Principles of Fluorescence Microscopy
Fluorescence microscopes are different from ordinary optical microscopes. Instead of observing specimens under the illumination of ordinary light sources, they use light of a certain wavelength (usually ultraviolet light, blue-violet light) to excite the fluorescent substances in the specimens under the microscope to make them emit fluorescence. Therefore, The light source of a fluorescence microscope does not function as direct illumination, but as an energy source that excites the fluorescent substances within the specimen. The reason why we can observe the specimen is due to the illumination of the light source, but the fluorescence phenomenon presented after the fluorescent substance in the specimen absorbs the excited light energy.
It can be seen that the characteristic of the fluorescence microscope is that its light source can supply a large amount of excitation light in a specific wavelength range, so that the fluorescent substance in the tested specimen can obtain the excitation light of the necessary intensity. At the same time, the fluorescence microscope must have the corresponding filter system.
Fluorescence microscopy is an essential tool in immunofluorescence histochemistry. It is composed of ultra-high-voltage light source, filter system (including excitation and suppression filter plate), optical system and photography system and other main components. It uses light of a certain wavelength to excite the specimen to emit fluorescence.
The way to excite fluorescence: According to the wavelength range of light, it can be divided into two types: UV excitation method (using ultraviolet lighting method) and BV excitation method (using blue-violet light). The UV excitation method uses near-ultraviolet light shorter than 400nm for excitation. There is no visible excitation light in this method, so the observed fluorescence presents the inherent fluorescence of the dye, and it is easy to distinguish the specific fluorescence on the specimen from the autofluorescence of the background tissue.
The BV excitation method is based on 404nm and 434nm for excitation from ultraviolet to blue light. This method uses blue light to irradiate the specimen, so the cut-off filter of the fluorescence observation system must use a filter that can completely block the blue light and fully pass the required green and yellow fluorescence. Fluorescent dyes for fluorescent antibody assays. The maximum absorption wavelength of excitation light is relatively close to the maximum emission wavelength of fluorescence, so the filter used in the BV excitation method must use a sharp cut-off filter. This method can use blue light as the excitation light, so the absorption efficiency of the fluorescent pigment is higher, and a brighter image can be obtained. Its disadvantage is that the fluorescence below 500nm cannot be seen, and the fluorescence above 500nm makes the whole image appear yellow. In the fluorescent antibody method, the specificity of the fluorochrome is often judged by the color unique to the fluorochrome. Therefore, when discussing subtle specificity, the above-mentioned shortcomings of the BV excitation method often have a great influence.
To sum up, the illumination of the fluorescence microscope can be considered according to the following three points according to the structure of the condenser and the wavelength of the excitation light.
① From the perspective of the contrast requirements of the fluorescent image, the UV excitation dark field concentrator is used for illumination.
② Considering the brightness of the image, BV excitation filter has the highest dark field observation efficiency.
③The characteristics of UV excitation filter dark field observation and BV excitation dark field concentrator illumination can be regarded as between these two illumination methods, but the former has stronger dark field properties and displays brighter images. The contrast is small; the latter retains the properties of the dark field light path, so the displayed image is darker, and the contrast is improved. When actually using a fluorescence microscope, the illumination method that best suits the requirements of the specimen should be used for observation.
It must be pointed out that even if the UV-excited dark field condenser with the best contrast is illuminated, part of the ultraviolet excitation light refracted or scattered by the specimen will enter the objective lens. Autofluorescence can worsen the image response. Therefore, a UV-absorbing filter must be used in front of the eyepiece as a cut-off filter.
