Differences Between Fluorescence Microscopes and Ordinary Light Microscopes
Fluorescence microscopes differ from conventional optical microscopes. Instead of observing specimens with light from an ordinary 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 slide, causing these substances to emit fluorescence. Therefore, the light source of a fluorescence microscope does not directly illuminate the specimen; it acts as an energy source to excite the endogenous fluorescent substances in the specimen. We can see the specimen not because of illumination from the light source itself, but because the fluorescent substances inside the specimen absorb the excitation light energy and then emit fluorescence.
It follows that the key feature of a fluorescence microscope is that its light source can supply excitation light of a specific wavelength band, enabling the fluorescent substances in the inspected specimen to emit fluorescence of sufficient intensity. Meanwhile, a fluorescence microscope must be equipped with a corresponding filter system. It is an essential instrument for fluorescence histochemistry. A fluorescence microscope is mainly composed of an ultra-high pressure light source, a filter system (including excitation filters and barrier filters), an optical system and a photographic system. It works by using light of a specific wavelength to excite fluorescent substances.
① Methods of Fluorescence ExcitationAccording to the wavelength range of the excitation light, there are two types: UV excitation method (ultraviolet illumination method) and BV excitation method (blue-violet illumination method).The UV excitation method uses ultraviolet light shorter than 400 nm for excitation. It generates invisible fluorescence, so the observed fluorescence comes from the specimen itself. This method easily distinguishes specific fluorescence from the autofluorescence of the background.
② BV Excitation MethodThe BV excitation method uses excitation light ranging from violet to blue centered at 404 nm and 434 nm. Since blue light is used to irradiate the specimen, the excitation filter system for this method must completely block blue light and only allow the required green and yellow light to pass through.For fluorescent antibody staining, the maximum absorption wavelength of the excitation light is close to the maximum emission wavelength of the fluorescent dye. For this reason, the filter set for BV excitation must use a cut-off barrier filter. This method uses blue light for excitation, so the fluorescence collection efficiency is relatively high and brighter images can be obtained. Its disadvantage is that fluorescence above 500 nm cannot be detected, and only yellow-green images can be seen above 500 nm. In fluorescent antibody staining, judgment of specificity relies heavily on color information, so the drawback of the BV excitation method can be a major limitation when discussing subtle specificity.
