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FV1000
The Olympus FluoViewTM FV1000 is a next-generation imaging system designed for high-resolution, confocal observation of both fixed and living cells. The FV1000 offers advances in confocal system performance while providing the speed and sensitivity required for live cell imaging with minimal risk of damage to living specimens. In addition, the FV1000 offers a revolutionary synchronized laser scanning system called the SIM Scanner. While one laser stimulates, the second laser provides simultaneous imaging. This coordination of laser stimulation and imaging makes the FV1000 an ideal choice for FRAP, FLIP, photoactivation, photoconversion and uncaging experiments.
Features & Benefits
Among the advanced features of the FV1000 is the simultaneous application of two laser scanners (SIM scanner) to enable synchronization of specimen excitation and observation. Highly efficient ion deposition interference filters increase the instrument's sensitivity, while high-speed galvanometers and spectroscopic detection combine to yield precision scans with 2-nanometer wavelength resolution.
FV1000 Scanning Unit
The newly developed FV1000 scanning units offer a high-sensitivity detection system coupled to high-speed scanning and laser feedback to enable the capture of high-quality images of living organisms undergoing rapid change. Images may be scanned in any pixel array size up to 4096 x 4096, with each channel digitized to 4096 gray levels (12-bit resolution), permitting observation of fine image detail. The FV1000 scanning unit employs a new spectral imaging technology using diffraction gratings to provide distinct wavelength separation with precise resolution.
FV1000 Operating Software
Enhanced with specialized applications modules, the FV1000 confocal operations software is designed to operate on standard Windows computer systems with an intuitive, menu-driven interface. Images can be displayed side-by-side in single channels, merged, tiled, and presented with details of the acquisition mechanism. In addition, the software contains look-up tables for individual colour settings and pseudo-colour, as well as providing the ability to add graphical and text input for comments.
FV1000 Specifications
The Olympus FV1000 specification sheet includes information about laser light sources, the scanning unit, microscope combinations, external illumination sources, host computer configuration and requirements, the applications software, and power consumption statistics.
SIM (SIMultaneous) scanner system
Simultaneous laser light stimulation and imaging experiments
Assured capture of reactions immediately following laser light stimulation.
The compact design incorporates two laser scanners, one for confocal imaging and the other for simultaneous bleaching or stimulation. They can be controlled separately and independently, making it possible to stimulate the specimen during observation. As a result, the rapid cell reactions that occur right after laser stimulation can be accurately and reliably captured, making the FV1000 ideal for such applications as FRAP, FLIP, photoactivation and photoconversion.
Stimulation area can be changed during imaging.
Two laser beams, one for imaging and laser light stimulation, can be controlled independently and separately. The stimulation point or area can be relocated in the cell during the imaging time course, making the system powerful for a variety of experiments.
Wide choice of different bleaching modes.
Various scan modes can be used for both observation and stimulation modes. This enables free-form bleaching of designated points, lines, free-lines, rectangles and circles.
Multi-laser combiner enables 2-channel laser output.
Laser light is branched in the laser combiner, and each laser wavelength provided in the system can be selected and used for imaging and stimulation.
Tornado scanning for highly efficient bleaching.
Conventional raster scanning cannot always complete photobleaching quickly. Tornado scanning makes the procedure much more efficient by significantly reducing unnecessary scanning.
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Tornado scan method
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Stimulus setting window
This enables selection of the laser wavelength, intensity, scanning mode and other parameters for the stimulation laser. When using the SIM scanner, control can be done in linkage with imaging in µs units.
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Laser stimulation and live cell imaging.
In contrast to bleaching, light stimulation can be harnessed to induce a variety of sensitive changes to cells, with minimal laser input. Live cell imaging is dramatically enhanced by monitoring the diffusion, transfer or interaction of fluorescence-labelled molecules, or by optically highlighting a cell with the aid of laser stimulation. The unique FV1000 SIM system enables precise synchronisation of stimulation and imaging settings, minimizing image/ data loss and allowing creative configuration of live cell experiment protocols.
Even with the main scanner only, it is possible to perform laser light stimulation during a time course by instantaneously switching the laser scanning mode (laser wavelength, laser irradiation range). However, images cannot be acquired during laser light stimulation.
Example: Kaede
This fluorescent protein is derived from Trachyphyllia geoffroyi. It emits a strong green light after synthesis, but when stimulated by ultraviolet laser illumination, changes its colour from green to red, like a maple tree in autumn. Its name is derived from this characteristic ("kaede" means "maple tree" in Japanese). When violet (or ultraviolet) laser illumination is directed onto a Kaede-expressing cell, diffusion of the reddish Kaede can be monitored throughout the whole area of the cell, providing an easy and accurate method for whole cell labelling. The FV1000 allows this to be done while observation is in progress.
When Kaede is expressed in the cytoplasm of a live cell, it shows a high-speed diffusion coefficient value (about 30µm2/s) in spite of its tetramic structure. Taking advantage of this property, the movements of Kaede molecules in the cell can be observed more easily than molecules in general GFP/FRAP experiments. Since Kaede photoconversion requires only slight laser light, less intense than that used in ordinary photobleaching, labelling can be completed in a very short time.
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Kaede-expressing astroglia cells are stacked on the Kaede-expressing neurons. By illuminating two colonies with a 405nm 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. Data courtesy of : Dr. Hiroshi Hama, Ms. Ryoko Ando and Dr.Atsushi Miyawaki RIKEN Brain Science Institute Laboratory for Cell Function Dynamics.
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Live cell imaging - Advantages with FV1000
Time Course Experiment Design
The Time Controller allows image acquisition conditions to be changed easily while observation is in progress. The experiment protocol can be entered from the taskbar in the protocol schedule area. Settings can be entered freely by clicking and dragging the mouse on the column of items to be scheduled. In addition to experiments such as FRAP and FLIP, the following protocols are supported:
- Image acquisition while storing data on the hard disk.
- Changing time-lapse intervals during the course of an experiment.
- Image acquisition while changing the excitation laser in mid-procedure.
- Data output from a specified point, using an external trigger.
- After acquisition of reference images, laser intensity and excitation area can be changed.
Wide variety of line scanning modes.
Line scanning for a straight line, slanted line or free curve enables easy analysis of changes over time at the msec order. Observation of complex time lapse combined with Z or λ element is also possible.
Trigger function.
The system also has a trigger function for synchronizing scanning with external devices. Scanning can be started/stopped with a trigger signal, and it is also possible to gather frames for each external trigger.
Laser monitoring function.
Feedback is applied to the laser output so that the sample is always exposed to a fixed level of excitation light. There is no need to pay attention to variation in laser output, and the amount of fluorescence can be measured accurately.
Multi- Area Acquisition - Long term time lapse of live cells.
By equipping the system with a motorized XY stage, repeated image acquisition of multiple points located over a wide range is performed automatically. Furthermore, time lapse observation of the cells under different conditions is accomplished by using a well plate. These functions dramatically improve throughput of experiments requiring long-term observation.
- Repetitive operation can be done in sequence for multiple registered points, by simply setting the cells or specific points you wish to observe.
- Cell observation using well plates can be done more efficiently.
- Tiling image acquisition: After automatic registration of the neighboring visual field, a wide observation area can be automatically acquired while maintaining high resolution. (Separate software is needed to integrate the acquired images.)
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Superior Spectral Performance
Original spectral system.
An independent photomultiplier is incorporated into the original optics for each channel. The special spectral scanning system uses a diffraction grating, which has excellent linear performance with no wavelength deviation and ensures high-precision, high-resolution, high-speed spectroscopy.
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High-speed spectroscopy.
High-speed galvanometer diffraction grating delivers high wavelength change speed (100nm/ms), enabling very fast acquisition of XYλT images.
High-precision spectroscopy.
Cross talk derived from two fluorochromes with similar emission wavelength peaks can be clearly separated by the system's highly accurate 2nm wavelength resolution. Even when working with specimens whose fluorescence emission wavelength is similar to the excitation wavelength, it is possible to obtain images unaffected by the excitation light.
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Variable Barrier Filter (VBF).
With the spectral detection system, once a fluorochrome combination is selected within the software, the ideal wavelength spectrum is automatically selected. Of course, manual adjustment of the wavelength range is also possible according to the fluorescence emission wavelength peaks. The spectral scan unit can be used to optimize image acquisition since the sensitivity of each channel can be adjusted with the same responsiveness as the filter version.
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High sensitivity detection system.
High-sensitivity, high S/N ratio optical performance is achieved through integration of a pupil projection lens within the optics and employment of a highsensitivity photomultiplier and analog processing circuit with minimal noise.
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Photon counting detection mode.
The Olympus original Hybrid Photon Counting Mode (HPCM) can successfully capture images even when fluorescence emission is weak. This mode optimizes photomultiplier control conditions to acquire images with minimum noise and high quantitative performance.
Spectral unmixing.
Fluorescence can easily be separated through two modes (Normal and Blind). In Normal mode, separation is performed based on a designated ROI fluorescence spectrum using already-known fluorochrome wavelength data, or data derived from acquired images. In Blind mode, the separation uses an iterative process to derive the best fit of a given number of fluorescence spectra.
- 2nm spectral resolution allows two fluorochromes with similar emission peaks to be clearly separated
- Spectral unmixing is successful even when there are emission intensity differences in each fluorochrome
- A fine diffraction grating is used to gain precise separation for unmixing.
New PLAPON60xOSC objective
With low chromatic aberration, to deliver even better precision for colocalization analysis.
The new high NA oil-immersion objective,PLAPON60xOSC minimizes chromatic aberration in the 405–650 nm region for enhanced imaging performance and image resolution at 405 nm. It 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.
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Low Chromatic Aberration Objective PLAPON60xOSC
Magnification: 60xNA: 1.4 (oil immersion) W.D.: 0.12 mm Chromatic aberration compensation range: 405–650 nm Optical data provided for each objective.
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Lateral and Axial Chromatic Aberration
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Chromatic Aberration Comparison for
PLAPON 60×OSC and UPLSAPO 60×O
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PLAPON 60×OSC and UPLSAPO 60×O
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Diffusion Measurement Package
Analysis of the molecular interaction within cells.
This optional software module enables data acquisition and analysis to investigate the molecular interaction and concentrations 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.
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Diverse analysis methods for intracellular molecular interactions.
RICS (Raster Imaging Correlation Spectroscopy).
Raster image correlation spectroscopy (RICS) is a 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.
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Application
RICS can be used to designate and analyze regions of interest based on acquired images. 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 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 simultaneously determine changes in diffusion coefficient, for detailed analysis of various intracellular signaling proteins.
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Point FCS (Point scan Fluorescence Correlation 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.
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FRAP(Fluorescence Recovery after Photobleaching) analysis.
The Axelrod analytical algorithm is installed as a FRAP(Fluorescence Recovery after Photobleaching) analysis method. The algorithm is used to calculate diffusion coefficients and the proportions of diffusing molecules.
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Fluorescence Recovery after Photobleaching
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Compatible with laser light stimulation and spectral unmixing.
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Suitable for multiple users
For the convenience of multiple researchers, each user may create an independent log in which contains individual settings such as the display window and toolbar. To change researchers, the new user simply logs in at software start-up.
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Wide choice of scanning modes
Several scanning modes are available, including ROI, point and high-speed bi-directional scanning. These can be used together freely, in such combinations as XYZ, XYT, XYZT, XYλ, XYλT, etc.
Easy designation of scan area
The observation field of view and scanning area are displayed graphically, and settings can be altered while confirming the magnifications with the scroller. The operator can move the image scan area at will using the Pan button.
Rotation scanning of the image field is also possible.
Eliminate cross talk by using
Both frame and line sequential modes can be selected. The order of image acquisition, or image combinations, can be freely changed.
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Free emission wavelength selection
When selecting fluorochromes from within the software, the fluorescence spectra are displayed and the ideal detection wavelengths are automatically selected. Naturally, manual adjustment of the wavelength emission range, in as little as 1nm step increments, is also possible in order to optimize acquisition of specific fluorescence emission peaks.
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One-touch exchange between confocal and fluorescence observation
Since the FV1000 is fully automated, one-touch exchange between confocal and fluorescence observation is possible. In addition, microscope settings can be easily changed through the Microscope Controller.
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Easy image acquisition for 3D, 4D and 5D series
Multi-dimensional image acquisition, such as λ series, Z series and timelapse, is easily performed.
Automatic contrast
Automatic contrast selects optimal photomultiplier voltage settings for each detection channel, simplifying image acquisition.
Real-time emission intensity graph display
For image acquisition in real time, live emission intensity is displayed in a graph. Since the images are captured using the full 12-bit capacity, this is also convenient for setting sensitivity levels.
Easy image search
Explorer software provides simultaneous thumbnail image display, making it easy to search for previously stored data.
Data manager
The data manager provides thumbnail displays and different types of file information.
2D image display
The 2D control panel enables free manipulation of the image display. Tiling display or multi-dimensional image display can also be freely selected.
Re-use function
Previously-set scanning conditions can be recalled and applied to new or subsequent experiments.
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File Input/Output
OIB (Olympus Image Binary format) is employed to acquire both scanning and microscope setting conditions together with image acquisition data. This software also handles all widely used image formats (TIF, BMP, JPEG, AVI, MOV etc) with high interchangeability
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FV1000-ZDC
Corrects for thermal drift during confocal time-lapse imaging.
During long time-lapse observations, temperature changes around the microscope and drug administration during the observation cause focal drift, resulting in a loss of focus on the target. For confocal laser scanning biological microscopes with high resolution in the Z-direction, even slight focal drift can impair image acquisition to the point that images are no longer useful to researchers. Olympus is the world leader in equipping a confocal laser scanning biological microscope with zero thermal drift compensation.
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TIRFM (Total Internal Reflection Fluorescence Microscopy)
High S/N images near the cell surface: automated control of necessary volume of laser filtering light enables easy reproduction of TIRFM observation.
This special TIRFM unit employs the FV1000's laser for TIRFM illumination. The incident angle of the excitation laser toward the specimen is controlled through FV1000 software FV10-ASW, to set up the necessary laser filtering light volume. The optimum light path length is provided automatically through the selection of excitation wavelength and the objective. Since TIRFM observation can be done by exchanging confocal observation, protein localization on the cell surface and cross section images of the cell interior may be acquired simultaneously. A CCD is required for TIRFM image acquisition and image-capturing software is required. Note that time-lapse imaging by interchanging CCD and confocal images and then overlapping them is not possible.
* Cannot be incorporated with SIM scanner
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TIRFM
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LSM
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| Features |
Acquisition of high S/N images at 50-200nm depths from the specimen surface (surface observation only) |
Allows observations from the surface to the intracellular structure
Capable of 3D observation for each Z-axis position
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| Acquisition of high S/N images with minimal influence from the background |
Capable of composing 3D images from slice images |
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Imaging through CCD camera
Frame rate depends on camera performance |
Point scanning
Detector incorporates photomultiplier |
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Scanning Modes
Live tile mode, Look back function.
This allows the user to check the acquired images during time lapse experiment. Adjustment of focus and/or image brightness can be done during rest time.
High-speed image acquisition (4KHz/line).
High-speed scanning mode can capture confocal images at 16 frames per second with 256 x 256 pixel resolution. In combination with clip scanning, images can be acquired even faster than video rate.
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Live plot function
Changes in fluorescent intensity in a region designated as ROI are plotted in real-time during image acquisition.
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Wide variety of line scanning modes.
Line scanning for a straight line, slanted line or free curve enables easy
analysis of changes over time at the msec order. Observation of complex time lapse combined with Z or λ element is also possible.
Trigger function.
The system also has a trigger function for synchronizing scanning with external devices. Scanning can be started/stopped with a trigger signal, and it is also possible to gather frames for each external trigger.
Laser monitoring function.
Feedback is applied to the laser output so that the sample is always exposed to a fixed level of excitation light. There is no need to pay attention to variation in laser output, and the amount of fluorescence can be measured accurately.
3D Selectable rendering modes.
Using the laser light stimulation setting function (Stimulus Setting), laser light stimulation experiments can be done with either the main scanner (image scanner) or SIM scanner. Even with the main scanner only, it is possible to perform laser light stimulation during a time course by instantaneously switching the laser scanning mode (laser wavelength, laser irradiation range). However, images cannot be acquired during laser light stimulation.
Interactive volume rendering.
Using the interactive volume rendering method with the 3D display function, the angle of a 3D rendered image can be freely changed to the direction you wish to see by operating the mouse. A variety of display functions are available, including the ability to display a cross section at an arbitrary location, and extend focus images.
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