Using Fluorescence Microscopy: Techniques and Skills
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
Use skill method
(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 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.
