Comparison and Features of Fluorescence Microscope vs. Ordinary Light Microscope
Fluorescence microscopes differ from ordinary light microscopes. Instead of observing specimens under illumination from a conventional light source, they use light of a specific wavelength (usually ultraviolet or blue-violet light) to excite fluorescent substances within the specimen on the microscope, 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 illumination from the light source, but due to the fluorescence phenomenon produced after the fluorescent substances in the specimen absorb the excitation light energy.
It follows that the key feature of a fluorescence microscope is that its light source delivers abundant excitation light within a specified wavelength range, so that fluorescent substances in the test specimen can receive excitation light of sufficient intensity. Meanwhile, a fluorescence microscope must be equipped with an appropriate filter system. The fluorescence microscope is an essential tool for fluorescence histochemistry. It consists of major components including an ultra-high-pressure light source, a filter set (including excitation and barrier filters), an optical system and a photographic system. It works by exciting specimens with light of a certain wavelength to induce fluorescence emission.
Fluorescence excitation methods: Classified by wavelength range into two types: UV excitation (ultraviolet illumination method) and BV excitation (blue-violet light method). The UV excitation method uses near-ultraviolet light shorter than 400 nm for excitation. No visible excitation light is present with this method. Consequently, the observed fluorescence shows the inherent fluorescence of the dye, making it easy to distinguish specific fluorescence on the specimen from autofluorescence of the background tissue.
BV excitation method: Excitation is performed using light ranging from ultraviolet to blue, centered at 404 nm and 434 nm. Since this method irradiates the specimen with blue light, the cut-off filter of the fluorescence observation system must fully block blue light while sufficiently transmitting the required green and yellow fluorescence. It is applied to fluorochromes used in fluorescent antibody assays. The maximum absorption wavelength of the excitation light is relatively close to the maximum emission wavelength of fluorescence, so sharp-cut filters are mandatory for the BV excitation method. Blue light can be used as the excitation light in this approach, which yields high absorption efficiency of fluorochromes and relatively bright images. Its disadvantage is that fluorescence below 500 nm cannot be detected, while signals above 500 nm render the whole image yellow. In fluorescent antibody assays, specificity is mostly judged by the characteristic colour of fluorochromes. Therefore, when subtle specificity is under evaluation, the drawbacks of the BV excitation method often exert a significant adverse impact.
