Comparison and Features of Fluorescence Microscopes and Conventional Optical Microscopes
A fluorescence microscope differs from an ordinary optical microscope. Instead of observing specimens under illumination from a conventional light source, it uses light of a specific wavelength (usually ultraviolet or blue-violet light) to excite fluorescent substances within the specimen, causing them to emit fluorescence. Therefore, the light source of a fluorescence microscope does not serve for direct illumination; it acts as an energy source to excite endogenous fluorescent substances in the specimen. We are able to observe the specimen not because of lighting from the source, but due to the fluorescence emitted by the fluorescent substances in the specimen after absorbing the excitation light.
It follows that the key feature of a fluorescence microscope is its light source, which can supply abundant excitation light within a specific wavelength range to provide sufficient excitation intensity for the fluorescent substances in the test specimen. Meanwhile, a fluorescence microscope must be equipped with a corresponding filter system. As an essential tool for fluorescence histochemistry, it mainly consists of an ultra-high-pressure light source, a filter set (including excitation filters and barrier filters), an optical system and a photographic system. It works by exciting specimens with light of a certain wavelength to produce fluorescence.
Fluorescence excitation methods: Classified by wavelength range into UV excitation (ultraviolet illumination method) and BV excitation (blue-violet light method). UV excitation uses near-ultraviolet light shorter than 400 nm for excitation. No visible excitation light is involved, so the observed fluorescence shows the inherent fluorescence of the dye. This makes it easy to distinguish specific fluorescence on the specimen from autofluorescence of the background tissue.
BV excitation method: Excitation is carried out with light ranging from ultraviolet to blue light, centered at 404 nm and 434 nm. Since blue light irradiates the specimen in this method, the cut-off filter of the fluorescence observation system must completely block blue light while fully transmitting the required green and yellow fluorescence. This method is applied to fluorochromes used in fluorescent antibody techniques. The maximum absorption wavelength of the excitation light and the maximum emission wavelength of the fluorescence are relatively close, so sharp-cut filters are required for BV excitation. Blue light is used as excitation light here, which brings high absorption efficiency of fluorochromes and yields brighter images. Its drawback is that fluorescence below 500 nm cannot be detected, and signals above 500 nm give the whole image a yellow tint. In fluorescent antibody assays, specificity is mostly judged by the characteristic color of the fluorochrome. Therefore, when evaluating subtle specificity, the above disadvantage of the BV excitation method can have a significant impact.
