Using immunofluorescent techniques makes the choice of a microscope very important. Results can depend on microscope quality making it an important investment.
We have used an Olympus FluoView™ 500 confocal laser scanning fluorescence microscope for a long time been and have been rather satisfied with its performance. This is a basic model which nevertheless, offers the features necessary for optical sectioning, 3D construction and time-series observation, as well as a variety of image processing and analysis functions. Several of its features are noteworthy.
The scan unit can be used in common with an upright or inverted microscope. We have used this confocal microscope in combination with the BX50WI inverted microscope from Olympus.
Two modes of acquisition of signal are available: sequential or simultaneous. A sequential scan is essential to limit crossover between different fluorescent labels. A total of 5 image channels allow for simultaneous acquisition of up to 4 fluorescence images and 1 transmitted image. Our laboratory opted for 3 lasers: an Argon laser (488 nm excitation), a Helium-Neon green laser (543 nm excitation), and a Helium-Neon red laser (633 nm excitation). These 3 lasers allow triple-labelling with common fluorescent labels, emitting green, red and far red light.
The transmitted light is detected by a photomultiplier; its detector has 12-bit resolution and makes the Olympus FluoView™ 500 microscope capable of detecting small changes in the fluorescence inside cells.
The system has high resolution: 2048 x 2048 pixels, and 10-fold digital zoom is available to help in seeing minor details. However, when you use high-speed scanning (4 images per second), useful in time-lapse experiments, the resolution will be less (512 x 512 pixels).
The auto gain adjustment feature eliminates the need for complicated gain adjustment operation. This rapidly gives a general idea of the staining. Thereafter, you can proceed to minor manual adjustment.
The confocal aperture is continuously variable and the optimum confocal aperture for
each objective is set automatically, as are the dichroic mirrors for excitation and spectroscopy.
In addition to faster scanning, scanning modes such as vector scanning and oblique
scanning are provided to meet a wide range of applications. When using the system with acousto-optical tunable filter (FV5-COMBA), you can benefit from additional functions, such as line Kalman scanning, line sequential scanning, and image acquisition in the REX/Bleach mode (FRAP experiment).
The FluoView™ software is organized by 2 kinds of panels: the function panel and
display panel. Different sections of the former (Acquire, File, Tile, Process, Analyze and
Visualize) help in transitioning rapidly to the necessary function. The latter panel shows either the live panel or the panel image loaded from a file (filename panel).
Finally, I should say that using Olympus FluoView™ 500 microscope is simple and intuitive. You can easily control and modify the acquisition process and obtain nice images provided that the emission spectra of the fluorescent labels you are using are sufficiently spaced out.
Alexandra Khomitch-Baud, PhD
Cellular Imaging Department