Special filter functions permit convenient database searches.
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Specification LSM 5 PASCAL
Microscopes Models
Upright: Axio Imager.Z1, M1, Axioskop 2 FS MOT; Inverted: Axiovert 200 M BP (BasePort) or SP (SidePort)
Z drive
Upright: Stepper motor, smallest increment 10 or 25 nm Inverted: DC motor with optoelectronic coding; smallest increment 25 nm
XY stage (option)
Motorized XY scanning stage with Mark & Find (XYZ) and Tile Scan (mosaic scan) functions; smallest increment approx. 1 µm
Accessories
High-resolution digital AxioCam microscope camera; integration of incubation chambers, micromanipulators, etc.
Scanning module Scanner
Two independent galvanometric scanning mirrors for rotation, zoom, offset
Scanning resolution
4 x1 to 2048x2048 pixels, user-definable
Scanning speed
13 x 2 speeds; line frequencies from 4 to 2600 lines/sec, 5 fps with 512x512 pixels (max. 77 fps with 512x32 pixels)
Scanning zoom
0.7x to 40x, variable in steps of 0.1
Scanning rotation
Free rotation through 360°, variable in steps of 1°
Scanning field
Field diagonal 18 mm (max.) in the intermediate image plane, homogeneous field illumination
Pinhole
1 confocal pinhole, continuously variable diameter, preadjusted
Detection
1 or 2 confocal channels (R/FL), 1 optional external transmitted-light channel (DIC-capable); each channel equipped with high-sensitivity PMT detectors
Data depth
Selectable between 8 and 12 bit
Laser Modules Lasers
Ar laser (458, 488, 514 nm) 25 mW; HeNe laser (543 nm) 1 mW; HeNe laser (633 nm) 5 mW; diode laser (405 nm) 25 mW (specification valid to the end of life time)
Fiber optics
Polarization-preserving single-mode fibers
Attenuation
Individual and variable intensity setting of all laser lines by means of MOTF or AOTF
Electronics Module LSM 5 Control
Control of microscope, laser modules, scanning module and other (accessory) components, using a high-performance digital signal processor (DSP)
Computer
High-end PC with ample RAM and hard disk storage capacity; with ergonomic high-resolution monitor or TFT flat panel display; many accessories. Windows XP operating system with multi-user capability
Image Browser
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Free software package for the display, editing, sorting, printing and export/import of LSM 5 images
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Standard Software System configuration
Convenient control and configuration of all motor-driven microscope functions, the laser modules and the scanning module; saving and retrieval of application-specific configurations
ReUse function
Restoration of acquisition parameters with just a single mouse click
Acquisition modes
Spot, line/spline, frame, Z stack, time series and combinations: xy, xyz, xyt, xyzt, xz, xt, xzt, spot-t. On-line computation and presentation of ratio images; averaging and summation (linewise or framewise, configurable); Step Scan (for higher frame rates, configurable)
Crop function
Convenient selection of scanning ranges (simultaneous zoom, offset and rotation)
ROI scan
Scanning of up to 99 regions of interest (ROIs) of any shape, and blanking of the lasers by means of AOTF
Spline scan
Scanning along a freehand defined line
MultiTracking
Signal crosstalk in multifluorescence acquisition minimized by fast change between excitation lines
Image processing
Image processing options for any kind of mathematical procedures, such as crosstalk correction by selective channel subtraction
Visualization
Orthogonal view (xy, xz, yz in one view), 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 variation, off-line interpolation for Z stacks, selection and modification of color lookup tables (LUTs), drawing functions for documentation
Analysis, measurement Advanced tools for colocalization and scatter plot analysis with individual parameters and options, profile measurement on straight lines and curves of any shape, measurement of lengths, angles, areas, intensities, and many other capabilities Image operations
Addition, subtraction, multiplication, division, ratioing, shift, filters (low pass, median, high pass, etc., or user-definable)
Data archiving, export, import
LSM image database with comfortable functions for managing images together with the associated acquisition parameters; Multiprint function for creating assembled image-plus-data views; more than 20 file formats (TIF, BMP, JPG, PSD, PCX, GIF, AVI, Quicktime, ...) for compatibility with all common image processing programs
Software Options Physiology
Comprehensive software for the analysis of time series, graphic mean-of-ROI analyses, on-line and off-line calibration of ion concentrations
FRAP
Menu prompting for recording FRAP experiments (Fluorescence Recovery After Photobleaching)
FRET
Acquisition of FRET image data (Fluorescence Resonance Energy Transfer) with subsequent analysis and computation of FRET efficiency. Methods supported: Acceptor Photobleaching and Sensitized Emission
Visual Macro Editor
Creation and editing of macros using representative icons for programming routine procedures
LSM Image VisArt
Fast 3D and 4D reconstruction and animation (various modes: shadow projection, transparency projection, surface rendering)
Multiple Time Series
Complex time series with varied application-specific configurations
Topography Package
Visualization of 3D surfaces (fast rendering modes), plus many measurement functions (roughness, surface areas, volumes)
Stitch Art plus
MXZ and MXYZ stitching software for reflection (motor-driven XY scanning stage required)
3D Deconvolution
Image restoration based on computed point spread functions (modes: nearest neighbor, maximum likelihood, constraint iterative)
3D for LSM
3D presentation and 3D measurement of volume data records
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System Overview LSM 5 PASCAL
Diode laser, 405 nm, 25 mW HeNe laser, 543 nm, 1 mW
Ar-laser, 458, 488, 514 nm, 25 mW
Laser module Argon Laser module Argon/HeNe
ECU for LSM 5 PASCAL Scan modules LSM 5 PASCAL: Scan module 1 channel//1 fibre Scan module 1 channel/2 fibres Scan module 2 channels/2 fibres Scan module 2 channels/1 fibre
Addition laser module 633 nm HeNe laser, 633 nm, 5 mW
Laser module AOTF UGB Scan module 2 channels/2 fibres
ECU for LSM 5 PASCAL AOTF
Laser module AOTF RGB Scan module AOTF RGB
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Scan module LSM 5 PASCAL
PIEZO objective focus (not for laser modules AOTF)
HBO 100 illuminator with lamp mount and collector HBO 100 illuminator self-adjusting, with lamp mount and collector Microscope stand Axio Imager.Z1 with TFT monitor and light control mot
Scanning stage 130x85 PIEZO for upright stand
Power supply unit for HBO 100 FluoArc variable intensity lamp control for HBO 100 X-Cite 120 fiber coupled illuminator
AxioCam HRm AxioCam HRc AxioCam MRm
Scanning stage 225x85 PIEZO for upright stand
HAL 100 illuminator with collector 1:1
XY-stage controller PIEZO
Halogen lamp 12 V 100 W
Axiovert 200 M SP
XY-Joystick for stage controller PIEZO
Axiovert 200 M BP Several solutions for incubation will be offered. Switching mirror mot.
Scan module LSM 5 PASCAL
Scanning stage DC 120x100 with mounting frame Scan module LSM 5 PASCAL
Transmitted-light channel for LSM 5
AxioCam HRm AxioCam HRc AxioCam MRm
Motor control MCU 28
2-axes control panel
Power supply for Halogen lamp 12 V DC 100 W, stabilized 04
08
01
07
02
06
03
05
AxioCam HRm AxioCam HRc AxioCam MRm
100
90
Axioskop 2 FS mot
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System table with breadboard Wide: 1000x750mm (1200x950 overall) Narrow: 750x1000mm (950x1200 overall)
Table for host computer width 1200 mm, height 750 mm, depth 800 mm
Large LSM 5 system table width 1500 mm, height 780 mm, depth 800 mm with vibration absorption Granite slab
Small LSM 5 system table width 650 mm, height 780 mm, depth 800 mm with vibration absorption Granite slab
Micro 60 active antivibration system (recommended for Axio Imager.Z1) table surface: 60 cm x 60 cm
Host computer
Monitor 21" (50 cm)
LCD TFT-Flatscreen 18"
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Functions
Glossary
of the LSM 5 PASCAL from Carl Zeiss
MultiTracking
AOTF
Acousto-Optical Tunable Filter
Scanning mode in the LSM 5, generates multifluorescence
CFP
Cyan Fluorescent Protein
DDS
Dual Direction Scan
DSP
Digital Signal Processor
EC
Enhanced Contrast
FP
Fluorescent Protein
FLIP
Fluorescence Loss in Photobleaching
FRAP
Fluorescence Recovery After Photobleaching
FRET
Fluorescence (or Förster)
images without crosstalk of emission signals by means of fast switching between excitation and detection. Spline Scan Scanning along a freehand-defined line for capturing fast (physiological) processes, e.g., along neuronal appendices. Spot Scan Scanning mode in which the signal intensity in a confocal spot can be monitored with extremely high time resolution. Step Scan Fast overview scan, in which intermediate lines are supplemented by interpolation. Tile Scan Acquisition of a mosaic of partial images of a large object, to improve resolution.
Reliable Service is Part of Our Tradition
To ensure smooth operation of your LSM 510 PASCAL, we offer you the following services: Our competent regional consultants and technicians will provide technical support to assist you in your research. After every system installation your LSM users are given comprehensive training. Carl Zeiss also offers training courses and workshops, which provide in-depth know-how about practical topics and applications in laser scanning microscopy.
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Resonance Energy Transfer GFP 2
Green Fluorescent Protein
IC S
Infinity Contrast and Color Corrected System
LCI
Live Cell Imaging
MOTF
Mechano-Optical Tunable Filter
NIR
Near InfraRed
PA
PhotoActivation
ROI
Region of Interest
YFP
Yellow Fluorescent Protein
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LSM 5 PASCAL US Patents: 5127730, 6037583, 6167173, 6462345, 6563632, 6665068, 6848825 German Patents: 19702752C2, 69131176T2
Carl Zeiss Advanced Imaging Microscopy 07740 Jena, Germany Phone: +49 36 41-64 34 00 Fax: +49 36 41-64 31 44 E-Mail:
[email protected] www.zeiss.de/lsm Subject to change.
Printed on environment-friendly paper, bleached without the use of chlorine.
For further information, please contact:
45-0058 e/09.05
