Confocal microscopy is a powerful tool in cellular biology that offers insights into the organization and function of cells and even organisms. Confocal microscopy is superior to conventional fluorescence imaging as it records fluorescence generated from the focal plane within the sample, while rejecting all other light coming from above or below that focal plane. The Olympus FluoView™ FV1000 is a leader among point-scanning, point-detection, confocal laser scanning microscopes. Its point-scan/pinhole-detection confocal optics produce high-contrast images with unparalleled resolution.
With the advance of imaging of biological specimens and of fluorochromes that can mark cell structures, there is a need for multiple laser lines. The FV1000 incorporates two laser scanners in a single compact design that enables the researcher simultaneous excitation and observation. In addition, this system minimizes specimen damage and thus enables the visualization of the specimen for long periods of times without reduction in imaging quality.
The FluoView confocal microscopes can be equipped with a variety of laser systems in several different configurations: argon-ion and helium-neon, as well as argon-krypton, helium-cadmium, and blue diode lasers. In our imaging core facility, we have both a blue diode and an argon-krypton system.
The Olympus system is very versatile and it can be adapted to tungsten-halogen and arc-discharge lamps for simultaneous imaging in fluorescence, confocal brightfield and differential interference contrast techniques. I always used the sequential scanning mode which minimizes the crosstalk between fluorophore channels as the system is able to excite each dye individually and then reassemble the images into a final montage. The microscope has the UIS infinity optics which give an impressive performance.
The Olympus FluoView FV1000 has a user-friendly image acquisition and image analysis software as well. I found that one can easily master the software with limited training time. Data saved as TIFF and AVI can be easily used and exported. With a few steps, one can generate composite and three-dimensional views of optical section data once it is acquired as z-series image stacks. I also used the software to measure various parameters such as length, volume, and depth. One can also easily add annotations and scale bars onto the acquired images. I also used extensively the Olympus FluoView FV1000 program to collect temporal data for various time-lapse experiments using cultured smooth muscle cells from rat arteries following wounding. The time-lapse images collected for 6 hours were analyzed in order to determine the speed of migration for smooth muscle cells to close the wound after various treatments. One can also conduct real time acquisition in smaller frames for short periods of time as well as use cells expressing fluorescent markers.
I found the Olympus FluoView acquisition software easy to use also for FRAP (fluorescence recovery after photobleaching experiments). This technique is extremely useful for studying cytoskeleton dynamics, a main focus of my studies. First a laser is used to photobleach fluorophores within a region of choice in the cell, then the software monitors the fluorescence intensity recovery in the bleached region over time. This is very useful for studying tubulin flux in microtubules as a measure of their dynamics, as well as of actin dynamics in actin filaments.
As an option, one can also perform Fluorescence Resonance Energy Transfer (FRET) by adding a helium-cadmium (HeCd) laser system to the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) combination.