The LSM 5 Family Laser Scanning Microscopes Localizing, Imaging and Analyzing Biomolecules

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1 Microscopy from Carl Zeiss The LSM 5 Family Laser Scanning Microscopes Localizing, Imaging and Analyzing Biomolecules

2 The LSM 5 Family The fifth generation of Carl Zeiss Laser Scanning Microscopes offer efficient solutions for every LSM application. The family comprises a wide range of models from a single-user instrument to the revolutionary LSM 510 META to the multiphoton microscope and the fluorescence correlation spectrometer.

3 Confocal Analysis Methods 4 System Concept 6 LSM 5 PASCAL 8 LSM LSM 510 META 12 LSM 510 NLO 14 ConfoCor 2 LSM LSM 5 Software 18 Procedures and Methods 20 Functions and Tools 22 Brochures 23

4 The LSM 5 Family Laser Scanning Micro Cellular Functions Clearly Identified Cellular Dynamics Optimally Detected Fluorescence Signals Reliably Separated Deep Insights Carefully Gained Efficient Analysis of Molecular Interactions 2

5 scopes without Compromise Experience Pays Off Carl Zeiss The claim to creativity Continuous development and optical perfection at Carl Zeiss have already resulted in many pioneering accomplishments in fundamental biomedical research. Four years later, the revolutionary Emission Fingerprinting method of the LSM 510 META opened the doors to new and exciting capabilities in the life sciences. The LSM 44 launched by Carl Zeiss in 1982 was the first commercial laser scanning microscope. In 1996, Zeiss introduced ConfoCor 1, the first fluorescence correlation spectrometer, which paved the way for novel research results for biochemists worldwide. In recognition of this innovative method, the LSM 510 META was awarded the coveted R&D 100 Award in Innovation at Carl Zeiss is continuous. Expect more in the future. 3

6 Confocal Analysis Methods Clear Images in All Planes Measurements in Confocal microscopy for maximum optical resolution In the non-confocal image, the information of interest from the specimen s focal plane is blurred by out-of-focus light; specimen details stained with differently colored dyes appear as a color mix. In the confocal presentation, object details blurred in the non-confocal image are clearly visible, and image contrast is enhanced. Triple-stained tissue (section through the intestine of a mouse). Principle. Conventional microscopes detect not only signals from the focal plane but also the interfering light from the specimen regions above and below that plane. Confocal microscopes use a pinhole to selectively collect the signal from the focal plane only while obstructing light from extrafocal specimen regions. Image formation. Two-dimensional confocal images are formed by pointwise and linewise scanning of the specimen with a focused laser beam. To generate spatial images, the focal plane is successively shifted between image acquisitions, resulting in a stack of images. Computer reconstruction of the stack creates images of three and more dimensions. Advantages over conventional light microscopy: High contrast High resolution Spatial data Detector Laser light source Open pinhole in the confocal plane Collimator Main dichroic beamsplitter Scanning mirrors Objective Specimen 4 Focal plane

7 Smallest Volumes Multiphoton microscopy for depth information Fluorescence correlation spectroscopy for functional analysis Principle. For fluorescence excitation, multiphoton microscopes use pulsed infrared light, which is less absorbed and less scattered than visible light by biological materials. At the same time, multiphoton microscopy excites the flourochrome exclusively at the focus of the infrared laser beam. Unlike confocal laser scanning microscopes, multiphoton microscopes thus detect fluorescent signals emitted deep inside heavily light-scattering or absorbing specimens. No pinhole is required to select the fluorescent light coming from the focal plane. Image formation. The specimens are scanned as in a confocal laser scanning microscope. Advantages over single-photon excitation: Imaging of structures deep inside a specimen Lowest possible light load on the specimen Improved sensitivity Principle. Fluorescence correlation spectroscopy (FCS) uses the transient fluorescence of mobile molecules passing through the confocal volume to quantitatively analyze molecule mobilities and concentrations. Unlike confocal laser scanning microscopy, the method does not yield image data but generates and analyzes spectroscopic information known as correlation functions. FCS is a method for studying dynamic processes in solutions and cells. Advantages: Quantitative analysis of molecule diffusion processes Investigation of biochemical interactions between molecules (binding constants) Determination of nanomolar concentrations Laser light source Detector The confocal volume has the size of a bacterium (1 2 femtoliters). Molecules diffusing through the confocal volume cause intensity fluctuations (δi) versus time (t). 1 Collimator Scanning mirrors Longpass filter Focal plane Objective Specimen Detector 2 2 Detector Shortpass filter Detector I Mirror t 5

8 System Concept A Perfect Kit of Building Blocks for Maximum Microscope: The body Laser module: The source The laser scanning microscopes of the LSM 5 family can be configured around different Carl Zeiss research microscopes to suit different applications. Each of these research microscopes is fully motordriven and conveniently controllable through the LSM software. Their integration into a uniform overall optical concept warrants optimum performance and prime imaging quality in a word, optical perfection. Depending on the application, the laser scanning microscopes of the LSM 5 family are equipped with a variety of lasers from UV to the visible region to the near infrared. With convenient control through the LSM software, laser intensities are switched and varied with the speed and exactitude needed to minimize photodamage even in highly sensitive specimens. 6

9 Experimental Flexibility Scanning and detection module: The heart LSM 5 software: The brain Independently movable scanning mirrors in the scanning and detection module exactly direct the laser beam across the specimen, permitting optimum adaptation of the scanfield to the specimen requirements in terms of shape, size and orientation. Depending on model and application, the scanning and detection module may have up to four photomultiplier detectors. For the requirements of multifluorescence microscopy, in particular, Carl Zeiss designed the META detector. This is a multichannel detector used for recording the spectral emission profile of every spot of the specimen ( Spectral Imaging ). In conjunction with intelligent software algorithms, the META detector exactly separates combinations of fluorochromes having extremely similar emission properties. The laser scanning microscopes are controlled by the LSM software. It coordinates the work of the system modules and monitors all motor-driven components. The LSM software supports a wide range of functions from image acquisition to image analysis, and from data extraction to image archiving. This functionality is supplemented by optional software packages tailored to specific applications. Additional licenses for multi-user workstations are, of course, available. 7

10 Compact Class Entry-Level Confocal Microscopy The LSM PASCAL* is a powerful confocal system for demanding routine tasks in biomedical laboratories, education and research. With full motorization, superb image quality, multitracking, and comfortable software, it is unique in its class. Configuration Microscopes Upright: Axioskop 2 MOT, Axioskop 2 FS MOT, Axioplan 2 Imaging MOT Inverted: Axiovert 200 M Laser modules Wavelength ranges: 405 nm 543 nm or 458 nm 633 nm Scanning modules LSM 5 PASCAL scanning module with one or two detection channels and an adjustable pinhole Option: Transmitted-light channel Software Basic software for LSM 5 PASCAL Options: Physiology, Image VisArt, Deconvolution, 3D for LSM Benefits Easiest handling of multiple fluorescence measurements with the ReUse software function Clear separation of superimposed fluorescence signals (crosstalk) by the multitracking concept Observation of fast biological processes thanks to high scanning speeds and flexible image formats Finest image quality due to adjustable and variable pinhole 8 * LSM 5 PASCAL US Patents: , German Patent: C2

11 LSM 5 PASCAL 1) 2) The extraordinary variability of the LSM 5 PASCAL gives you unprecedented freedom of application. Whether multiple fluorescence imaging in cellular biology and molecular genetics, 3D structural analysis in neurobiology or 4D time-lapse series of intracellular ion concentrations in physiological studies: with the LSM 5 PASCAL you can meet any experimental challenge. Applications 3D analyses of cell and tissue structures Multiple fluorescence and quantitative colocalization experiments, e.g. neurogenetic studies (Fig. 1) Monitoring of fast physiological processes by Fast Ion Imaging, e.g. when measuring calcium transients (Fig. 2) Identification and quantification of molecule interactions by FRET microscopy 1 Atrium of a guinea pig. Actin (BODIPY FL phallacidin: green) and NeuroPeptide-Y (Alexa 594: red). Specimen: Prof. Y. Satoh, Iwate Medical University, Japan 2 Salivary gland of the fly Calliphora vicina. Hormoneinduced increase in cytosolic calcium concentration, imaged with Fluo-4 in the intact organ. Specimen: Dr. B. Zimmermann, Potsdam University, Germany 9

12 Top Class That Extra Bit of Individuality The LSM 510* is ideal for solving complex analytical problems in cross-disciplinary research teams, multi-user facilities, microscopy service centers, or in the central analytical lab. Wherever you need that extra bit of system performance, flexibility or add-on modularity. With its excitation wavelengths in the UV range, fast, pixel-accurate scanfield definition, a proven multiple pinhole concept and many more features, the LSM 510 is bound to convince you. Configuration Microscopes Upright: Axioskop 2 FS MOT, Axioplan 2 Imaging MOT Inverted: Axiovert 200 M Laser modules Wavelength ranges: 351/405 nm 633 nm or 458 nm 633 nm Scanning modules LSM 510 scanning module with up to four detection channels and up to four adjustable pinholes Option: Transmitted-light channel Software Basic software for LSM 510 Options: Physiology, Image VisArt, Deconvolution, 3D for LSM, Multiple Time Series, Macro-Edit Benefits Fast acquisition of image stacks of multiply labeled specimens in up to four detection channels Selective bleaching and imaging of a specimen segment with the RealROI concept Precise results in multiple fluorescence experiments due to identical detection volumes High flexibility in using fluorochromes thanks to a broad spectrum of excitation wavelengths 10 * LSM 510 US Patents: , , , , , , , , German Patents: C2, C2, C2, C2, C2, C2, C2, C2, C2

13 LSM 510 1) 2) Given the virtuoso performance of the LSM 510, you are no longer restricted to mere microscopic imaging. You can track motions in cells, cell aggregates or organisms with targeted photoactivation or advanced photobleaching experiments. Observe molecular changes or interactions by means of energy transfer between suitable pairs of fluorochromes. Use the flexibility of an intelligent laser scanning microscope from Carl Zeiss. Applications Efficient analysis of complex multiple fluorescence signals with a broad spectrum of fluorochromes, e.g., the structural relationship between cell compartments in organs (Fig. 2) Direct observation of transport processes in live cells and organisms by targeted photoactivation of novel fluorochromes such as Kaede (Fig. 1) Study of diffusion and transport mechanisms by targeted photobleaching experiments Determining the molecular interactions of biomolecules by acceptor bleaching in FRET microscopy 1 Photoconversion of Kaede transfected living cell by repeated local irradiation at 405 nm. Specimen: Prof. A. Miyawaki, Riken, Japan 2 Morphology of a plant root. Epidermis (blue), mitochondria (red) and cell walls (green). Orthogonal sections of a Z stack. Specimen: Prof. P. C. Cheng, New York State University, Buffalo, USA 11

14 Top Class and More Spectral Dimensions Add another dimension: The LSM 510 META* Laser Scanning Microscope, an efficient upgrade from the LSM 510, recognizes fluorescence signals by their unique spectral signatures. The Emission Fingerprinting technique provided by Carl Zeiss positively and precisely separates spectrally overlapping fluorescence signals. Rather than merely sorting fluorescent proteins by their color, the system analyzes the entire spectrum. Configuration Microscopes Upright: Axioskop 2 FS MOT, Axioplan 2 Imaging MOT Inverted: Axiovert 200 M Laser modules Wavelength ranges: 351/405 nm 633 nm or 458 nm 633 nm Scanning modules LSM 510 META scanning module with two conventional and 32 spectral detection channels and three individual pinholes Option: Transmitted-light channel Software Basic software for LSM 510. Options: Physiology, Image VisArt, Deconvolution, 3D for LSM, Multiple Time Series, Macro-Edit Benefits Time-saving, specimen-preserving spectral imaging by parallel data acquisition in up to 8 META channels Unambiguous, reliable separation of fluorescent signals by the proprietary Emission Fingerprinting technique Detection of the spectral properties of fluorochrome and autofluorescence signals On-line separation of fluorescences during measurements by the Online Fingerprinting technique 12 * LSM 510 META US Patent: German Patent: C2

15 LSM 510 META 1) 2) Relative intensity Wavelength (nm) The LSM 510 META is predestined for all multiple fluorescence labelings in which heavy crosstalk or spectrally close emission signals prevent separation into classical detection channels. In genetic experiments with the LSM 510 META whether it s gene expression in the study of protein functions, or in situ hybridization in chromosome analysis your multi-labeled specimens will show themselves in their true colors. 1 Autofluorescent pigments of the coral Lobophyllia corymbosa. Spectral signatures in the indicated ROIs show GFP-type coral proteins and chloroplasts of dinoflagellate symbionts. Specimen: Dr. A. Salih, Australian Key Centre for Microscopy & Microanalysis, University of Sidney, Australia 2 HeLa cells with Trans Golgi network (GCC-GFP: blue), microtubules (Cy3: green), actin (Texas Red: red), visualized by Emission Fingerprinting. Specimen: Prof. J. Stow and J. Lock, University of Queensland, Australia Applications Exact separation of fluorochromes with extremely overlapping emission spectra, such as Texas Red and Cy3 (Fig. 2) Gene expression studies and protein localization in biological systems, especially by Spectral Imaging of multiple fluorescent proteins Spectral imaging of autofluorescences, as in corals (Fig. 1) Examination if protein-protein interactions by FRET microscopy in conjunction with Emission Fingerprinting Observation of dynamic processes and imaging of ion concentrations by measurement of the spectral changes of fluorescence signals Precise analyses of multiple fluorescence in situ hybridization (M-FISH) 13

16 In a Class of Its Own Deep Insights The LSM 510 NLO* is ideal for the gentle in-depth analysis of live specimens, including whole organisms. The LSM 510 NLO is outstanding for its unparalleled selectivity: Even low concentrations of fluorochromes are registered thanks to tunable multiphoton excitation and non-descanned detection. With the depth-selective excitation unique to the LSM 510 NLO, your bleaching experiments will succeed with pinpoint accuracy. Configuration Microscopes Upright: Axioskop 2 FS MOT, Axioplan 2 Imaging MOT Inverted: Axiovert 200 M Laser modules Wavelength range: 458 nm up to 1064 nm Scanning modules LSM 510 scanning module with one or two detection channels Option: LSM 510 META scanning module with two conventional and 32 spectral detection channels and three individual pinholes Option: Transmitted-light channel, NDD Software Basic software for LSM 510 Options: Physiology, Image VisArt, Deconvolution, 3D for LSM, Multiple Time Series, Macro-Edit Benefits Highly informative imaging of fluorescence signals in tissue layers up to 500 µm deep by multiphoton excitation Low load on specimens thanks to near infrared excitation Efficient detection of weak fluorescence signals by non-descanned detection (NDD) in reflection and transmission Homogeneously illuminated optical sections by automatic variation of laser output with increasing specimen penetration depth Flexible use of various fluorochrome combinations by elimination of excitation crosstalk using Excitation Fingerprinting 14 * LSM 510 NLO US Patents: , German Patent: C2

17 LSM 510 NLO 1) 2) 3) Utilize the innovative potential of the LSM 510 NLO to cope with thick tissue sections and live specimens. Structural or functional analysis of finest nerve cells in neurobiology, or of whole embryos in developmental biology: the LSM 510 NLO will enhance your views of life. 1 Zebra fish embryo. Optical section through head and eye, double labeled with antibodies against axonal cell surface components. Specimen: Prof. M. Bastmeyer, Jena University, Germany 2 Rat cerebellum. GFAP-labeled astrocytes, immunocytochemical staining (Cy3). Specimen: Dr. O. Baumann, Potsdam University, Germany 3 Spinal cord of a living transgenic zebra fish embryo expressing GFP in motoneurons and their processes. Specimen: Dr. M. Marx, Prof. M. Bastmeyer, Jena University, Germany Applications 3D microscopy of thick tissue sections and complete organisms, e.g. in the study of structures in a zebra fish or in the cerebellum of a rat (Figs. 1, 2) Live cell microscopy of specimens with high photosensitivity, as of a live zebra fish (Fig. 3) Highly 3D-localized uncaging of biologically active molecules from caged compounds Z-selective bleaching experiments thanks to the 3D effect of multiphoton excitation Separation of fluorescence signals by their fluorescence lifetimes: Fluorescence Lifetime Imaging Microscopy (FLIM) 15

18 A Unique Combination Analysis of Moving Molecules Its high sensitivity and statistical reliability make the ConfoCor 2 LSM 510 a unique motion detector at the level of single molecules. Select the desired location inside the cell with nanometer accuracy, and start the automatic measurement and analysis run. The system detects tiny signal fluctuations and quantifies them in terms of molecular dynamics. Configuration Microscopes Inverted: Axiovert 200 M Laser modules Wavelength ranges: 351 nm*/458 nm 633 nm/1064*nm Scanning modules FCS detection module with two detection channels, plus up to four imaging channels for LSM 510 Option: LSM 510 META scanning module with two conventional and 32 spectral detection channels and three individual pinholes Software Basic software for FCS, plus basic software for LSM Options: Physiology, Time Series, Image VisArt, Deconvolution, 3D for LSM Benefits Integrated solution for confocal imaging and spectroscopic analysis Maximum sensitivity and time resolution through sensitive high-performance detectors Real-time analysis with intelligent algorithms High information density of molecule measurements: localization + concentration + interaction + speed * LSM only, not for use with FCS 16

19 ConfoCor2 LSM 510 1) 2) Count [khz] Position [mm] 3) 4) Cross correlation G(t) Time [µs] The ConfoCor2 LSM 510 precisely analyzes and localizes molecular interactions, even in a tiny sample volume. Whether in biophysics or biochemistry, cellular biology or molecular diagnostics, the ConfoCor2 derives enough information from a femtoliter of sample to better understand and classify complex processes with functional biomolecules. Applications Analysis and localization of molecular interactions within a cell Measurement of extremely low molecule concentrations In vivo studies of molecular mobility to analyze cellular diffusion and transport processes Investigation of association and dissociation processes for the functional characterization of cellular proteins Determination of binding kinetics in biochemistry and biophysics 1 Z scan through an adherent cell and positioning of the confocal volume onto the upper plasma membrane. 2 Equatorial section (left) through a cell expressing the A1 adenosine receptor (fused to TOPAZ). 3 Determination of the diffusion time of the A1 adenosine receptor (fused to TOPAZ) by correlation analysis. 4 Upper cell membrane. Specimen: S. Briddon, Nottingham University, UK 17

20 The Class Act of Software Control Mature Concept for Exacting Demands The LSM 5 software endows Carl Zeiss laser scanning microscopes with intelligence and creativity. It configures, directs and monitors all motor-driven system components with intuitive logic, user-friendliness and reliability. The basic LSM 5 software package can be supplemented with optional modules tailored to specific applications. Additional licenses and add-on modules are available, allowing you to conveniently process your data at a separate workstation for optimum system utilization. Off-line software Image Browser The Image Browser is an optional software package available free of charge. Download it from web site Use the browser for viewing, editing, sorting, printing and exporting LSM 5 images. Additional licenses An additional license includes the full functionality of the LSM 5 software and allows you to set up another workstation. Image Examiner The Image Examiner is part of every basic software package. It provides full functionality (except image acquisition) for another computer. Benefits Straightforward system control and configuration via the graphic user interface Individual adaptation to the task by intuitive user-defined configuration Reproducibility by a mouse click the ReUse function quickly calls up all image acquisition parameters Clearly structured data visualization and analysis with easy-to-handle tools Convenient archiving of images and acquisition parameters in a well-structured database 18

21 LSM 5 Software 1) 2) 1 Image VisArt software: Lachrymal gland of a mouse. 3D visualization of endpieces, labeled with phalloidin and ethidium homodimer 1. 2 Physiology software: Salivary gland of a Calliphora fly. Time series of the cytosolic Ca 2+ concentration (Fluo-4, green) and the mitochondrial membrane potential (TMRE, red). Specimen: Dr. B. Zimmermann, Potsdam University, Germany Optional software packages 3D for LSM Visualization and analysis of threedimensional data records. Visualization of 3D images Measurement in 3D images Deconvolution Image restoration from threedimensional image data. Image analysis based on computed point spread functions Visualization of the computed image data Multiple Time Series Image acquisition for the investigation of complex dynamic processes. Simultaneous automatic image acquisition in several positions Image acquisition with different configurations for every ROI, also in combination with bleaching Visualization of complex time series Image VisArt fast reconstruction and animation of multidimensional imagery. Silhouette projection, surface rendering of 3D and 4D image stacks Presentation and animation of the images Macro-Edit Creation of individual measurement and analysis routines. Programming of automated image acquisition runs Definition of automated analysis functions Physiology image acquisition and data analysis for studying simple dynamic processes. Acquisition of time series, also in combination with local bleaching Data analysis, e.g., change of intensity vs.time within a defined region (mean-of-roi analyses) Calibration and measurement of ion concentrations 19

22 Techniques and Methods For Your Application LSM 5 PASCAL LSM 510 LSM 510 META LSM 510 NLO ConfoCor 2 LSM 510* 3D Investigations Added information in three spatial dimensions (X,Y,Z) by using the confocal volume: Acquisition, visualization and measurement of 3D image stacks. Multifluorescence Imaging Image generation from specimens with multiple fluorescence labels: Crosstalk-free detection and presentation of multiple fluorescences. Quantitative Colocalization Detection of the coincidence of two fluorescence-labeled molecules in the confocal detection volume. Investigation of neighborhood relations and interactions: Definition of parameters, image presentation and data analysis (colocalization coefficients). Time Series Added information on simple dynamic processes by acquisition of image series, also in combination with local bleaching: Acquisition, visualization and analysis of time series (X,Y,t or X,Y,Z,t). FRET by Sensitized Emission (Fluorescence Resonance Energy Transfer) Investigation of molecule interactions by energy transfer between fluorescence-labeled donor and acceptor molecules spaced at 1 10 nm: Direct registration of FRET by detecting acceptor fluorescence intensity after donor excitation. Ion Imaging Measurement and imaging of ion concentrations by means of selective fluorescence markers: Image acquisition, measurement and calibration of ion concentrations. Transmitted-Light Microscopy Image generation in transmitted light: Brightfield, phase and DIC images in the LSM mode with optional transmitted-light detector. FLIP and FRAP Microscopy (Fluorescence Loss in Photobleaching; Fluorescence Recovery after Photobleaching) Investigation of diffusion and transport mechanisms by targeted bleaching under observation: Image acquisition and analysis of the bleaching experiments. 20 * ConfoCor2 without LSM 510 is specially tailored to FCS.

23 LSM 5 PASCAL LSM 510 LSM 510 META LSM 510 NLO ConfoCor 2 LSM 510* FRET with Acceptor Bleaching (Fluorescence Resonance Energy Transfer) Investigation of molecule interactions by energy transfer between fluorescence-marked donor and acceptor molecules spaced at 1 10 nm: Indirect registration by detection of acceptor and donor fluorescence intensities after acceptor bleaching. Complex Time Series Added information about complex dynamic processes by simultaneous automated acquisition of image series in several freely defined ROIs, also in combination with local bleaching: Acquisition (at variable intervals, in different positions, with different scanning modes and configurations), visualization and analysis of complex time series. Uncaging, Photoactivation, Photoconversion Activation or uncaging of biologically active substances by intense irradiation: targeted, pixel-accurate irradiation in a region defined in spatial and temporal terms, and imaging. Emission Fingerprinting Spectral unmixing of emission signals by means of reference spectra. Clear separation of heavily overlapping fluorescence and autofluorescence signals: Acquisition of lambda stacks, spectral unmixing, presentation as a multichannel image (on-line and off-line). Spectral Imaging Acquisition of spatially resolved fluorescence emission spectra: Acquisition of lambda stacks, presentation and saving of fluorescence spectra. ( ) ( ) ( ) ( ) FLIM Microscopy (Fluorescence Lifetime Imaging Microscopy) Measurement of the fluorescence lifetime as a fluorochrome characteristic: Spatially resolved measurement of fluorescence intensity as a function of time (in the ps range). Multiphoton Microscopy Fluorescence excitation through multiphoton processes. For the penetration of thick specimens and specimen-sparing long-time experiments: Excitation with pulsed infrared laser, image acquisition, and data analysis. FCS (Fluorescence Correlation Spectroscopy) Statistical analysis method for the quantitative study of molecule interactions, based on the fluctuation of fluorescence-marked molecules in the confocal volume: Data acquisition and analysis. ( ) for the META detector option 21

24 Functions and Tools Standard Features That Deliver Excellence Software Functionality for all Systems of the LSM 5 Family Auto Z On-line adaptation of acquisition parameters for Z stacks to equalize brightness in the various Z sections. Crop Convenient graphical selection of scanning regions; zoom and rotation for the recording of fast processes. Expert Mode Software mode with access to all functions for flexible experimenting. Multitracking Sequential excitation of the specimen to obtain multiple fluorescence images without emission crosstalk. Real ROI Scan Acquisition and imaging of freely defined specimen regions, with laser blanking for minimum light load on the specimen (only with LSM 510, LSM 510 META, LSM 510 NLO, ConfoCor 2 LSM 510). ReUse Restitution of acquisition parameters by a mouse click, for reproducible experiments. ROI Bleach Defined bleaching in freely defined specimen regions, for applications such as FRAP, FRET, photoactivation, photoconversion, uncaging (only with LSM 510, LSM 510 META, LSM 510 NLO, ConfoCor 2 LSM 510). Routine Mode Software mode with access to defined functions for routine work. Spline Scan Scanning along a freely defined line for capturing fast processes. Spot Bleach Defined bleaching in a specimen spot, for observation of extremely fast dynamic movements. Spot Scan Scanning with extremely high temporal resolution of signal intensity in a confocal spot. Step Scan Fast overview scanning, with skipped rows replaced by interpolation. Tile Scan Acquisition of a mosaic consisting of partial images, for recording larger objects. Z Bleach Bleaching in a defined specimen plane for targeted manipulation. Specification of all Systems of the LSM 5 Family Scanning resolution: 2048x2048 pixels Scanning speed: 5 frames/s at 512x512 pixels, or 2600 lines/s Maximum frame rate: 77 frames/s at 512x32 pixels Scanning rotation: Free 360 rotation in steps of 1 Data depth: Choice of 8 or 12 bits 22

25 Ask for the following brochures which give detailed information on each system of the LSM 5 family: LSM 5 PASCAL Laser Scanning Microscope Cellular Functions Clearly Identified LSM 510 Laser Scanning Microscope Cellular Dynamics Optimally Detected LSM 510 META Laser Scanning Microscope Fluorescence Signals Reliably Separated LSM 510 NLO und LSM 510 META NLO Multiphoton Laser Scanning Microscopes Deep Insights Carefully Gained ConfoCor 2 LSM 510 Efficient Analysis of Molecular Interactions Further information: 23

26 Salivary gland of a cockroach Jaw and tooth of a mouse Retina of a fruit fly Head of a Bristle-worm Principle of FCS For further information, please contact: Carl Zeiss Advanced Imaging Microscopy Jena GERMANY Phone: Telefax: micro@zeiss.de Subject to change e/12.03