The Olympus FluoView^TM 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 simultaneously provides high-resolution imaging. This coordination of laser stimulation and imaging makes the FV1000 an ideal choice for FRAP, FLIP and photoactivation.

Olympus FV1000 Confocal Microscope Features

Confocal microscopy can improve conventional fluorescence images by recording fluorescence generated from the focal plane within the sample, while rejecting all other light coming from above or below the focal plane. The efficient point-scan/pinhole-detection confocal optics of the FluoView^TM systems virtually eliminate out of focus light to produce high-contrast images with superb resolution. The features listed below are available on all Olympus FV1000 microscope system configurations.

• The FV1000 systems are fully integrated workstations that incorporate user-friendly image acquisition and image analysis software with high-resolution confocal optics that require no user alignment. Olympus offers a choice of several optional scanning module configurations, including a 4-channel, non-confocal, SIM (dual laser) scanning, and fiber port unit.
• An intuitive, Windows-based graphic user interface allows new users to quickly generate images in a wide variety of common scan modes, such as X-Y, X-Z, X-T, X-Y-Z, X-Y-T, and X-Y-Z-T. In addition, scanning heads equipped for multi-spectral analysis can acquire X-l, X-Y-l, X-l-Z, X-l-T, and X-l-Z-T, and X-Y-l-T lambda stacks. Standard image formats, including grayscale, JPEG, BMP, TIFF and AVI (24/32/48 bit full color), permit easy, direct export of FluoView^TM images to off-line analysis packages.

• The FV1000 features a specially designed optical system with an independent photomultiplier for each channel. The spectrometry detector system utilizes diffraction gratings for high-speed x-y-l-t acquisitions. The 2-nanometer resolution makes it possible to clearly separate crosstalk produced by two fluorochromes with similar emission spectral profiles. The spectrometer operates in Normal Mode, which requests the input of fluorophore spectral profile data, and a Blind Mode that performs the separation automatically based on information derived from the specimen image.
• The SIM scanning system can synchronize laser light stimulation and imaging to capture rapid changes in living cells immediately following fluorophore excitation. In addition, the tornado scanning mode (available only on the SIM scanning unit) can be used to efficiently photobleach defined specimen areas, thus minimizing the waste areas experienced with conventional reciprocating systems.
• Lateral x-y scanning is performed with a pair of high-speed (16 frames per second) galvanometric mirrors, yielding a wide scanning range up to a field number of 18. The optical zoom (up to 50x magnification, in 0.5x increments) can be performed by narrowing the scanning range while maintaining the maximum pixel resolution of 2048 x 2048 pixels (the range can be extended from 64 x 64 to 4096 x 4096 with applications software).

• Fully automated Olympus microscope platforms can be interactively controlled through the FluoView^TM software, with an internal stepper motor z-resolution of 0.01 micrometer.
• A common pinhole for all channels can be adjusted in aperture size between 50 and 800 micrometers in photomultiplier detection channels, and between 50 and 300 micrometers in spectrometer channels (adjustments in 0.5 micrometer increments). The aperture is software-controlled for an optimal match to the objective magnification, numerical aperture, and emission wavelength settings.
• The optional SIM scanner unit features two galvanometer mirror scanners, a pupil projection lens, built-in laser shutter, and a single laser port. The 4-channel detector unit allows multiple fluorophores to be imaged simultaneously, while the non-confocal detector features 2-channel detectors with ion deposition dichromatic mirrors and barrier filters for wavelength selection. The fiber port enables fluorescence output with a 100 to 125 micrometer fiber core. Photomultipliers are capable of operating either in analog accumulation or photon counting mode. The FV1000 scanning units feature an integral transmitted light detector, which is connected to an external light source via a fiber channel. Brightfield and differential interference contrast (DIC) images can be simultaneously recorded with the confocal fluorescence images. The transmitted light images, while non-confocal, are very useful when superimposed to the confocal image.
• Time-lapse observation is possible on the FluoView^TM with imaging rates of up to 16 frames per second at 512 x 512 pixels and line scan intervals at 0.5 milliseconds per line. A feedback mechanism monitors laser light in the scanner and uses this data to control the AOTF to ensure excitation light of constant intensity. A Time Series Controller is employed to maintain the precision of time-lapse experiments in microseconds and also allows very accurate control of synchronization to external devices (such as shutters). Kinetic and ratiometric analysis is available for quantitative analysis of fluorescence changes in live cells over time. Real time intensity plotting and ratiometric image display are also possible.
• Multi-parameter experiments can be quickly designed and executed through the powerful Programmable Acquisition Protocol Processor (PAPP) software, an integrated component of the FluoView^TM software package. With the PAPP software, advanced time-lapse protocols involving photobleaching, photoactivation, or multi-location acquisition can be easily generated and saved as routine protocols.
• Transistor-transistor logic (TTL) input and output signals can be generated to coordinate experiments timed with external instrumentation.

Versatility in Scanning Modes

The FluoView^TM confocal microscopes are equipped with several efficient scanning modes, including point, line, free line, and rectangle, which are especially suited for many time-lapse applications.

• XY scanning - Acquires a single confocal optical image. Rotation of the image through 360° is available.
• XZ scanning - Acquires a single cross section image that cannot be obtained with a conventional microscope. The cross section image may also be rotated 360° or drawn as a free line (free line-Z).
• XT scanning - Acquires a single line image over time for time-lapse analysis with high temporal resolution. The cross section image may also be rotated 360° or drawn as a free line (free line-T).

• Point scanning - Acquires a series of intensity changes at a single point within the image to produce the highest temporal resolution.
• Xl scanning - Acquisition of line data as a function of wavelength. The line may be rotated by 360° or drawn as a free line (free line-l). The spectral system is required.
• XYZ scanning - Acquires a series of confocal optical XY images through the thickness of the sample.
• XYl scanning - Acquires a single confocal optical two-dimensional (XY) image as a function of wavelength. Image rotation through is available. The spectral system is required.
• XYT scanning - Acquires a single XY confocal optical image over time, at an interval that can be arbitrarily chosen. This scanning mode permits observation and analysis of live cell kinetics, such as changes in intracellular calcium ion concentrations, pH, and membrane potential.
• XlT scanning - Acquires a single confocal line image as a function of wavelength and time (for time-lapse analysis). The line may be rotated over 360° or drawn as a free line (free line-T). The spectral system is required.
• XlZ scanning - Acquires a single confocal cross sectional (XZ) image through the thickness of the sample as a function of wavelength. The cross section image may also be rotated throught 360° or drawn as a free line (free line-Z). The spectral system is required.
• XYZT scanning - Acquires an XYZ series over time, permitting observation and analysis of three-dimensional changes over time in live cells.
• XlZT scanning - Acquires a single confocal cross sectional (XZ) image through the thickness of the sample as a function of wavelength and time. The section may be rotated through 360° or drawn as a free line (free line-T). The spectral system is required.
• XYlT scanning - Acquires a series of confocal optical XY images as a function of wavelength and time for time-lapse analysis. The image may be rotated through 360°. The spectral system is required.
• Automated Scanning Stage - Multi-location image acquisition is available for most imaging modes, including XYZ, XYT and XYZT, with addition of an optional automated scanning stage.

Laser Systems Offer Large Selection of Wavelengths

Laser systems designed for the Olympus FV1000 confocal microscopes have a broad spectrum of available wavelengths with intensities selectable by the acousto-optic tunable filter (AOTF) controller for simultaneous or automated-sequential collection of multi-channel images. The 440-nanometer blue-violet diode laser is available for fluorescent protein (cyan and yellow, CFP/YFP) resonance energy transfer applications, providing optimal excitation of CFP and spectral separation from the YFP. The shutters and light intensity can be controlled via the system computer.

• Blue argon-ion (488 nanometers) laser
• Multi-line argon-ion (457, 488, and 514 nanometers) laser

• Green helium-neon (543 nanometers) laser
• Red helium-neon (633 nanometers) laser
• Yellow krypton-ion (568 nanometers) laser
• Violet and blue-violet diode (405 and 440 nanometers) lasers
• Ultraviolet argon-ion (351 nanometers) laser
• Infrared diode (750 nanometers) laser

The 405-nanometer diode laser, a low cost alternative to water-cooled ultraviolet lasers, can be used for most ultraviolet imaging applications, including nuclear imaging with DAPI and the Hoechst DNA probes. Unique laser modulation of the 405-nanometer diode laser permits high-speed control of the light intensity, which can be critical for live-cell photoactivation and photobleaching studies. The exceptional light stability of the 405-nanometer diode laser is also important for time-lapse studies of living cells.

Additional Features

The newly designed FV1000 high-sensitivity detection system coupled to the high-speed scanning and laser feedback mechanism enables the capture of extremely rapid changes occurring in living organisms. The confocal microscope employs a new spectral technique using diffraction gratings to provide more distinct wavelength separation with very precise resolution. A proprietary algorithm allows easy separation in the event of unexpected fluorescence emission crosstalk.

In the FV1000, as many as three high-sensitivity photomultiplier tubes can be incorporated directly within the confocal fluorescence emission light path for high sensitivity detection of the fluorescence signal. A fourth detector, dedicated for transmitted light imaging, may be used for the simultaneous detection of high-resolution brightfield or DIC images with which the confocal fluorescence images may be overlaid.

All FV1000 filters (dichromatic mirrors and barrier filters) feature a multi-layer interference coating applied by means of ion deposition technology. This provides sharp cut-on/cut-off characteristics that cannot be achieved using conventional vacuum deposition methods. Another key advantage is the filters' total coverage of all the fluorescence wavelength ranges of fluorescent proteins, which enables observation under conditions of very weak excitation illumination, thus minimizing damage to living cells.

Depending on configuration, up to four of the detection channels may be imaged simultaneously. Images may be scanned in any pixel array size up to 2048 x 2048 pixels, with each channel digitized to 4096 gray levels (12-bit), permitting observation of fine image detail. Images may also be acquired in an automated sequential mode in order to reduce spectral crosstalk between channels for multi-color images.

The FV1000 microscopes incorporate a common pinhole aperture for all channels that is continuously variable between 50 and 100 micrometers for software-controlled matching of the diameter to objective magnification, numerical aperture, and emission wavelength settings.

Automated software control of the FluoView^TM confocal microscope laser excitation, emission dichroics, and barrier filters provide a simplified setup of the system's hardware. Simply select the combination of fluorochromes to be imaged and the software automatically configures the complete imaging pathway for each channel to be recorded.

Flexibility in the selection of the microscope base, laser lines, and optical filters permits researchers to customize their Olympus confocal system for a broad range of research applications.

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