The Olympus FluoView^TM FV500 is a point-scanning, point-detection, confocal laser scanning microscope designed for biological research applications. Excellent resolution, efficiency of excitation, intuitive user interface and affordability are key characteristics of the Olympus FV500. A fully automated confocal system, the FV500 may be configured with ultraviolet through infrared lasers and permits simultaneous collection of up to 5 detection channels. The FV500 scanning unit can be coupled to either the IX2 inverted or the BX2 upright research microscope platforms.

Olympus FV500 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 FV500 microscope system configurations.

• The FV500 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.
• An intuitive, Windows-based graphic user interface allows new users to quickly generate images in various scan modes, such as x-y, x-z, x-t, x-y-z, x-y-t, and x-y-z-t. Standard image formats, including TIFF and AVI, permit easy, direct export of FluoView^TM images to off-line analysis packages.
• Lateral x-y scanning is performed with a pair of galvanometric mirrors, yielding a wide scanning range to cover up to a field number of 18. The optical zoom (up to 10x magnification) can be performed by narrowing the scanning range while maintaining the maximum pixel resolution of up to 2048 x 2048 pixels.
• Fully automated Olympus microscope platforms can be interactively controlled through the FluoView^TM software, with internal stepper motor z-resolution of 0.01 micrometer.
• Independent confocal apertures for each photomultiplier detection channel are continuously variable and software-controlled for an optimal match of each aperture diameter to the objective magnification, numerical aperture, and emission wavelength settings.
• 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 4 frames/second at 512 x 512 pixels, and line scan intervals at 0.5 milliseconds per line. 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 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.
• XYZ scanning - Acquires a series of confocal optical XY images through the thickness of the sample.
• 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).
• 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.
• 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 with the highest temporal resolution.
• XYZT scanning - Acquires an XYZ series over time, permitting observation and analysis of three-dimensional changes over time in live cells.
• 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 FV500 confocal microscopes have a broad spectrum of available wavelengths with intensities selectable by individual neutral density filters or the acousto-optic tunable filter (AOTF) controller for simultaneous or automated-sequential collection of multi-channel images. A choice of the 440-nanometer diode laser and the 442-nanometer helium-cadmium 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
• Blue-violet helium-cadmium (442 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. Unique laser modulation of the 405-nanometer diode laser permits high-speed control of the light intensity, which can be important for 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

In the FV500, as many as four high-sensitivity photomultiplier tubes can be incorporated directly within the confocal fluorescence emission light path for high sensitivity detection of the fluorescence signal. A fifth photomultiplier 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.

Depending on configuration, up to five 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 FV500 microscopes incorporate independent confocal apertures in front of each photomultiplier fluorescence detector. Each confocal aperture is continuously variable and software-controlled for an optimal match of each aperture diameter to the 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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