Real-Time Imaging Solutions. xcellence, cell^tirf and cell^frap Life Sciences

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1 Real-Time Imaging Solutions xcellence, cell^tirf and cell^frap Life Sciences The Olympus Real-time Imaging Solutions for Life Sciences

2 Real-Time cell Imaging The three color channels of widefield (top) and TIRF (bottom) images: red (phalloidin): actin; green (FITC): tubilin, blue (Alexa405): SHC. Courtesy by M. Faretta, Eup. Inst. Oncology (IEO-IFOM), Milan, Italy Fast, Better and more Flexible The Olympus real-time imaging Solutions Olympus always strives to provide systems that don t just do the job; they do it faster, better and with greater flexibility. As a result, the xcellence rt system and dedicated hardware solutions like cell^tirf and cell^frap covers an amazing array of advanced fluorescence techniques, presenting users with an easy-to use interface and peerless options. SOLID FOUNDATION - XCELLENCE RT The Olympus XCELLENCE RT imaging platform is the perfect starting point for advanced fluorescence imaging at an unprecedented level of speed and accuracy. WITHOUT COMPROMISE - CELL^TIRF With 4 independent light paths the cell^tirf system provides true online multi-line detection with exactly the same penetration depth for each wavelength. OPTICALLY ADVANCED - CELL^FRAP The cell^frap system provides a versatile platform for all your study needs, from basic bleaching to advanced photocontrol protocols.

3 XCELLENCE Solid Foundation - Xcellence RT The Olympus xcellence platform takes microscopy to a new level with peerless speed and precision. The xcellence platform integrates the finest components with unique control technology and the latest imaging techniques. Experiment Manager With full control of the microscope and all the components, the Olympus xcellence rt Experiment Manager provides a universal experiment planning and execution tool enabling even the most complex tasks to be completed with ease. In the graphical interface the most complex experiments are set up by intuitive drag-and-drop assembly of command icons for acquisition, z- stacks, time-lapse and hardware control. Once set, the complete experiment plan is visible at a glance and is stored with the data in the archival database. As a result, key imaging tasks can be repeated with great ease simply by loading the experiment plan. High Speed and Intelligent Control For complex multi-device experiments xcellence rt uses a Real-Time Controller board offering µsec timing precision. Additionally the board controls multiple processes (as set up in the Experiment Manager) in parallel. This guarantees smooth hardware interplay and efficiency, resulting in shorter light exposure to your sample and high speed acquisition of your images. This control system ensures that experiment flow and timing precision are maintained while changing hardware settings. For example, filter wheels, attenuators and shutters are optimally synchronized with the camera, as well as the optional piezo z-drives and other devices. By using an independent plug-in CPU board, xcellence rt does not rely on the imaging computer to control the hardware which is essential for high speed imaging. The xcellence rt workstation comes with a 7 port TTL panel in order to add to the flexibility of controlling a number of peripheral components. Image Acquisition A broad range of monochrome CCD and EMCCD cameras are supported by the software. What is more, the xcellence rt platform has been developed to leverage huge capabilities of each camera, enabling features such as pixel-binning and partial readout area. The software also supports advanced features such as overlapping readout and EM gain control as well as real gain control. Additionally for acquisition for ratio images (Ca ratio or FRET) the DualView and DualCam TM options are supported. Fast switching and highly stabilized light source Fast switching between excitation wavelengths is crucial for many applications, e.g., dual excitation ratio measurements or fast multi-color time-lapse experiments. Therefore the xcellence rt system comes with an all-in-one illumination system, MT20. This excitation system includes a light shutter, attenuation and filter switching (58 ms). The integrated attenuators switch between the intensity (14 positions) at even faster speeds than the filter wheel. Furthermore, the built-in shutters have exceptional on/off intervals of only 1 ms eliminating photo-bleaching when not acquiring an image; a fundamental prerequisite for imaging living specimens. In combination with the parallel processing of the Real Time board it provides high speed functionality. Time-Lapse imaging: Fura2-labelled HeLa cells stimulated with ATP. Top left: dual excitation image, others: false color ratio images revealing increasing calcium concentration.

4 Real-Time cell Imaging Image analysis and Handling Measurements The xcellence rt software program offers a unique measurement environment suitable for all type of measurements in 2D to 6D dimensions. Results can be presented as graphs and the statistics can be represented immediately or as 3D rendered images. Spectral unmixing: enhanced color resolution Spectral overlap of the excitation and emission characteristics of certain fluorescent dyes often prevents their combination within one sample. Linear spectral unmixing ascertains the contribution of each fluorochrome to the total signal and, by chromatic redistribution of the intensity, restores a clear signal for each color channel, undisturbed by the emission from other fluorochromes. Bleaching Correction The xcellence rt software can be used to apply bleaching correction algorithms across time series to ensure that during long term imaging, the effect of any unwanted bleaching is corrected. Programming Besides the graphical user interface of the Experiment Manager additional specific tasks can be programmed using the programming language Imaging C. This includes a programming library with unrestricted access to the wide range of image acquisition and processing functions. Imaging C consists of a compiler, an interpreter, a macro recorder, a multiple-document (MDI) text editor, a symbol and function browser, and an interactive debugger with single step processing, conditional breakpoints as well as a browser for variables and constants. ZDC2 Spectral unmixing: GFP-labeled vesicles and YFP-labeled membranes proteins: YFP displayed in red for clarity. Left: original image; right: after spectral unmixinig. Courtesy of Y. Okada, Gra. School of Medicine, Univ. Tokyo, Japan. Fluorescence analysis and kinetics More complex analysis routines required for processes such as ion imaging are included. Online intensity kinetics, co-localization, ratio and ΔF/F analyses. Results are provided as false-color images, (online) graphs of regions of interest and data sheets. Deconvolution The deconvolution algorithms included in the xcellence rt software, provide fast and accurate deconvoluted images whilst ensuring outstanding usability. The xcellence rt software include no neighbor, nearest neighbour and Wiener algorithms as well as the optional Fast Maximum Likelihood Estimation for the various widefield of confocal microscopic techniques. Comprehensive constraint iterative deconvolution: for crisp and detailed results. Image provided courtesy by Peter Gutierrez in Prof. Dr. Alex Hajnal s laboratory at the Institute of Zoology, University of Zürich, Switzerland Tracking Tracking of cells, cell organelles and molecules provide key insights into the dynamics of biology and biochemistry. The tracking feature detects moving objects either automatically or manually and the results are presented as object tracks, accompanied by charts and histograms of parameters such as velocity, direction, path length and total distance. During long time-lapse observations, temperature changes can cause focal drift, resulting in a change of focal plane. This can be a serious problem, for example in TIRF microscopy, because of the high z-resolution required (100 nm). The Olympus ZDC2 unit (Z Drift Compensation) automatically corrects for thermal drift prior to imaging. (Note: ZDC2 not simultaneous with cell^frap) High Speed Z Acquisition The xcellence rt system supports the Nosepiece PIFOC, which provide piezo control over the focal depth enabling high speed acquisition of z-stack and with the 10nm resolution. Alternative illumination Systems To provide more flexibility, the xcellence rt systems can integrate with specialized light-sources such as the multi-color LED illumination system precisexcite and the Monochromator Polychrom5000. These systems offer switching times of less than 5 ms and are controlled with µs precision by the xcellence rt realtime controller board. Other light sources can be used as well e.g. in combination with Olympus fast filter wheels, fast turrets for IX microscopes and fast shutters. Simultaneous Emission Detection The DV2 Dual-View Micro-Imager beam-splitting device can be added to allow the simultaneous acquisition of two chromatically separated fluorescence images. With the Quad-View system, up to 4 colors can be separated and simultaneously acquired with the xcellence rt software. For simultaneous imaging of the full field of view the xcellence rt system is capable of synchronized acquisition with two cameras using the DC2 Dual-Cam TM.

5 cell^tirf Cell^TIRF - Without compromise Total internal reflection fluorescence microscopy (TIRF) has firmly established itself over the last few years as a key technique in the investigation of molecular interactions or near the cell surface. Olympus has been at the forefront of providing advanced TIRF solutions and the motorized multiline cell^tirf illuminator takes this to the next level with a series of peerless features. Motorized Multiline TIRF Illuminator Olympus cell^tirf offers 4 laser channels with independent, wavelength dependent beam paths. Each path has individual motorized angle control which enables simultaneous excitation and emission at the exact same penetration depth for true online multiline TIRF experiments. Highest Specification Objectives This superior multi-laser independent beam path technology, combined with the broadest selection of TIRF objectives available, ensures that cell^tirf delivers a new level of sensitivity and precision in multi-color TIRF. Olympus offers 6 dedicated TIRF objectives of exceptional optical quality, including the highest resolution available with a numerical aperture of 1.69 NA, as well as the highest magnification at 150x (1.45 NA). A choice of 14 compact, silent and long-life diode and DPSS lasers are available covering wavelengths from 405 to 640 nm. The lasers, available with powers of up to 150 mw, self configure within the xcellence rt software. In addition, shuttering and attenuation can be performed in real-time from the Experiment Manager. Newly available objectives offering increased numerical apertures of 1.49 at 100x and 60x magnification ensure exacting angle control and precise knowledge of the depth of TIRF penetration, which is highly advantageous for cell membrane studies. Exceptional mechanics along with these objectives mean that users can precisely and automatically adjust laser penetration with 1 nm accuracy at the click of a button.

6 Real-Time cell Imaging Intelligent & intuitive TIRF control The intuitive and intelligent system software ensures that with one button one can switch between critical angle, defined penetration depth and laser widefield illumination. Furthermore, fine adjustments are made via the mouse or keyboard, removing the need for screwdriver adjustments. Complete integration within the Experiment Manager software allows full flexibility in your experiments. HILO Due to the X,Y adjustable field-stops for each laser beam, the xcellence rt cell^tirf can be used as source of for highly inclined and laminated optical sheet (HILO) illumination. The laser beam is converted into a thin sheet that passes through the centre of the specimen at an oblique angle. As a result, HILO illuminates targets at depths of tens of microns and three-dimensional reconstructions can be generated within the software. Point FRAP Olympus cell^tirf has integrated point-frap optics for the first laser line. This enables the user to make FRAP and photo switching experiments simultaneously with TIRF and widefield imaging. Double labelling of f-actin cytosceleton (red) and the focal adhesion kinase (FAK, green).the TIRFM image reveals that the actin fibers are spanned between the FAK-containing adhesion structures. Courtesy of G. Pilarczyk, U. Joos, T. Biskup, O. Ernst, I. Westphal, Fraunhofer Institut für Biomedizinische Technik, Berlin; H. Kahl, Olympus Germany. TIRFM image of FITC-labelled actin and DyeMer605-labelled EPS8. Courtesy of M. Faretta, Eur. Inst. Oncology (IEO-IFOM), Milan, Italy.

7 cell^frap Optically Advanced - Cell^FRAP The OLYMPUS xcellence rt systems are designed with high-speed components, so that sample bleaching does not happen during long-time experiments. This is why the systems are the perfect starting point for conducting experiments that require accurate bleaching of specific parts of a sample. cell^frap is ideal for such sophisticated photo manipulation processes. Pattern Bleaching System The Olympus cell^ FRAP system is capable of bleaching a large number of points in a defined pattern at high speed. The cell^frap system uses an independent light path enabling simultaneous fluorescence imaging and bleaching. The cell^frap module can also integrate seamlessly with other advanced fluorescence imaging modules such as the cell^tirf. Full integration in the xcellence real time imaging system and the software control through the Experiment Manager gives the researchers all the flexibility in their experiments. Subcellular Laser Cutting The optics are also UV compatible, enabling the use of pulsed short wavelength lasers to cut cellular components such as growth cones and filopodia, as well as intracellular structures such as actin fibres, microtubules, ER or mitochondria. This opens the door for a new world of direct functional analysis. Intelligent scanning modes By scanning just the region of interest, the bleaching is up to 10x faster and multiple areas within the same frame can be bleached very quickly. Furthermore, the spot size can be adjusted easily with the smallest size restricted only by the refraction limit of the optics, enabling the bleaching of very small structures precisely and larger areas more quickly. Point and Shoot With cells being very dynamic environments, some bleaching protocols cannot necessarily be performed automatically. The xcellence rt cell^frap system enables the user to bleach a target by clicking on it in a live image using the mouse. Multiple points can be bleached and the resultant images can be captured during or immediately after the bleach.

8 System Details System Highlights xcellence Real-time high-speed controller for parallel high speed controls, high timing precision and data stream without jitter Unique graphical interface to set up and parameterize experiment by intuitive drag & drop assembly Integrated high speed illumination system with 8 position filter wheel (58ms filter switch) and 14 levels attenuation cell^tirf 4 Individually controlled inputs for simultaneous and wavelength optimized multi-color TIRF Integrated Point FRAP optics for 1st laser line allows diffraction limited photo activation and FRAP with TIRF Motorized control of TIRF angles for precise adjustment of incident angles and quick-set penetration depth cell^frap Precise fast multi-area bleaching by scanning only the ROI achieving bleaching rate up to 10x faster than standard methods Comes with its own dedicated laser system and light-path, allowing truly simultaneous imaging and photo-manipulation Precise µ second switching allows initial frames to be acquired as early as 150 µs after the FRAP experiment ends System Diagram illuminations systems Fast Shutter and Filterwheel Widefield illumination systems Two-port MT illumination coupling for IX2_MPITIR illumination combiners MT-CON Illumination coupling MT illumination coupling for IX2_MPITIR* illumination combiners IX2-SHA Motorised shutter U-FSHA Fast motorised shutter U-FWR Motorised reflected light filter wheel U-LH100HGAPO 100 W Mercury apo lamp housing (others possible) MT_FIB/P5 MT_FIB-WD/2m Single quartz Single quartz light fibre light fibre Pol 5000 Illumination MT20 system Illumination system CMR-IX2-MTA Flange to mount MT-CON-OG to IX2 microscope LH Illumination coupling Power supply unit POLY_ARC/XE Xenon arc burner, 150 W MT_ARC/XE Xenon arc burner, 150 W MT_ARC/HG Mercury xenon arc burner, 150 W cell^tirf 2L cell^tirf 4L Motorised TIRF-illuminator ADD_FLASH/C2 UV flash illumination system precisexcite LED illumination system CTR_TRIGOUT_5V TTL-to-5-V converter System control Cameras and observation equipment Stages DSU DSU-CAD C-mount adaptor IX2-DSU BX-DSU Disk-scanning unit Laser systems XCE-PC Imaging PC * CCD and EMCCD cameras: different models of different manufacturers IX2-SFR Cross stage with flexible handle FFWO-CMAD DSU Fast motorised observation filter wheel for DSU MT-CON-DSU cell^frap Controller cell^frap Scanning device FireWire or camera link board Camera trigger connection CMR-LAS D 405 nm, 60 mw diode laser CMR-LAS D 405 nm, 100 mw diode laser CMR-LAS nm, 100 mw solid state laser CMR-LAS nm, 50 mw solid state laser XCELLENCE rt Realtime controller board SW-XCELLENCE rt realtime software U-DPCAD Dual port tube with C-mounts U_FFWO Fast motorised observation filter wheel Dual-view microimager Quad-view microimager Beam splitting device U-CMAD3 C-mount adaptor Motorised stage IX2-UCB2 Microscope controller for IX81 IX81 Motorised inverted microscope or IX71 Inverted microscope Mirror turrets ADD_PFC or ADD_NPPFC/IX81/71 PIFOC objective or PIFOC nosepiece & controller CMR-LAS D 445 nm, 50 mw diode laser CMR-LAS D 473 nm, 25 mw diode laser CMR-LAS nm, 50 mw solid state laser CMR-LAS D 488 nm, 60 mw diode laser CMR-LAS nm, 150 mw solid state laser CMR-LAS nm, 50 mw solid state laser CMR-LAS nm, 50 mw solid state laser CMR-LAS nm, 50 mw solid state laser CMR-LAS nm, 100 mw solid state laser CMR-LAS nm, 150 mw solid state laser CMR-LAS nm, 50 mw solid state laser CMR-LAS D 640 nm, 100 mw diode laser STM U-TV1x-2 primary image video port tube IX2_FRFACA Fast motorised mirror unit turret IIX2-RFACA Motorised mirror unit turret IX2-RFAC Mirror unit turret Specifications are subject to change without any obligation on the part of the manufacturer. Soft Imaging System is a trademark of Olympus Soft Imaging Solutions GmbH Art. code: /2011 Johann-Krane-Weg Münster Germany Phone: Fax: info.osis@olympus-sis.com No. 2 Jurong East Street 21 #05-30, IMM Building, Singapore Singapore Phone: Fax: info.osis@olympus-sis.com