Optical Sectioning Microscopy Family

Transcription

1 Microscopy from Carl Zeiss Optical Sectioning Microscopy Family The most comprehensive family of techniques. Discover the right microscope solution for your research.

2 Life is 3D! All biological specimens from the thinnest cell monolayers to large whole organisms inherently have some level of thickness. Traditional microscopes have a depth of field, meaning a focal plane at a focal depth where the image is sharply focused to allow the best image quality. Parts of the specimen above or below this depth of field are described as out of focus but also form part of the acquired image. Historically, the only way to get a clear sharp image of a thicker sample was to physically cut thin sections of the sample, a technique which was both time-consuming, likely to introduce artefacts, and not conducive to imaging of living samples. During the last few decades a large number of techniques have been developed that allow the contributions from these out of focus regions to be removed optically, leaving a clear image of the sample in the focal plane without having to physically harm the sample. This family of techniques is most commonly referred to as optical sectioning microscopy. Carl Zeiss has always pioneered advances in the field and continues to do so. Today, by introducing nextgeneration techniques, Carl Zeiss has the largest family of optical sectioning microscopy techniques. This portfolio ensures tailored system solutions for your application. This leads to the question: Which technique is best for my science? This brochure will give an overview of all of these optical sectioning techniques and point out their comparative strengths. Cover and back page image: Cobea (pollen grain) Z-stack-rendered using surface rendering for the lower half, a cut plane for the optical section of interest, and light transparency rendering for the upper half. Back = depth-coded transparency rendering

3 Total Internal Reflection Laser TIRF 3 4 Widefield Deconvolution Cell Observer 6 Structured Illumination ApoTome 8 Spinning Disk Cell Observer SD 10 Line Scanning LSM 5 LIVE 12 Single Point Laser Scanning LSM 700 LSM Multiphoton LSM 710 NLO / LSM 7 MP 18 System feature summaries Technique comparisons 20 22

4 Types of Optical Sectioning Microscopy Total Internal Reflection Widefield Deconvolution Structured Illumination Total Internal Reflection Fluorescence (TIRF) microscopy uses a specially modified excitation beam path to create an evanescent wave that only penetrates approximately 100 nm into the sample immediately adjacent to the coverslip. This technique does have the restriction that only this single optical section can be imaged but this technique is extremely sensitive and offers the highest background discrimination. Widefield fluorescence microscopes acquire both the in-focus and out of focus contributions from the sample. This makes them one of the most sensitive systems for live cell imaging be cause no emitted light is thrown away. The majority of the out of focus light is derived from the point spread function (PSF). A precise model for the PSF can be used to deconvolve image stacks. It is both a very efficient and cost-effective approach to generate 3D data. Deconvolution can also be used to enhance other optical sectioning techniques outlined in this brochure. The ApoTome system uses a tech nique called structured illumination to project a grid into the focal plane of a widefield fluorescent microscope. The grid is precisely moved and several images are acquired at each focal plane, each with the grid in a slightly different location. Since parts of the sample in focus vary more significantly than those out of focus, it is possible to automatically generate an optical section free of out of focus blur. Whilst the time to acquire is a little slower than a deconvolution only approach this technique offers greater levels of out of focus discrimination. 2

5 Confocal Confocal means having the same focus. Confocal microscopy refers to the technique of focusing the excitation light into parts of the focal plane and then focusing the returning emission though precisely positioned apertures which physically reject light coming from out of focus regions of the sample. The area(s) in which the light is focused is rapidly moved across the sample to form the full image. Lasers are used in order to produce the highly focused light necessary for this family of techniques. These last two points mean that confocal microscopes are most commonly referred to as Laser Scanning Microscopes (LSMs). A number of different variations of confocal microscopy are available. Spinning Disk Line Scanning Single Point Laser Scanning Multiphoton Spinning disk microscopes pro ject hundreds of circular apertures simultaneously into the sample and collect the returning emission though the same apertures. The beams are not actually scanned but produced by spinning the disk rapidly to illuminate the whole field of view. Out of focus light is almost entirely rejected by the disk, meaning the acquired image is already optically sec tioned wit hout the need for software processing. Spinning disks sys tems are a form of parallel confocal, meaning they work on multiple areas of the sample at the same time to trade a little resolution for a lot of speed. Line scanners like the LSM 5 LIVE are also parallel LSMs like spinning disk microscopes. Instead of projecting hundreds of spots, line scanners project a single line of illumination into the sample which is swept across the sample. The returning emission is filtered through a slit aperture to reject out of focus light before being detected on a line detector. The line scanners are more complex systems than the spinning disk systems, but offer slightly better discrimination of out of focus light and additional flexibility in scan modes. For low pixel dimension images line scanners offer the highest possible frame rate. The most widely used form of optical sectioning microscope today is the spot scanner. A single spot is focused into the sample and very rapidly moved across the field of view. The re turning emission is filtered through a pinhole aperture to reject the out of focus light and the remaining light detected by Photon Multiplier Tubes (PMTs) to form an image. The single spot approach is slower than parallel scanning techniques but offers great flexibility in terms of image size and imaging techniques, as well as offering the highest optical discrimination of all routine optical sectioning techniques. Multiphoton microscopes (MPMs), also commonly called Two Photon (2P) or Non Linear Optics (NLO), also rapidly scan a single beam across the sample and are a form of LSM. Multiphoton microscopes utilize a pulsed infrared laser to focus light into the sample. In the focal plane the photon density reaches a point where two or more photons can excite a fluorophore in a similar way to a single photon with half the wavelength. The pulsed light is too weak out side of the focal plane to create emission, and so no pinhole is necessary to reject out of focus light. Because there is no need for a pinhole, NLO systems are technically not confocal. 3

6 Laser TIRF 3 The Laser TIRF 3 enables extremely sensitive measurements at the cell/coverslip border with unparalleled rejection of out of focus light to enable interaction studies that are simply impossible any other way. Total Internal Reflection Imaging at the Boundaries of Cell Science Configuration: TIRF can provide the field of Life Sciences with new knowl edge about the molecular mechanisms at the boundary to the cell interior. This high-speed, high-contrast imaging technique has allowed the study of even the fastest membrane processes with very weak signals. Microscopes Inverted: Axio Observer.Z1 or D1 Excitation options Any combination of lasers: 405 nm (50 mw), 488 nm (20 mw and 100 mw), 532 nm (75 mw / 20 mw) or 561 nm, (40 mw / 20 mw), 635 nm (30 mw) DG4, HBO, or Colibri Options Cameras: AxioCam HRm, MRm or HSm EMCCD (QuantEM from Photometrics) Manual and motorized TIRF sliders Software AxioVision, and options, e.g. Measurements, FRET, Fast Acquisition, Physiology, HDR, Colocalization, etc. 4

7 B16/F1 melanoma cells (mouse) TIRF illumination Blue: CFP-actin. Excitation 458 nm, Green: dsred-clathrin Light Chain A. Excitation 514 nm Plan-Fluar 100x/1.45 oil Oberbanscheidt, van den Boom, Bähler, Institute of General Zoology and Genetics, University of Münster, Germany B16/F1 melanoma cells (mouse) Multi-color TIRF in combination with DIC Green: CFP-actin. Excitation 458 nm, red: YFP-myrpalm. Excitation 514 nm Plan-Fluar 100x/1.45 oil Oberbanscheidt, van den Boom, Bähler, Institute of General Zoology and Genetics, University of Münster, Germany Benefits: Applications: Fast acquisition observe dynamic processes as higher laser powers, more sensitive cameras, and efficient filters lead to very short exposure times. Measure cell adhesion sites with crystal-clear clarity and resolution. High speed multi-color imaging highly efficient multiline filters mean that only the laser lines and piezo controlled TIRF angle needs to be changed between colors. Easily reproducible angle setting adapting the wavelength and TIRF angle due to a revolutionary new TIRF slider mechanism with 0.1 degree accuracy. Image vesicles undergoing endocytosis or exocytosis with ultimate speed. Perform single molecule imaging with ultimate background suppression for extreme contrast. Observe cell-to-cell interactions with ultimate sensitivity and precision. High image quality specially designed filter sets for TIRF and a new apochromatic optics TIRF slider allow outstanding contrast and precision. Single molecule detection sensitivity due to maximum sensitivity beampath and no excitation of out of focus light. 5

8 Cell Observer and Deconvolution Modern life science research calls for a powerful imaging system that can be used flexibly, is easy to operate, and is equipped with a whole host of functionalities. A convincing example of such a system is Cell Observer from Carl Zeiss. Widefield Deconvolution Platform for Live Cell Imaging Configuration: Whilst offering the best components for the task, care has been taken to ensure seamless integration of all components. This allows imaging techniques from simple time lapse to complex heterogeneous experiments with the multidimensional Smart Experiments functionality. Reliable long-term imaging as well as high-speed imaging using streaming and precise trigger synchronization are all available within one single platform. This platform can be built upon when adding other techniques such as a spinning disk confocal unit or the ApoTome sectioning device. Microscopes Upright: Axio Imager, Axio Examiner Inverted: Axio Observer Excitation options Various HBO or Xenon arc lamps; fast switchable Xenon lamp, innovative Colibri LED light source. Hardware options Fast piezo focusing, filter wheels, shutters, scanning stages, flexible incubation setups, high-resolution and sensitive CCD cameras, EM-CCD cameras for ultimate sensitivity. Dual Camera option for maximum speed and efficiency. Software AxioVision, and options, e.g. Multidimensional Acquisition, Fast Acquisition, Physiology, 3D Deconvolution, Inside4D (rendering), 3D Measurements, Assaybuilder (high content analyses) Colocalization, etc. 6

9 Four dictyostelium cells shortly after spore germination in the vegetative stage, stably transfected with a GFP construct. The pseudopodia growth that is typical for this stage is clearly visible. Cells: Dr. Ralph Gräf, University of Potsdam, Germany HeLa cell transfected with reggie-1/fl otillin-2-egfp (green). Mitochondrial staining with MitoTracker Red. Time lapse of 21 z-planes in two channels, every 3.5 seconds. Z-stack, deconvolved using measured PSFs and rendered using Inside4D with a mixture of isosurface and volume rendering. Benefits: Applications: Fast and efficient acquisition observe the faintest signals in live cells at high speed using LEDs as excitation light source with the least phototoxicity. Live cell imaging with the whole range of available fluorescent proteins with extreme sensitivity. Perfect integration unique workflow combining fast acquisition, 3D deconvolution and rendering, and various measurement methods save time. Advanced technology TTL-controlled hardware synchronization, innovative dual camera option prevents time delay between channels. Broad Scope of applications from long-term time-lapse imaging to superfast 3D acquisition with just one instrument. Quantitative imaging of intracellular trafficking events at high speed. Perform simultaneous quantitative ion or FRET imaging with dual camera acquisition. Capture morphology changes in high resolution using deconvolution microscopy. Quantify cell cycle studies with long-term time-lapse microscopy. Platform for further 3D techniques add structured Illumination or a spinning disk unit for efficient 3D timelapse microscopy without losing any functionality. 7

10 ApoTome ApoTome introduces optical sectioning capabilities to your widefield imaging system simply by inserting the ApoTome slider into the field stop position. Using structured illumination, out of focus blur is efficiently removed, resulting in brilliant optical sections of highest contrast and optimal resolution. Structured Illumination Suddenly Everything Looks Different Configuration: ApoTome achieves optical sectioning by using the principle of structured illumination microscopy (SIM). A grid pattern is projected onto the focal plane and an optical section is calculated online from three images with different grid positions. Thus, ApoTome maintains the ease of use of your widefield system but efficiently removes out of focus blur even in thicker samples, making ApoTome the system of choice for easy multicolor imaging of tissue sections. Microscopes Upright: Axio Imager.D1 or Z1 Inverted: Axio Observer.D1 or Z1 Excitation options HBO, HXP 120, or Colibri Options Different grids for optimal use of different objectives; Decon volution of SIM images for optimal resolution. Software AxioVision, and options, e.g. Multidimensional Acquisition, Fast Acquisition, Physiology, 3D Deconvolution, Inside4D (rendering), 3D Measurements, Assaybuilder (high content analyses) Colocalization, etc. 8

11 Rat hippocampus, triple fluorescence, maximum intensity projection of a 3D image stack, Plan APOCHROMAT 63x/1.4. Right side = Pure widefield and Left side = Processed ApoTome image. E. Fuchs and S. Bauch, DPZ Goettingen, Germany Platynereis dumerili, triple fluorescence (DAPI, phalloidin-alexa488, alpha-tubulin-alexa555), maximum intensity projection of a 3D image stack, plan Apochromat 20x/0.8. Top right corner = Pure widefield and Lower left corner = Processed ApoTome, V. Wilkens, University of Osnabrueck, Germany Benefits: Applications: Simple integration add optical sectioning to your widefield system by simply inserting the ApoTome slider into the field stop position. Neuronal branching in brain sections captured in brilliant multicolor images. Optimal performance with all objectives three different grids allow optimal sectioning over the full range of objectives and magnifications. Highest flexibility use white light sources for free choice of fluorescent dyes or the Colibri LED light source for unmatched contrast. Studying tissue formation in embryo development with low bleaching. Capture subcellular structures in three dimensions with an easy-to-use setup and short acquisition times. Analyze fluorescent markers in pathological sections with out out of focus blur. Fast acquisition optical sections are calculated online. Advanced software control raw data acquisition allows offline processing of SIM images and combination with specialized deconvolution algorithms for optimal contrast and resolution. 9

12 Cell Observer SD Cell Observer SD combines the speed, modularity, and reliability of the ZEISS Cell Observer with the fast confocal image acquisition of the Yokogawa CSU-X in a seamlessly integrated modular system for fast confocal live cell imaging. Spinning Disk An Unbeatable Team Configuration: The ZEISS Cell Observer SD offers precise hardware con trol in the millisecond range and fastest image acquisition due to direct image streaming. The Yokogawa CSUX1 is a trusted instrument for fast confocal imaging. The combination of these two systems, both groundbreaking in their technology, yields a perfectly integrated system of unmatched functionality. Microscopes Axio Observer.Z1 including incubation Excitation options Up to four laser lines: 405 / 488 / 532 or 561 / 635 nm Different power options are available. Options Fast emission filter wheel Dual camera acquisition Scaleable and lasersafe incubation solutions EMCCD cameras for best detection sensitivity Software AxioVision, and options, e.g. Multidimensional Acquisition, Fast Acquisition, Physiology, 3D Deconvolution, Inside4D (rendering), 3D Measurements, Assaybuilder (high content analyses) Colocalization, etc. 10

13 Depth coded maximum intensity projection of a GFAP-labelled rat brain section (depth scale in μm), 63x/1.4 PlanAPOCHROMAT Mitosis in LLC-PK1 cells, tubulin-egfp + Histon 2b-mCherry, 63x/1.4 PlanAPOCHROMAT Benefits: Applications: Fast confocal acquisition observe dynamic cellular pro cesses with outstanding frame rates using streaming technology. Image subcellular trafficking in 3D over time with maximum acquisition speed. Visualize cytoskeletal dynamics with highest sensitivity. Perfect integration all components are seamlessly integrated and are controlled with millisecond precision. Modular system to suit your needs choose from the wide variety of peripheral components to build a system matching your applicational needs. Functional imaging of cellular signal transduction with high temporal resolution. Long-term confocal live cell imaging with lowest phototoxicity. Reliable service and support a complete system from a renowned partner with service you can rely on. 11

14 LSM 5 LIVE The LSM 5 LIVE is a revolutionary parallel scanning confocal design that offers new levels of speed and sensitivity to capture life in motion like never before. The line scanning approach allows image dimensions and parameters to be perfectly balanced for every sample. Line Scanning New Visions of Life Configuration: The versatile combination of sensitivity and speed available on the LSM 5 LIVE allows imaging phenomena that were previously impossible to observe. Be it imaging calcium transients at hundreds of frames per second or fine structural changes at several Z-stacks per second, the LSM 5 LIVE is extremely versatile and efficient. Microscopes Upright: Axio Imager.Z1, M1 Axio Examiner.Z1 Inverted: Axio Observer.Z1 Excitation options Combination of four lasers: 405 or 440, 488, 532 or 561, 635 nm HBO, HXP 120, or Colibri for widefield Scanning modules LSM 5 LIVE scanning module with one or two high-sensitivity CCD line detectors. Option: additional LSM DuoScan unit (for ROI photomanipulation with laser). Software ZEN 2009 Options: Physiology, Image VisArt plus, 3D for LSM, Multiple Time Series, VBA Macro recorder / editor, FRET plus, FRAP, Visual Macro Editor 12

15 Zebrafish embryo. Erythrocytes (dsred : Red) and endothelial cells (egfp : Green) 2 Channels captured simultaneously at 33 frames / second. Specimen: Dr. S. Hermanson and Dr. S. C. Ekke, University of Minnesota, USA Rat, ventricular cardio myocytes (Fluo-4, Ca2+ ion indicator) and mean ROI plot captured at 300 frames / second. Specimen: Dr. J. Lederer, Medical Biotechnology Center, Baltimore, USA Benefits: Applications: Ultrafast acquisition observe dynamic processes with millisecond time resolution. Capture fast moving structures such as blood cells or vesicles with fast 4-dimensional image acquisition. Imaging precision adjustable confocal apertures for outstanding 3-dimensional resolution. Confocal high-speed imaging in structural and developmental biology even with low magnification and dipping lenses. Sensitivity and flexibility AchroGate main beamsplitter with 95% efficiency, SK filters for best transmission, and double bandpasses for fast four-label acquisition. Effortless data handling of gigabytes of 4D data with real-time electronics and 100 MByte/sec computing capacity. Perform physiology measurements perfectly matched to the biological timescales. Visualize fine cell structures at high resolution with the necessary sensitivity. Study subcellular dynamics by pixel-precise bleaching, photoactivation, and conversion with the LSM 5 LIVE DuoScan. 13

16 LSM 700 The LSM 700 is a member of the seventh generation of confocal microscopes from Carl Zeiss a family characterized by a wealth of genuinely innovative ideas and technologies. The LSM 700 concept combines convincing Carl Zeiss quality and exceptional ease of operation. Single Point Laser Scanning Fresh Impetus to Your Research Configuration: Utilizing the same top-grade system components and software across the family ensure superior performance and an uncompromising approach to image quality, result ing in an excellent price/performance ratio. A novel new beampath ensures outstanding laser suppression and maximum emission collection for breathtaking images. The new scanning module incorporates a radically new way to capture fluorescence. Sophisticated tasks, like spectral acquisition, are achieved very easily; all with excellent image quality and rapid scan speed. Microscopes Upright: Axio Imager.Z1, M1 Axio Examiner.Z1 Axio Scope.A1 Inverted: Axio Observer.Z1 Excitation options Up to four PTC laser ports: 405 or 445, 488, 555 and 639 nm HBO, HXP 120, or Colibri for widefield Scanning modules LSM 700 scanning module with one or two PMTs with spectral flexibility. Option: T-PMT Software ZEN 2009 Options: Physiology, Image VisArt plus, DCV, 3D for LSM, Multiple Time Series, VBA Macro recorder / editor, FRET plus, FRAP, Visual Macro Editor 14

17 50um section of Mouse stomach. Blue: Plasma membrane, stained with Alexa Fluor 350 WGA, Red: Actin, stained with Alexa Fluor 488 Phalloidin, Green: Nuclei, stained with Sytox Green - Objective: Plan-Apochromat 20x / 0.8, Zoom 1 Platynereis dumerilii (Bristle Worm) Red: Nervous system, stained with Alexa 555, Green: Muscles: Alexa 488, Blue: Nuclei, stained with Draq5TM, Grey: Transmitted light. Objective: EC Plan-Neofluar 20x / 0.5, Zoom 1.5 Benefits: Applications: Easy operation via the ZEN software and the Smart Setup function allow the LSM 700 to be used intuitively after short training times. Up to four color signals can be acquired in a (quasi-)simultaneous mode for multifluorescence colocalization analyses in cell and molecular biology. Space saving the compact setup fits onto many standard worktables, with low noise and heat output. Long, in depth 3D and 4D timelapse studies in developmental biology and in vivo examinations. Integration of up to four solid-state lasers and the Laser Life Extender technology make the LSM 700 a futureoriented investment. Live Cell Imaging, Ion Imaging in physiology, FRET, FRAP, and FLIP analysis of molecules, including photoactivation and photoconversion. Revolutionary beampath design including new spectrally flexible secondary beamsplitter ensures maximum sensitivity and innovative spectral detection principle. Sensitivity, accuracy, and flexibility of scan modes allow single molecule analysis with innovative RICS software. 15

18 LSM 710 The new LSM 710 is the logical evolution of the successful LSM series from Carl Zeiss. The LSM 710 combines and surpasses the advantages and capabilities of all existing confocal systems. Single Point Laser Scanning The Power of Sensitivity Configuration: Enhanced sensitivity and reduced background noise are prerequisites for every demanding application in laserscanning microscopy. The excellent sensitivity of the LSM 710 comes with outstanding suppression of noise and excitation laser light to deliver the best image results and protection of the sample. Working closely together with leading scientists worldwide, we have created an instrument that reflects the latest ideas and technological possibilities that will accompany you in your research experiments. Microscopes Upright: Axio Imager.Z1, M1 Axio Examiner.Z1 Inverted: Axio Observer.Z1 Excitation options Eight PTC laser ports for 10+ lines: 405 (ps/cw), 440 (ps/ cw), 458, 488, 514, 543 or 561, 594, 635 nm, IR tunable Ti:Sa HBO, HXP 120, or Colibri for widefield Scanning modules LSM 710 scanning module with 2, 3 or 34 spectral PMT detectors. Option: T-PMT and ConfoCor 3 for FCS and imaging with high sensitivity APD detectors. Software ZEN 2009 Options: Physiology, Image VisArt plus, DCV, 3D for LSM, Multiple Time Series, VBA Macro recorder / editor, FRET plus, FRAP, Visual Macro Editor 16

19 Perfect 3D results in superior samples resulting from perfect adjustments; Submandibular gland of a mouse, labelled with ZO-1 antibody and YFP. S. Sheu, MCB, Harvard University, Boston, USA Mouse Embryo, labelled with Blue: DAPI, Green: H2B-GFP, Red:myr-TdTomato. Dr. S. Shankar, University of Oxford, UK Benefits: Applications: Highly sensitive QUASAR detector with lowest noise possible and digital gain control. Multifluorescence imaging in cell and molecular biology, including colocalization analysis and simultaneous spectral imaging of fluorescent proteins. TwinGate exchangeable main beam splitter for 50 laser combinations and outstanding laser light suppression. Spectral recycling loop for low-loss spectral separation; beam guides for unlimited flexibility of detection bands. Upgradeable PTC laser ports (V, VIS, IR) for outstanding excitation flexibility. 3D examinations in vivo and in developmental biology examinations, 3D in-depth imaging and 405 or multi photon uncaging in neurobiology. Live Cell Imaging, Ion Imaging in physiology, FRET, FRAP, and FLIP analysis of molecules, including photoactivation and photoconversion. Single molecule analysis with innovative RICS software, FLIM with pulsed lasers in the 405/440 or IR range, aniso tropy imaging in molecular biology. 17

20 LSM 710 NLO / LSM 7 MP LSM 710 NLO and LSM 7 MP are perfect for sensitive in-depth analysis of live specimens, including whole organisms. Both systems are outstanding in their unmatched sensitivity: Precise settings of the femtosecond laser and most effective non-descanned detection provide efficient excitation and imaging deep within tissue. Bleaching and manipulation experiments will succeed with pinpoint accuracy due to 3D-defined excitation. Multiphoton Looking at the Perfection of Life Configuration: Full choice between the most versatile or the most dedicated system that your applications demand ensure the system will perfectly match your experiment: Both provide multiphoton imaging technology without compromises easy-to-use stable femtosecond lasers with or without precompensation unit for efficient excitation, newest detection technology for the very best imaging results. Microscopes Upright: Axio Examiner.Z1 Axio Imager.Z1 Inverted: Axio Observer.Z1 Excitation options Any combination of a femtosecond laser with: 405, 440, 458, 488, 514 / 543 or 561 / 594, 635 nm HXP 120/ XCite 120, HBO, or Colibri Options, e.g. scanning modules LSM 710 NLO scanning module with 2, 3 or 34 detection channels LSM 7 MP with NDD detection Options: Up to 5 NDD detectors in transmission and reflection (depending on microscope). Software ZEN 2009, same options as LSM

21 Principal (projection) neurons in cortex of a transgenic mouse expressing YFP A region of the caudal part of the cortex was imaged from the transverse cut surface to a depth of 260 microns using multiphoton excitation (930 nm). Specimen: S. Turney, Harvard University, USA Brine shrimp stained with eosin. Depth coded transperency projection of 120 single images. Benefits: Applications: Deep tissue imaging to observe labeled structures up to 1 mm inside tissue. Developmental studies over long time periods with minimized damage to the sample. 3D-defined manipulation for volume defined manipulation with low or high power. Combine electrophysiology and imaging to get best images from patched cells within a tissue slice or animal. First-class sensitivity with new detector technology for highest signal/noise ratio keep finest structures visible. Precise results from uncaging experiments due to exactly defined uncaged volumes. Minimal bleaching and phototoxicity effects restricted to the focus only, ideal for long-term imaging of large 3D specimens. 19

22 ApoTome Cell Observer SD LSM 5 Live LSM LSM 710 NLO / LSM 7 MP Cell Observer 1 2 LSM 710 Laser TIRF 3 Technical Specifications Detector type Camera, including AxioCam MRm, HRm, HS and EMCCD Line detector Photo Multiplier Tube = PMT Non Descanned Detector = NDD Excitation source Visible lasers Widefield sources, e.g. HBO, Colibri Femtosecond pulsed IR laser Spectral capability Sequential imaging and channel unmixing Sequential spectral detection Simultaneous spectral detection Software AvioVision ZEN Photomanipulation capability Stripe bleach Sequential arbitrary regions of interest (ROI) with scanners Optional simultaneous ROI scan with DUO or DuoScan systems Number of detection channels or 34

23 Applications and Techniques All of the systems in this brochure can perform the following techniques and applications: 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). Multifluorescence Imaging Image generation from specimens with multiple fluorescence labels: crosstalk-free detection and presentation of multiple fluorophores. Measurement Imaging Measurement and imaging of ion concentrations by means of selective fluorescence markers: image acquisition, measurement and calibration of ion concentrations. 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). Transmitted-Light Microscopy Image generation in transmitted light: brightfield, phase and DIC images in the LSM mode with optional transmitted-light detector or via an internal channel on the LSM 5 LIVE. 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. 21

24 Relative Performances of the Techniques Most optical sectioning microscopes are very flexible and can be used to image a wide range of sample types. The exact performance of a system for a particular application is very hard to describe in terms of exact numbers. For example: You cannot definitively say A multiphoton system will always have three times the resolution of an ApoTome system when imaging 100 µm into a sample. If the sample is very sparse and cleared the images may be comparable, whereas in dense highly scattering tissue a multiphoton system may be able to image at depths where the ApoTome is unable to acquire any image. With that in mind the tables presented over the next two pages are included to give a very rough indication of how the different optical sectioning microscopes may perform relative to each other. Exact imaging conditions vary from sample to sample, and so the relative performance can also vary significantly. The graph on this page indicates roughly the relative performances of the techniques from a purely technical level. There is no linearity to the scale used, i.e. a 4-point score is better than a 2-point score but not necessarily by twice as much. Performance Specification Technique TIRF Example ZEISS System Depth Penetration Laser TIRF 3 X Widefield Deconvolution Cell Observer Structured ApoTome Illumination Spinning Disk Cell Observer SD Line Scanning LSM 5 Live LSM 700 Single Point 22 LSM LSM 710 Multiphoton LSM 710 NLO and LSM 7 MP Maximum Speed Out of Focus Discrimination Lateral Resolution Axial Resolution Spectral Flexibility Simplicity

25 Performance in Typical Experiments The table below shows how the technical performance of a system effects its relative suitability for performing some example real world experiment types. As in the last table the relative scores shown below could change according to exact conditions but should serve as an initial guideline as to the relative strengths and weaknesses of the optical sectioning techniques. We encourage you to talk to your local Carl Zeiss representative to determine which type of system is really best for your samples, your application and ultimately your science. Example experiments Technique TIRF Example ZEISS System High Speed Time lapse for Vesicle tracking Colocalisation studies in 30 µm tissue sections Structural imaging of Zebrafish or Drosophila Simultaneous FRET imaging Very high speed ion imaging, e.g. Calcium imaging Spectral flexibility and removal of Auto fluorescence Neuronal imaging deep in live brain tissue Photoactivation and/or bleaching, e.g. FRAP Laser TIRF 3 X Widefield Deconvolution 4D imaging of Mitotic Spindle division X X Cell Observer Structured ApoTome Illumination Spinning Disk Cell Observer SD Line Scanning LSM 5 Live X X X X X X X X X LSM 700 Single Point LSM LSM 710 Multiphoton LSM 710 NLO and LSM 7 MP 23

26 Other Brochures of Interest Fore more detailed information on the products described in this brochure, please refer to the following brochures: Laser TIRF 3: Cell Observer: ApoTome: Cell Observer SD: LSM 5 LIVE: LSM 700: LSM 710: LSM 7 MP: Link for brochures downloads:

27 /e printed BioSciences Jena Location Phone : Telefax : micro@zeiss.de Subject to change. Printed on environmentally friendly paper, bleached without the use of chlorine. Carl Zeiss MicroImaging GmbH Jena, Germany