Olympus Fluoview FV1000 User's Manual
Contents
1. Dr Hiroshi Hama Dr Atsushi Miyawaki RIKEN Brain Science Institute Laboratory for Cell Function Dynamics Caged compound Bhcmoc Glutamate presented by Dr Toshiaki Furuta Department of Science Toho University Using multi stimulation software the user can configure continuous laser light stimulation of multiple points with simultaneous imaging which is effective for applications such as uncaging experiments involving laser light stimulation of several spines in neurons 18 Multi Dimensional Time Lapse The FV1000 can be used for ideal multi dimensional time lapse imaging during confocal observation using multi area time lapse software to control the motorized XY stage and focus compensation Equipped with motorized XY stage for repeated image acquisition from multiple points scattered across a wide area The system efficiently analyzes changes over time of cells in several different areas capturing large amounts of data during a single experiment to increase the efficiency of experiments Microplates can be used to run parallel experiments which significantly improves throughput for experiments that require long term observation Point 2 Glin aki GO E Focal Plane 4 Focal Plane 3 Focal Plane 2 Supports repeated image Focal Plane 1 acquisition from multiple areas in a single microplate well Point 4 M ulti P oint Time Lapse Software Point 6 apse Imaging The IX81 ZDC Zero Drift Compensa2. OLYMPUS i ces Mio coe Your Vision Our Future EV 4 07070 FLUOVIEW V4 World leading optics FLU O VIEW Always Evolving FLU O VIEW From FLUOVIEW More Advanced than Ever The Olympus FLUOVIEW FV1000 confocal laser scanning microscope delivers efficient and reliable performance together with the high resolution required for multi dimensional observation of cell and tissue morphology and precise molecular localization The FV1000 incorporates the industry s first dedicated laser light stimulation scanner to achieve simultaneous targeted laser stimulation and imaging for real time visualization of rapid cell responses The FV1000 also measures diffusion coefficients of intracellular molecules quantifying molecular kinetics Quite simply the FLUOVIEW FV1000 represents a new plateau bringing imaging to analysis Olympus continues to drive forward the development of FLUOVIEW microscopes using input from researchers to meet their evolving demands and bringing imaging to analysis o Quality Performance with Innovative Design FV10i Olympus is Open Imaging to Analysis ing up New Worlds e From Imaging to Analysis FV1000 e m oll my a 1 Advanced FLUOVIEW Systems Enhance the Power of Your Research Evolving Systems Meet the Demands of Your Application Upgradeable system with optional hardware and software to meet the demand
3. 16 Laser Light Stimulation The SIM scanner system combines the main scanner with a laser light stimulation scanner Control of the two independent beams enables simultaneous stimulation and imaging to capture reactions during stimulation Multi stimulation software is used to continuously stimulate multiple points with laser light for simultaneous imaging of the effects of stimulation on the cell Fluorescence loss in photobleaching FLIP combines imaging with continuous bleaching of a specific region to observe the diffusion of a target protein within a cell The changes in the image over time make it possible to observe the location of structural bodies that inhibit the diffusion of the molecule Esse O EP SEE EEN O 10 000 20 000 30 000 40 000 50 000 60 000 70 000 80 000 90 000 100 000 Time ms Specimen HeLa cell GFP free 488 nm excitation multi argon laser Image acquisition time 100 ms bleach time 100 s continuously 405 nm bleaching Exposure of fluorescent labeled target proteins to strong laser light c causes their fluorescence to fade locally Fluorescence recovery after photobleaching FRAP is used to observe the gradual recovery of fluorescence intensity caused by protein diffusion from the area surrounding the bleached region By examining the resulting images itis possible to characterize the diffusion speed of the molecule and the speed of binding and release between the molecule and cell struct
4. Medical and Dental University Jonteto iate P Combines the main scanner with a dedicated laser light labeled molecules and marking of specific live cells Simultaneous Laser Light Stimulation and Imaging Performs simultaneous laser light stimulation and imaging to acquire images of immediate cell responses to stimulation in photobleaching experiments Modifiable Stimulation Area During Imaging The stimulation area can be moved to a different position on the cell during imaging providing a powerful tool for photoactivation and photoconversion experiments Wide Choice of Bleaching Modes Various scan modes can be used for both the observation area and stimulation area Enables free form bleaching of designated points lines free lines rectangles and circles stimulation scanner for investigating the trafficking of fluorescent SIM Scanner Unit for Simultaneous Laser Light Stimulation and Lasers are used for both imaging and laser light stimulation Unique Tornado Scanning for Efficient Bleaching Conventional raster scanning does not always complete photobleaching quickly Tornado scanning greatly improves bleaching efficiency by significantly reducing unnecessary scanning Tornado scanning only available for SIM scanner ROI region of interest scanning Tornado scanning Superfluous scanning areas s ROI region of interest scanning a _ 7 a STA Er E l OG E
5. Photo Detection Method Scanning speed 512 x 512 1 1 sec 1 6 sec 2 7 sec 3 3 sec 3 9 sec 5 9 sec 11 3 sec 27 4 sec 54 0 sec 256 x 256 bidirectional scanning 0 064 sec 0 129 sec RI Z A Line scanning Straight line with free orientation free line Point scanning 2 detection modes Analog integration and hybrid photon counting ANTA Line scanning Straight line with free orientation free line Point scanning Pinhole Single motorized pinhole pinhole diameter 0950 300 um 1 um step Single motorized pinhole pinhole diameter 0950 800 um 1 um step Field Number NA Optical Zoom 18 1x 50x in 0 1x increment Z drive Integrated motorized focus module of the microscope minimum increment 0 01 um or 10 nm Transmitted Light Detector unit Module with integrated external transmitted light photomultiplier detector and 100 W Halogen lamp motorized switching fiber adaptation to microscope frame Motorized Microscope Inverted IX81 Upright BX61 Upright focusing nosepiece amp fixed stage BX61WI Fluorescence Illumination Unit External fluorescence light source with motorized shutter fiber adaptation to optical port of scan unit Motorized switching between LSM light path and fluorescence illumination System Control PC PC AT compatible OS Windows XP Professional English version Windows Vista English version Memory 2 0 GB or larger CPU Core2Duo 3 0 GHz Hard disk 500 GB or la
6. live tile series Z T A LUT individual color setting pseudo color comment graphic and text input Interactive volume rendering volume rendering display projection display animation displayed save as OIF AVI or MOV format Free orientation of cross section display 3D animation maximum intensity projection method SUM method 3D and 2D sequential operation function OIB OIF image format 8 16 bit gray scale index color 24 32 48 bit color JPEG BMP TIFF AVI MOV image functions Olympus multi tif format 2 Fluorescence spectral unmixing modes normal and blind mode Filter type Sharpen Average DIC Sobel Median Shading Laplacian Calculations inter image mathematical and logical DIC background leveling Fluorescence intensity area and perimeter measurement time lapse measurement 2D data histogram display colocalization Review station software Off line FLUOVIEW software for date analysis Motorized stage control software Diffusion measurement package Multi stimulation software Multi area time lapse software Programmable Scan Controller 2D Image Display 3D Visualization and Observation Image Format Spectral Unmixing Image Processing Image Analysis Statistical Processing Optional Software Objectives for BX2 and IX2 using U UCD8A 2 IX2 LWUCDA2 and U DICTS Objectives for fixed stage upright microscope using WI UCD WI DICTHRA2 Objectives DIC prism Revolving nosep
7. molecules move faster compared with large lT molecules that move relatively slowly The FV1000 acquires O us 10us 20ps 30us 40ps 50us nus information on the movement of these diffusing fluorescent labeled molecules as image data together with morphological ois information about the cell The image data obtained for each pixel was sampled at different times so the data for each pixel is affected by the passage of time in addition to its spatial XY information By analyzing this image data with a new Statistical algorithm for spatial correlation the diffusion coefficients and Scan in Y Axis Direction molecule counts can be calculated for molecules moving within the cell Spatial Correlation Algorithm When the spatial correlation algorithm is applied between pixels a higher correlation is obtained as the speed of movement of the molecule nears the scanning speed When calculating the spatial correlation in the X direction because the scanning speed in the X direction is fast a higher correlation is obtained for fast moving molecules than for slow moving molecules When the scanning speed in the Y direction is slow a higher correlation is obtained for slow moving molecules RICS using LSM images scans in both X and Y directions so it can be used to analyze the movements of a wide range of molecules both fast and slow RICS Analysis Method LSM Image Spatial Correlation Theoretical Formula Used for Fitting Calculation
8. over a wide area 3D construction can also be performed by acquiring images in the X Y and Z directions Tiled images can be enlarged in sections without losing resolution CNS markers in normal mice Objective PLAPON6Ox Zoom 2X Image acquisition numbers XY 32 x 38 48 slices for each image Courtesy of Dr Mark Ellisman PhD Hiroyuki Hakozaki MS Mark Ellisman National Center for Microscopy and Imaging Research NCMIR University of California San Diego Multi area time lapse software automates the process from 3D image acquisition using the motorized XY stage to stitching The software can be used to easily register wide areas and the thumbnail display provides a view of the entire image acquired during the mosaic imaging process Coordinate Information Thumbnail 20 Expandability to Support Diverse Application Application Standard Functions Optional Functions Molecular interaction and Intracellular diffusion measurement molecular concentration Calculation of diffusion coefficients for intracellular molecules and analysis of analysis molecular binding and changes in molecular density Supports a wide range of methods RICS ccRICS point FCS point FCCS and FRAP Software Required Diffusion measurement package Laser Light stimulation Acquires images while rapidly switching SIM scanner system the built in laser between imaging and Performs simultaneous imaging and laser light stimulation Provides de
9. IRFM LSM GFP Pak K298A in HeLa cells Courtesy of Dr J M Dong of sGSK NPP laboratory Singapore UAPON100xOTIRF NA 1 49 oil immersion WD 0 1 mm UAPON150xOTIRF NA 1 45 oil immersion WD 0 08 mm APON60xOTIRF NA 1 49 oil immersion WD 0 1 mm Apo100xOHR NA 1 65 oil immersion WD 0 1 mm Customized cover glass and immersion oil dE Brighter and Deeper Imaging with Finer Resolution The FV1000 is upgradeable to multiphoton excitation capability 4 by adding a dedicated laser and multiphoton optical system Optical design is optimized for multiphoton principles for brighter gt P imaging of features deep within living specimens at higher resolutions than previously possible i t Special Multiphoton Objective with Outstanding o Brightness and Resolution Olympus offers a high NA water immersion objective designed for a wide field of view with improved transmittance at near infrared wavelengths A correction collar compensates for spherical aberration caused by differences between the refractive indices of water and specimens forming the optimal focal spot even in deep areas without loss of energy density The objective Icy is designed to collect scattered light over a wide field of view for 3 dimensionally constructed images of neurons expressing EYFP in the cerebral neocortex of a p mouse under anesthesia maximum image brightness i Cour
10. Tornado scanning uT cn ba g ry ek is ein z i z Ti wi 15 i ini Cell membrane stained with DIO and subjected to both conventional ROI and tornado scanning Multi Purpose Laser Combiner All lasers can be used for both Imaging and laser light stimulation LD405 LD635 AOTF AOTF LD473 LD559 Laser Sharing with Main Scanner Dual fiber laser combiner provides laser sharing between the SIM scanner and main scanner eliminating the need to add a separate laser for stimulation New Objective with Low Chromatic Aberration Delivers World Leading Imaging Performance NEw Low Chromatic Aberration Objective Best Reliability for Colocalization Analysis A new high NA oil immersion objective minimizes chromatic aberration in the 405 650 nm region for enhanced imaging performance and image resolution at 405 nm Delivers a high degree of correction for both lateral and axial chromatic aberration for acquisition of 2D and 3D images with excellent and reliable accuracy and improved colocalization analysis The objective also compensates for chromatic aberration in the near infrared up to 850 nm Lateral and Axial Chromatic Aberration Low Chromatic PLAPON60xOSC Aberration Objective Magnification 60x NA 1 4 oil immersion W D 0 12 mm Chromatic aberration compensation range 405 650 nm Optical data provided for each objective Chromatic Aberration Comparison for PLAPON 60xOSC and UPLSAPO 60xO Axi
11. al chromatic Ou UPLSAPO60xO aberration Z direction Lateral chromatic aberration X Y direction at FN6 Performance Comparison of PLAPON 60xOSC and UPLSAPO 60x0 PLAPONGOXOSC UPLSAPOGOO Axial chromatic aberration Z direction Compared for PSF fluorescent Chromatic aberration values are design values and are not guaranteed values beads 405 nm 633 nm a Lateral chromatic aberration X Y direction Improved Flatness and Resolution at 405 nm Better flatness reduces the number of images for tiling Compared for PSF fluorescent beads 405 nm 488 nm 633 nm Approx 0 1 um 3D image Tubulin in Ptk2 cells labeled with two colors 405 nm 635 nm and compared Exceptional Resolution for Imaging of Cytoplasmic Membrane and Areas Deep Within Living Specimen Fluo rescence Microscope System Switchable between Confocal and TIRFM Imaging Switchable between confocal and TIRFM imaging for localization of proteins on the cytoplasmic membrane surface and acquisition of sectioning images within cells Software Control of TIRF Illumination Built in laser provides TIRF illumination Software can be used to tune the angle of incidence of excitation light and calculates the penetration depth of the evanescent wave based on the TIRF objective used High Numerical Aperture Objectives for TIRF Illumination A line of high numerical aperture NA objectives is available for TIRF illumination T
12. curacy colocalization analysis are present locally in the same New 60x oil immersion objective offers image acquisition with exceptional ra locations positional accuracy coefficient Calculate of Pearson coefficients Equipment Required PLAPON 60xOSC overlap coefficients and colocalization indices SIM scanner and TIRFM scanner cannot be installed on the same system For more information about peripheral equipment contact your Olympus dealer e Vs o A Ls e o amp 2 S f Q anal o Ss es D Scanning Units Two types of scanning units filter based and spectral detection are provided The design is all in one integrating the scanning unit tube lens and pupil projection lens Use of the microscope fluorescence illuminator light path ensures that expandability of the microscope itself is not limited Visible UV and IR laser introduction ports are provided as well as a feedback control system Laser Systems The multi combiner enables combinations with all of the following diode lasers 405 nm 440 nm 473 nm 559 nm and 635 nm The system can also be equipped with conventional Multi line Ar laser and HeNe G laser Illumination Units Conventional illumination modules are designed for long duration time lapse experiments Since light is introduced through fiber delivery systems no heat is transferred to the microscope Optional Upgrade Equipments for FV1000 4th C
13. detector system Inverted motorized microscope TIRFM unit BX61WI BX61 Upright motorized microscope FV Power supply Microscope control unit FV power supply unit CO2 Incubator MIU IBC IF 2 MIU IBC I 2 Highly precise incubator control keeps the environment inside a laboratory dish completely stable at just below 37 C temperature 90 moisture and 5 CO concentration in this way live cell activity can be maintained for approximately two days Not available in some areas Software il i FV control unit Basic software Review station software Diffusion Measurement Package Multi Stimulation Software Multi Area Time Lapse Software High Precision Motorized Stage PRIOR H117 Multi point time lapse photography using a 35 mm glass bottom dish is easy to perform with this motorized stage which can reproduce previously set positions with extreme precision It also allows efficient photographing of multiple cells and detection of individual cells showing expected reactions Main Specifications Laser Light Scanning and Detection Microscope Ultraviolet Visible Light Laser Spectral Version Filter Version LD lasers 405 nm 50 mW 440 nm 25 mW 473 nm 15 mW 559 nm 15 mW 635 nm 20 mW Multi line Ar laser 458 nm 488 nm 515 nm Total 30 mW HeNe G laser 543 nm 1 mW AOTF Laser Combiner Visible light laser platform with implemented AOTF sy
14. fusion coefficients and the proportions of diffusing molecules Analytical methods Small molecules Proteins Diffusion of Lateral diffusion Protein Molecular complex according to molecule in solution in solution proteins in cell membrane trafficking formation in cell aggregation diffusion speeds ae point FCS apable range of measurement RICS FRAP 15 RICS Application and Principles GFP Fusion Proteins Near to Cell Membranes and In Cytoplasm RICS can be used to designate and analyze regions of interest At cytoplasmic membrane In cytoplasm based on acquired images Diffusion coefficient D 0 98 um s Diffusion coefficient D 3 37 um s EGFP is fused at protein kinase C PKC for visualization using live cells to analyze the translocation with RICS The diffusion coefficient close to cell membranes was confirmed to be lower a than in cytoplasm after stimulation with phorbol myristate acetate PMA This is thought to be from the mutual interaction between PKC and cell membrane molecules in cell membranes In addition to localization of molecules RICS analysis can D 0 98 simultaneously determine changes in diffusion coefficient for j D 3 37 detailed analysis of various intracellular signaling proteins i Sample image HeLa cells expressing EGFP fusion PKC after PMA stimulation Molecules of different sizes diffuse at different speeds within Scan in X Axis Direction cells Small
15. hannel Detector Unit Attaches to the optional port of either the filter or spectral type scanning unit and is used as a 4th confocal fluo rescence detection channel This is a filter based fluorescence detection unit SIM Scanner Second scanner dedicated for laser light stimulation synchronized to the FV1000 main scanner for simultaneous laser light stimulation and confocal image acquisition Independent fiber optic laser Scanning Unit for IX81 Inverted Microscope Dedicated mirror unit cassette is required Dual Type The multi combiner outputs laser light with two fibers Light can be used both for observation and laser light stimulation Fluorescence Illumination Unit Stand with Mercury lamp house motorized shutter and fiber delivery system for conventional fluorescence observation Light introduction via fiber optic port TIRFM Unit Confocal introduction port Dichromatic mirror within motorized optical port of the scan unit required for introduction of laser into main scanner 23 Enables control of the necessary volume of excitation light using FV1000 soft ware This unit enables TIRF imaging using the laser light source used with Scanning Unit for BX61 BX61WI Upright Microscopes Fluorescence illuminator integrated with scanning unit Single Type Single channel laser output AOTF is standard equipment Transmitted Light Detection Unit External transmitted light photomultiplier detect
16. ht bd PE E Before Stimulation After Stimulation Kaede expressing astroglia cells are stacked on the Kaede expressing neurons By illuminating two colonies with a 405 nm laser the Kaede color can be photoconverted from green to red The glial cells in contact with the neurons are observed while they are forming colonies and extending their processes and the nuclei of these colonies can also be observed The SIM scanner FV1000 makes it easy to change cell colors from green to red while conducting an observation and to control neutral colors between red and green Data courtesy of Dr Hiroshi Hama Ms Ryoko Ando and Dr Atsushi Miyawaki RIKEN Brain Science Institute Laboratory for Cell Function Dynamics A 405nm laser is optional for uncaging with the SIM scanner system Caged compounds can be uncaged point by point or within a region of interest while the main scanner of the FV1000 captures images of the response with no time delay F 1 i is a i 4 E ae WA 3 P b By TE SE t ne Ba E 0 5 000 10 000 15 000 20 000 25 000 30 000 35 000 40 000 45 000 50 000 55 000 b i Time ms i f s a i i Caged Glutamate Fluorescent calcium indicator Fluo 3 in HeLa cells Image acquisition at 1 second intervals Using the caged compound Bhcmoc Glutamate an increase in calcium ion concentration inside the cell can be observed in response to glutamate stimulation released via 405 nm laser illumination Data courtesy of
17. iece WI SSNP WI SRE3 WI SSNP WI SRE3 WI SSNP WI SRE3 WI SSNP WI SRE3 WI SSNP WI SRE3 WI SSNP WI SRE3 WI DICXLU20HR WI SNPXLU2 Condenser for BX2 U UCD8A 2 optical element Condenser for IX2 IX2 LWUCDA2 optical element Cover glass thickness mm U DICTS position Correction Immersion lee ring Description MPLN5X UPLSAPO4X UPLSAPO1 0X2 0 17 UPLSAPO20X 0 17 UPLSAPO20X0 a UPLSAP040X2 0 11 0 23 UPLSAPOGOXO 0 17 UPLSAPO60XW 0 13 0 21 UPLSAP0100XO 0 17 PLAPONGOXO 0 17 PLAPONGOXOSC 0 17 UPLFLN4OXO 0 17 APONGOXOTIRF 0 13 0 19 UAPON1 OOXOTIRF 0 13 0 19 UAPON1 50XOTIRF 0 13 0 19 Apo100XOHR 0 15 UMPLFLN10XW WI DIC10HR U DIC10 U DIC20 U DIC20 U DIC40 U DIC60 U DIC60 U DIC100 U DIC60 U DIC60 U DIC40 U DIC60 U DIC100 U DIC100 U DIC100 IX2 DIC10 IX2 DIC20 IX2 DIC20 IX2 DIC40 normal IX2 DIC60 BFP1 X2 DIC60 IX2 DIC100 normal IX2 DIC60 BFP1 IX2 DIC60 BFP1 IX2 DIC40 BFP1 IX2 DIC60 BFP1 IX2 DIC100 IX2 DIC100 IX2 DIC100 normal normal UMPLFLN20XW WI DIC20HR normal LUMPLFLN40XW WI DIC40HR LUMPLFLN60XW WI DICGOHR normal LUMFLNGOXW WI DIC60HR XLUMPLFLNZOXW 1 00 Note These conditions are not met in confocal microscopy normal normal normal Dimensions Weight and Power Consumption Dimensions mm BX61 BX61WI IX81 Microscope with scan unit Fluorescence illumination unit Lamp Power supply Transmitted light detection unit Microscope contro
18. ion and image acquisition matched to fluorescence wavelength peaks User adjustable bandwidth of emission spectrum for acquiring bright images with minimal cross talk fic Mina Precise Spectral Imaging The spectral detection unit uses a grating method that offers linear dispersion compared with prism dispersion The unit provides 2 nm wavelength resolution to high sensitivity photomultiplier tube detectors Fluorescence separation can be achieved through unmixing even when cross talk is generated by multiple fluorescent dyes with similar peaks Enhanced Sensitivity Three channel scan unit with detection system featuring hard coated filter base High transmittance and high S N ratio optical performance is achieved through integration of a pupil projection lens within the optics the use of a high sensitivity photomultiplier and an analog processing circuit with minimal noise High Performance Filters Deliver Outstanding Separation Special coatings deliver exceptionally sharp transitions to a degree never achieved before for acquisition of brighter fluorescence images DM488 543 633 Comparison Wavelength nm Conventional mirror unit High performance mirror unit EGFP dendrite EYFP synapse XYA Wavelength detection range 495 nm 561 nm in 2 nm steps Excitation wavelength 488 nm Courtesy of Dr Shigeo Okabe Department of Anatomy and Cell Biology Tokyo
19. l unit FV Power supply unit FV control unit PC 19 inch dual value per monitor Display 29 8 inch Power supply unit for laser combiner Laser combiner with Ar laser heads Laser combiner without Ar laser heads LD559 laser power supply Multi Ar laser power supply HeNe G laser power supply Recommended FV1000 system setup IX81 BX61 BX61WI unit mm 320 W x 580 D x 565 H 350 W x 750 D x 640 H 180 W x 320 D x 235 H 90 W x 270 D x 180 H 170 W x 330 D x 130 H 125 W x 332 D x 216 H 180 W x 328 D x 424 H 180 W x 420 D x 360 H 363 W x 216 D x 389 5 489 5 H 689 W x 254 7 D x 511 5 629 5 H 210 W x 300 D x 100 H 514 W x 504 D x 236 H 514 W x 364 D x 236 H 200 W x 330 D x 52 H 162 W x 287 D x 91 H 130 W x 224 D x 62 H ee ie RG e ES o e Weight kg Expandability Power consumption AC 100 240 V 50 60 Hz 1 6 A AC 100 120 220 240 V 50 60 Hz 3 5 A 1 5 A AC 100 120 220 240 V 50 60 Hz 4 0 A 2 0 A AC 100 240 V 50 60 Hz 497 5 W AC100 120 200 240 V 50 60 Hz 0 65 A 0 4 A AC100 120 200 240 V 50 60Hz 1 8 A 0 8 A AC 100 120 200 240 V 50 60 Hz 2 0 A 1 0 A AC 100 240 V 50 60 Hz 30 W AC 100 240 V 50 60 Hz 20 A AC 100 120 V 50 60 Hz 0 45 A 1 This product corresponds to regulated goods as stipulated in the Foreign Exchange and Foreign Trade Control Law An export license from the Japanese government is
20. la Courtesy of Dr Tetsuya Kojima Laboratory of Innovational Biology Department of Integrated Biosciences Graduate School of Frontier Sciences University of Tokyo Alpha Blend method Cultured nerve cells derived from the mouse hippocampus Courtesy of Dr Koji Ikegami Dr Mitsutoshi Setou Molecular Geriatric Medicine Mitsubishi Kagaku Institute of Life Sciences OLYMPUS www olympus com FLUOVIEW website OLYMPUS CORPORATION Shinjuku Monolith 3 1 Nishi Shinjuku 2 chome Shinjuku ku Tokyo Japan OLYMPUS EUROPA HOLDING GMBH Wendenstrasse 14 18 20097 Hamburg Germany OLYMPUS AMERICA INC 3500 Corporate Parkway Center Valley Pennsylvania 18034 0610 U S A OLYMPUS SINGAPORE PTE LTD 491B River Valley Road 12 01 04 Valley Point Office Tower Singapore 248373 www olympusfluoview com e OLYMPUS CORPOARATION is ISO9001 ISO14001 certified e Illumination devices for microscope have suggested lifetimes Periodic inspections are required Please visit our web site for details e Windows is a registered trademark of Microsoft Corporation in the United States and other countries All other company and product names are registered trademarks and or trademarks of their respective owners e Images on the PC monitors are simulated e Specifications and appearances are subject to change without any notice or obligation on the part of the manufacturer OLYMPUS AUSTRALIA PTY LTD 31 Gilby Road Mt Waver
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22. ntrations by calculating the diffusion coefficients of molecules within the cell Diverse analysis methods RICS ccRICS point FCS point FCCS and FRAP cover a wide range of molecular sizes and speeds Raster image correlation spectroscopy RICS is a new method for analyzing the diffusion and binding dynamics of molecules in an entire single image RICS uses a spatial correlation algorithm to calculate diffusion coefficients and the number of molecules in specified regions Cross correlation RICS ccRICS characterizes molecular interactions using fluorescent labeled molecules in two colors lation Spectroscopy point scan fluorescence correlation spectroscopy point FCS analyzes intensity fluctuations caused by diffusion or binding unbinding interactions of a protein complex point FCS uses an auto correlation function to carry out operations on fluorescence Signals obtained by continuous scanning of a single pixel on the screen point scan fluorescence cross correlation spectroscopy point FCCS analyzes the fluctuation of fluorescent labeled molecules in two colors The coincidence of fluctuations occurring in two detection channels shows that the two proteins are part of the same complex point FCS and point FCCS can now be performed with a standard detector eliminating the need for a special high sensitivity detector The Axelrod analytical algorithm is installed as a FRAP analysis method The algorithm is used to calculate dif
23. ocal PMT detectors and optional module with fourth confocal PMT expandable up to four PMT channels Spectral Scanning Unit Filter Scanning Unit Two Versions of Light Detection System e Spectral detection for high precision spectroscopy with 2 nm resolution e Filter detection equipped with high quality filter wheels icity Specimen kg EE o Optical System ecimens Motorized Microscopes UIS2 Objectives Supports a Wide Range of Samples and Compatible with Olympus IX81 inverted Olympus UIS2 objectives offer world Specimens microscope BX61WI focusing nosepiece leading infinity corrected optics that Tissue culture dishes slide chambers and fixed stage upright microscope and deliver unsurpassed optical performance microplates and glass slides can be used BX61 upright microscope over a wide range of wavelengths with live cells and fixed specimens gs E High S N Ratio Objectives with Suppressed Autofluorescence Olympus offers a line of high numerical aperture objectives with improved fluorescence S N ratio including objectives with exceptional correction for chromatic aberration oil and water immersion objectives and total internal reflection fluorescence TIRF objectives Two Versions of Light Detection System that Set New Standards for Optical Performance Flexibility and High Sensitivity Spectral detection using gratings for 2 nm wavelength resolut
24. or and 100 W Halogen conventional illumination integrated for both laser scanning and conventional transmitted light Nomarski DIC observation Motorized exchange between transmitted light illumination and laser detection Simultaneous multi channel confocal fluorescence image and transmitted DIC acquisition enabled Fiber Port for Fluorescence Output Confocal fluorescence emission can be introduced via fiber delivery system into external device Fiber port equipped with FC connector fiber delivery system not included FV1000 System Diagram Fluorescence illumination unit LD635 laser 635 nm LD559 laser 559 nm HeNeG laser 543 nm Select either laser COz2 incubator Scanning unit for IX81 Spectral type or Filter type detector system AOTF Laser combiner Single fiber type Multi Ar laser AOTF Laser combiner 458 488 515 nm LD473 laser 473 nm Select either laser LD440 laser 440 nm LD405 laser 405 nm Dual fiber type Riase gt gt Transmitted light detection unit H hon Fiber port for fluorescence output a 4th channel detector unit O ptional unit IX81 ZDC Focal drift compensation for long time lapse imaging Requires IX81 microscope For information about ZDC compatible objectives contact your Olympus dealer Motorized XY stage Scanning unit for BX61WI BX61 Spectral type or Filter type
25. orized XY stage TIRFM imaging Uses the laser from the laser combiner to provide evanescent illumination for imaging the movement of molecules near the glass surface such as cell membranes and adhesion factors Software and Equipment Required TIRFM unit TIRF objective high sensitivity CCD camera CCD camera control software Provides FRET analysis functions CFP YFP FRET Diode laser offers exceptional stability Ratio imaging and sensitized emission and long life Available 440 nm diode laser is optimized for CFP YFP FRET experiments Supports FRET efficiency methods measurements using acceptor Diode laser offers exceptional stability and long life photobleach method Equipment Required LD 440 nm Laser Multi color imaging Three channel detector for Imaging blue dyes simultaneous acquisition of Available 405 nm laser for image acquisition of multi stained samples labeled with fluorescence images from three V excitation fluorescent dyes such as DAPI Hoechst and Alexa 405 D different dyes Equipment Required LD 405 nm laser 2 Sequential mode for acquisition of fluorescence images without cross talk Fluorescence can also be separated using unmixing only available on spectral scan unit Simultaneous four color imaging Fourth channel detector can be easily added to simultaneously acquire images of four colors Equipment Required 4 channel detector Colocalization analysis Easily determine if labeled substances High ac
26. required when exporting or leaving Japan with this product 2 The performance and safety of this device is not guaranteed if it is disassembled or modified 3 This device is designed for use in industrial environments for the EMC performance IEC61326 1 Class A device Using it in a residential environment may affect other equipment in the environment Depth 990 Images are courtesy of the following institutions EE Brainbow mouse brain stem _ Courtesy of the laboratories of Jeff W Lichtman and Joshua R a a Sanes Harvard University MCB Department and the Center for pe Brain Science mF Hippocampal neurons Courtesy of Dr Shigeo Okabe Department of Cellular Neurobiology Graduate School of Medicine The University of Tokyo j Cultured nerve cells derived from the mouse hippocampus Courtesy of Dr Koji Ikegami Dr Mitsutoshi Setou Molecular Geriatric Medicine Mitsubishi Kagaku Institute of Life Sciences Cerebellum Purkinje cell Courtesy of Dr Tetsuro Kashiwabara Assistant Professor and Dr Akira Mizoguchi Professor Neuroregenerative medicine course Mie University School of Medicine Drosophila Stage 14 Courtesy of Dr Tetsuya Kojima Laboratory of Innovational Biology Department of Integrated Biosciences Graduate School of Frontier Sciences University of Tokyo ts Mouse brain section Courtesy of Mr Masayuki Sekiguchi Section Chief Department of Degenerative Neurological Di
27. rger Media DVD Super Multi Drive FV1000 Special I F board built in PC Graphic board conformity with Open GL Power Supply Unit Display Galvo control boards scanning mirrors and gratings Real time controller Galvo control boards scanning mirrors SXGA 1280X1024 dual 19 inch or larger monitors or WQUXGA 2560 x 1600 29 8 inch monitor Optional Unit SIM Scanner 2 galvanometer scanning mirrors pupil projection lens built in laser shutter 1 laser port Fiber introduction of near UV diode laser or visible light laser Optional 2nd AOTF laser combiner TIRFM Unit Available laser 405 633 nm Motorized penetration ratio adjustment Automatic optical setting for TIRFM objectives 4th CH Detector Module with photomultiplier detector barrier filter turret beamsplitter turret mounted with 3rd CH light path Fiber Port for Fluorescence Output port equipped with FC fiber connector compatible fiber core 100 125 um Software Image Acquisition Normal scan 64 x 64 128 x 128 256 x 256 320 x 320 512 x 512 640 x 640 800 x 800 1024 x 1024 1600 x 1600 2048 x 2048 4096 x 4096 Clip rectangle scan Clip ellipse scan Polygon clip scan line scan free line scan Point scan Real time image 2 dimension XY XZ XT and XA 3 dimension XYZ XYT XYA XZT XTA and XZA 4 dimension XYZT XZTA and XYTA 5 dimension XYZTA Time Controller function Each image display Single channel side by side merge cropping live tiling
28. s of your research Laser combiner Fiber Diode Laser Greater stability longer service life and lower operating cost are achieved using diode lasers Laser Feedback Control Scanner unit is equipped with laser power monitor for feedback control enhancing stable laser output Laser Compatibility Diode laser 405 nm 440 nm 473 nm 559 nm 635 nm Gas laser Multi line Ar laser 458 nm 488 nm 515 nm HeNe G laser 543 nm Broadband Fiber Broadband fiber connection for 405 635 nm lasers to achieve an ideal point light source with minimal color shift and position shift between images Excellent Precision Sensitivity and Stability FLUOVIEW Enables Precise Bright Imaging with Minimum Phototox Broadband fiber Laser Combiner Two versions available eSingle fiber type combiner is used for main scanner FV1000 with up to six lasers ranging from 405 to 635 nm eDual fiber type combiner is used for laser light stimulation with main and SIM scanner FV1000 Scanners Detection High Sensitivity Detection System High sensitivity and high S N ratio optical performance is achieved through the integration of a pupil projection lens use of a high sensitivity photomultiplier tube and an analog processing circuit with minimal noise Enables high S N ratio image acquisition with minimal laser power to reduce phototoxicity Up to Four PMT Channels Three integrated conf
29. seases National Institute of Neuroscience National Center of Neurology and Psychiatry Rudimentary limbs of larva in latter part of 3rd instar Courtesy of Dr Tetsuya Kojima Laboratory of Innovational Biology Department of Integrated Biosciences Graduate School of Frontier Sciences University of Tokyo Zebrafish Courtesy of Dr Toru Murakami Department of Neuromuscular amp Developmental Anatomy Gunma University Graduate School of Medicine Medaka embryogenesis somite stage Courtesy of Minoru Tanaka Hiromi Kurokawa National Institute for Basic Biology Laboratory of Molecular Genetics for Reproduction Pilidium larva of Micrura alaskensis Courtesy of Dr Svetlana Maslakova of the University of Washington and Dr Mikhail V Matz of the Whitney Laboratory for Marine Bioscience University of Florida 26 Osteoclast induced from rat monocyte in rat kidney pr a vi ry ce maa et ye Courtesy of Dr Keiko Suzuki Department of Pharmacology Showa University School of Dentistry Fucci Sliced mouse brain expressing S G2 M phases Courtesy of Dr Hiroshi Kurokawa Ms Asako Sakaue Sawano and Dr Atsushi Miyawaki RIKEN Brain Science Institute Laboratory for Cell Function Dynamics Immunolabeling of a transgenic mouse retina showing the major retinal cells types Courtesy of Dr Rachel Wong Mr Josh Morgan Dept Biological Structure University of Washington Seattle Wild type embryo in stage 17 of drosophi
30. stem Ultra fast intensity modulation with individual laser lines additional shutter control Continuously variable 0 1 100 0 1 increment REX Capable of laser intensity adjustment and laser wavelength selection for each region Fiber Scanner Module Detector Module Broadband type 400 nm 650 nm Standard 3 laser ports VIS UV IR Excitation dichromatic mirror turret 6 position High performance DMs and 20 80 half mirror Dual galvanometer mirror scanner X Y Motorized optical port for fluorescence illumination and optional module adaptation Adaptation to microscope fluorescence condenser Standard 3 confocal Channels 3 photomultiplier detectors Standard 3 confocal Channels 3 photomultiplier detectors Additional optional output port light path available for optional units Additional optional output port light path available for optional units 6 position beamsplitter turrets with CH1 and CH2 6 position beamsplitter turrets with CH1 and CH2 CH1 and CH2 equipped with independent grating and slit for fast and CH1 to CH3 each with 6 position barrier filter turret flexible spectral detection High performance filters Selectable wavelength bandwidth 1 100 nm Wavelength resolution 2 nm Wavelength switching speed 100 nm msec CH3 with 6 position barrier filter turret Filters High performance sputtered filters dichromatic mirrors and barrier filters Scanning Method 2 galvanometer scanning mirrors Scanning Modes
31. tailed laser light stimulation settings for laser light stimulation including position and timing Features tornado scanning for high Features tornado scanning for high efficiency bleaching using laser light efficiency bleaching using laser light stimulation stimulation Equipment Required SIM scanner laser combiner dual fiber version Multi point laser light stimulation system Register multiple points for laser light stimulation and program the respective stimulation order stimulation time and type of stimulation continuous laser light or pulse laser light Software Required Multi stimulation software Multi dimensional Long time lapse system time lapse imaging Microscopes equipped with zero drift compensation ZDC acquire each image at E a set focus plane The microscope CO2 incubator maintains cell activity for a long period for continuous imaging Equipment Required IX81 ZDC microscope CO2 incubator Multi point scanning system Register multiple points for repeated image acquisition Efficiently observe multiple cells in parallel on 35 mm dishes microplates or chamber slides Software and Equipment Required Multi area time lapse software motorized XY stage 3D mosaic imaging 3D mosaic imaging system Continuous imaging of adjacent fields of view and mosaic imaging to form a composite image Acquisition of adjacent Z series images for 3D mosaic imaging Software and Equipment Required Multipoint time lapse software mot
32. ter use Re Use Function Open previously configured scanning conditions and apply them to new or subsequent experiments Help Guide Comprehensive help guide describes the functions and usage for each command and overall sequence of operations ma a E a a um ta ma a a um a Ea ea m E Optional Software with Broad Functionality Diffusion Measurement Package For analysis of intracellular molecular interactions signal transduction and other processes by determining standard diffusion coefficients Supports a wide range of diffusion analysis using point FCS RICS and FRAP Multi Stimulation Software Configure multiple stimulation points and conditions for laser light stimulation synchronized with imaging for detailed analysis of the connectivity of cells within the stimulation area Multi Area Time Lapse Software Multi Area Time Lapse Software control of the motorized XY stage enables multiple measurement points in glass slides 35 mm dishes or individual microplate wells Repeated imaging of multiple cells improves the statistical power of time lapse experiments Mosaic Imaging A motorized XY stage is programmed with the use of a high magnification objective to acquire continuous images from adjacent fields of view to assemble a single high resolution image covering a wide area Three dimensional images can also be assembled using XYZ acq
33. tesy of Hiroaki Waki Tomomi Nemoto and Junichi Nabekura National Institute for Physiological Sciences National Institutes of Natural Sciences Japan XLPLN25xWMP Magnifications 25x NA 1 05 water immersion W D 2 0 mm Multiphoton Laser Light Stimulation Adding a multiphoton laser to the SIM scanner enables multiphoton laser light stimulation or uncaging confined to the focal volume The FLUOVIEW FV1000MPE is a class 4 laser product 10 User Friendly Software to Support Your Research Bj Configurable E Wavelength Select the dye name t filters and laser lines eS TATH id a SC i i iiri Hap rha aa fe ea a H de Anto E his PMs Lf2stoms F amp STis 5 56660min E Alim Flin 46 CHNS are Aspect Rahn Alea Flyer Gay z 4 S12 by ge CR a alii yoi Er hlin Tiaj Ai ia LO Ain hii AFE o Mina bre JA un Elm Pare Ldn Configurable Excitation Laser Power Bj aa this SS Easily adjust the optimum laser power for each specimen live cells and fixed pah specimens 11 Image Acquisition by Application User friendly icons offer quick access to functions for image acquisition according to the application XYZ XYT XYZT XYA XYAT Deh Time Time Controller Precisely synchronizes different experimental protocols including FRAP FLIP and FRET by acceptor photo bleaching and time lapse Save and open settings for la
34. tion system corrects loss of focus caused by temperature changes around the microscope and other factors during long time lapse observation The thermal drift compensation eliminates the need to take images at several Z planes minimizing live cell exposure to irradiation Offset Objective IR Laser for focal Baseline focal plane focal plane detection plane Set target Over time the Laser detects Immediately observation objective focal the glass returns to initial plane plane drifts from surface before offset plane as offset the observation imaging for focal drift plane compensation Proprietary CO2 incubator control keeps the environment inside the tissue culture dish completely stable The environment is precisely maintained at 37 C with 90 humidity and 5 CO2 concentration rss i z e nu A o e Do Uz E E Human lymphoblast cells TK6 Courtesy of Masamitsu Honma Dir Biological Safety Research Center Div of Genetics and Mutagenesis National Institute of Health Sciences 19 3D Mosaic Imaging Mosaic imaging is performed using a high magnification objective to acquire continuous 3D XYZ images of adjacent fields of view using the motorized stage utilizing proprietary software to assemble the images The entire process from image acquisition to tiling can be fully automated Composite images are quickly and easily prepared using the stitching function to form an image
35. uisition 12 Broad Application Support and Sophisticated Experiment Control m Measurement Diffusion measurement and molecular interaction analysis F Light Stimulation FRAP FLIP P hotoactivation Photoconversion Uncaging Multi Dimensional Time Lapse Long term and multiple point m 3D Mosaic Imaging High resolution images stitched to cover a large area Multi Color Imaging Full range of laser wavelengths for imaging of diverse fluorescent dyes and proteins 3D 4D Volume Rendering One click 3D 4D image construction from acquired XYZ T images Change the angle of 3D image with a single click Colocalization Configurable threshold values for fluorescence intensities on the scatterplot Accurate colocalization statistics and visualization of colocalized area on image FRET Configuration wizard simplifies the setting of FRET experimental procedures Optimal laser excitation wavelengths for CFP YFP FRET 1 1 1 1 15 000 20 000 25 000 30 000 35 000 40 000 Time ms Image of variations in calcium concentration of HeLa cells expressing YC3 60 when stimulated with histamine Reference Takeharu Nagai Shuichi Yamada Takashi Tominaga Michinori Ichikawa and Atsushi Miyawaki 10554 10559 PNAS July 20 2004 vol 101 no 29 14 Diffusion Measurement Package This optional software module enables data acquisition and analysis to investigate the molecular interaction and conce
36. ures Example Fluorescence recovery without interactions Example Fluorescence recovery with interactions If the protein can freely diffuse the bleached region recovers If the protein is strongly bound to a structure or forms part of a its fluorescence at a high speed due to Brownian motion large protein complex the bleached region recovers its fluorescence at a slower rate relative to the unbound state Fluorescent intensity gt o Q n o 5 LL a 3 F 3 P ee ee ee ee fi 41 Sits TH ah Ney Tas E7Sms 735 10Ams O Ls 64Sma E O Tic a ES E Ts free Specimen Hippocampal neurons Shank GFP stain 488 nm excitation multi argon laser oon iy Image acquisition time 100 ms Bleach time 80 ms 488 nm excitation Sapphire 488 laser ES eee ee RST ROT Re AT NT eee 0 10 000 20 000 30 000 40 000 50 000 60 000 70 000 80 000 Data courtesy of Dr Shigeo Okabe l i Time ms Department of Anatomy and Cell Biology Tokyo Medical and Dental University 17 The Kaede protein is a typical photoconvertible protein which is a specialized fluorescent protein that changes color when exposed to light of a specific wavelength When the Kaede protein is exposed to laser light its fluorescence changes from green to red This phenomenon can be used to mark individual Kaede expressing target cells among a group of cells by exposing them to laser lig
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