Fluorescence Microscopy Technical Parameters Fluorescence Microscopy Techniques and Methods
Fluorescence Microscope Technical Parameters 1. Wide-angle eyepiece 2. Achromatic objective lens 3. Four-hole objective lens converter 4. Epi-fluorescent device Blue (B) Green (G) excitation system 100W mercury lamp 5. Coaxial coarse and fine focus adjustment mechanism: adjustment Focus range: 15mm Micro-movement grid value: 0.002mm 6. Double-layer mechanical table Longitudinal movement range: 70mm Lateral movement range: 50mm
Fluorescence microscope technical parameters
1. Wide-angle eyepiece
2. Achromatic objective lens
3. Four-aperture nosepiece
4. Epi-fluorescence device blue (B) green (G) excitation system 100W mercury lamp
5. Coaxial coarse and fine focus adjustment mechanism: focus range 15mm fine movement grid value 0.002mm
6. Double-layer mechanical workbench
Vertical movement range: 70 mm Lateral movement range: 50 mm
Fluorescence Microscopy Skills and Methods
(1) Glass slide
The thickness of the glass slide should be between 0.8 and 1.2mm. A slide that is too thick will absorb more light on the one hand, and on the other hand, the excitation light cannot be concentrated on the specimen. Slides must be smooth, uniform in thickness, and free from obvious autofluorescence. Sometimes quartz glass slides are used.
(2) Cover glass
The thickness of the cover glass is about 0.17mm, smooth. In order to strengthen the excitation light, an interference cover glass can also be used, which is a special cover glass coated with several layers of substances (such as magnesium fluoride) that have different interference effects on light of different wavelengths, which can make the fluorescence go smoothly. Exciting light is passed through and reflected, and this reflected excitation light excites the specimen.
(3) Specimen
Tissue slices or other specimens should not be too thick. If it is too thick, most of the excitation light will be consumed in the lower part of the specimen, while the upper part directly observed by the objective lens will not be fully excited. In addition, cell overlapping or impurity cover, affect the judgment.
(4) Mounting agent
Glycerin is commonly used as a mounting agent, which must have no autofluorescence, colorless and transparent, and the brightness of the fluorescence is brighter at pH 8.5-9.5, and it is not easy to fade quickly. Therefore, an equal mixture of glycerol and 0.5mol/l carbonate buffer solution with a pH of 9.0 to 9.5 is commonly used as a mounting agent.
(5) mirror oil
Generally, when observing specimens with dark-field fluorescence microscopes and oil immersion immersion lenses, immersion oil must be used. It is best to use special non-fluorescent immersion oil. The above glycerin can also be used instead, and liquid paraffin can also be used, but the refractive index is low, which has a slight impact on image quality. Influence.
The principle and structural characteristics of fluorescence microscope
Fluorescence microscopy uses a point with high luminous efficiency to emit light of a certain wavelength (such as ultraviolet light 3650 in or purple blue light 4200 in) through the filter system as the excitation light to excite the fluorescent substances in the specimen to emit fluorescence of various colors After that, observe through the magnification of the objective lens and eyepiece. In this way, under a strong contrast background, even if the fluorescence is very weak, it is easy to identify and has high sensitivity. It is mainly used for the research of cell structure and function and chemical composition. The basic structure of a fluorescence microscope is composed of an ordinary optical microscope plus some accessories (such as a fluorescent light source, an excitation filter, a two-color beam splitter and a blocking filter, etc.). Fluorescent light source—generally use ultra-high pressure mercury lamp (50-200W), which can emit light of various wavelengths, but each fluorescent substance has an excitation wavelength that produces the strongest fluorescence, so an excitation filter ( Generally, there are ultraviolet, purple, blue and green excitation filters), which only allow excitation light of a certain wavelength to pass through and irradiate the specimen, while absorbing other light. After each substance is irradiated by excitation light, it emits visible fluorescence with a longer wavelength than the irradiation wavelength in a very short time. Fluorescence is specific and generally weaker than excitation light. In order to observe specific fluorescence, blocking (or suppression) must be added behind the objective lens and used in conjunction with it.
The difference between fluorescence microscope and ordinary microscope
1. The illumination method is usually episcopic, that is, the light source is projected on the sample through the objective lens;
2. The light source is ultraviolet light, the wavelength is shorter, and the resolution is higher than that of ordinary microscopes;
3. There are two special filters, the one in front of the light source is used to filter out visible light, and the one between the eyepiece and objective lens is used to filter out ultraviolet rays to protect the human eye.
Fluorescence microscope is also a kind of optical microscope, the main difference is that the excitation wavelength of the two is different. This determines the difference between the fluorescence microscope and the ordinary optical microscope in terms of structure and usage.
Fluorescence microscopy is an essential tool in immunofluorescent cytochemistry. It is composed of main components such as light source, filter plate system and optical system. It is to use a certain wavelength of light to excite the specimen to emit fluorescence, and to observe the fluorescence image of the specimen by amplifying the objective lens and eyepiece system.
