Features and Comparison of Fluorescence Microscope and Conventional Optical Microscope
A fluorescence microscope differs from an ordinary optical microscope. Instead of illuminating the specimen with ordinary light sources, it uses light of a specific wavelength (usually ultraviolet or blue-violet light) to excite fluorescent substances within the specimen to emit fluorescence.The light from the light source is not used for direct illumination, but serves as excitation energy for the fluorescent substances in the specimen. Therefore, we can observe the specimen not because of illumination from the light source, but because of the fluorescence emitted after the fluorescent substances in the specimen absorb the excitation light.It follows that the key feature of a fluorescence microscope is that its light source must provide excitation light of a specific wavelength, which enables the fluorescent substances in the specimen to emit fluorescence of sufficient intensity. Meanwhile, a fluorescence microscope must be equipped with a dedicated filter system.The fluorescence microscope is a fundamental instrument for immunofluorescence histochemistry. It mainly consists of an ultra-high pressure light source, filter sets (including excitation and barrier filters), an optical system and supporting components. It uses light of a specific wavelength to excite fluorophores.
There are two excitation modes classified by wavelength range: the UV excitation method (ultraviolet irradiation method) and the BV excitation method (blue-violet excitation method).
UV excitation method: Near-ultraviolet light shorter than 400 nm is used for excitation. No visible excitation light exists in this method. The observed fluorescence shows the intrinsic 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 with light centered at 404 nm and 434 nm, ranging from ultraviolet to blue-violet. Blue light irradiates the specimen in this method. Hence, the barrier filter of the fluorescence observation system must completely block blue light while transmitting the required green and yellow fluorescence. It is used for fluorochromes in immunofluorescence assays. Since the maximum absorption wavelength of the fluorochrome is close to the excitation wavelength of the BV method, blue light can be used as the excitation light, resulting in high light absorption efficiency of the fluorochrome and relatively clear images. Its disadvantage is that fluorescence at wavelengths below 500 nm cannot be observed, and only fluorescence signals above 500 nm can be displayed on the image. In immunofluorescence assays, fluorochromes are identified partly by their color. Therefore, when subtle specificity is discussed, the above drawback of the BV excitation method has a significant impact.
