Paraffin Section Making Process for Light Microscopy
In order to obtain better mirror image contrast, excess stray light should be shielded from entering the observation mirror field or imaging area as much as possible. When observing through the eyepiece with different magnification objectives, the field of view diaphragm can be adjusted to form a docking or slightly corresponding eyepiece mirror field. Later; in photomicrography or digital CCD imaging, the field of view diaphragm can be reduced according to the viewfinder frame and CCD imaging area, but do not shrink too much to cause the corner of the photo to be cut. When using the oil lens, it can also be properly zoomed out.
Condenser Numerical Aperture Setting
First of all, the numerical aperture (NA) range of the condenser lens should be basically equal to the NA value of the objective lens and marked with an NA scale. It is necessary to add oil to the top for high-magnification microscopic inspection. The function of the aperture condenser is to provide effective focused projected light corresponding to the numerical aperture of the objective lens. If the NA of the condenser lens is set larger than the NA of the objective lens, part of the light will be wasted. If the NA of the condenser lens is set too small, the projected light will be insufficient and due to the change of the exit angle, part of the light will irradiate the specimen obliquely and appear diffracted. Mirroring, both reduce the resolution of the mirroring. Therefore, certain corresponding setting principles should be followed. The empirical value is to adjust within 80--100% of the NA of the corresponding objective lens during microscope inspection. It is recommended to set it at 60-80% or 70% in order to obtain a more suitable contrast and depth of field during photography. - 80%. This means that every time you switch between different magnification objective lenses, you need to make corresponding adjustments. In practice, you can also make fine adjustments according to the thickness of the specimen and the depth of staining.
Poor coverage correction, the thickness of the standard cover glass is 0.17mm, and the thickness of the slide glass is 1.0mm. Due to the use of cover glass or slide glass that does not meet the standard and the uneven thickness of the slice and mounting agent, the coverage difference will occur, which will greatly affect the NA objective lens. mirror image quality. In order to correct the poor coverage, a correction collar (CC) is designed on the 40x, 60x or 20x interferometric objective lens. The correction range is generally 0.11--0.23 mm, and the long working distance objective lens can reach 2.0mm. You should master the essentials of CC adjustment carefully, otherwise it will cause trouble, especially when the previous experimenter made a calibration and the next experimenter's sample coverage is inconsistent. the way is,
First select the CC and align the 0.17 value line with the positioning reticle, and observe whether the mirror image can be clearly focused. If it is not very clear, turn the CC ring to the left and right directions until the mirror image reaches the best, and insist on changing the specimen every time Coverage correction and adjustment should be completed every time to ensure the quality of microscope inspection.
