Requirements for the preparation of routine samples for microscopy
Confocal microscope is an epoch-making high-tech new product developed in the 1980s, and it is one of the important analytical instruments in molecular biology and life science today. Confocal microscopy is based on fluorescence microscope imaging with a laser scanning device added, using a computer for image processing to obtain fluorescent images of the microstructure inside cells or tissues, and to observe changes in the morphology of many physiological signal machines at the subcellular level. Become a powerful research tool in the fields of morphology, molecular cell biology, neuroscience, pharmacology, genetics and other scientific fields.
Sample preparation for confocal microscopy is a critical step prior to detection. Samples need to be labeled with fluorescent probes (single, double, triple).
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Specimens can be fixed or living tissues, or fixed or living adherent cultures. Cells should be cultured on Confocal special small culture dishes or coverslips. Suspend cells, shake or drop slices, and cover with coverslips cover film. The Zda thickness of the sample is about 1~2mm, the thickness of the cover glass used should be less than 0.17mm, and the thickness of the slide glass should be between 0.8~1.2mm, and the surface is smooth, the thickness is uniform, and there is no obvious interference fluorescence. Mounting medium is often used for fixed samples, and glycerol prepared in PBS with a pH of 8.5-9 is commonly used for mounting.
Routine sample preparation requirements:
1. Selection of dyes
Since the confocal microscope uses a single-wavelength laser as the light source, the choice of fluorescent dyes should be based on the wavelength of the laser equipped with the confocal microscope. If there are multiple fluorescent dyes in the same sample, their emission wavelength should also be considered as much as possible. Do not overlap to avoid cross-color problems.
2. Selection of sample carrier
Generally, the high-power objective lens of confocal microscope is oil lens, its numerical aperture is small, and the working distance between the lens and the sample is required to be no more than 0.17mm. Slides and coverslips are fine. If it is suspended cells or suspended particles, a petri dish dedicated to a confocal microscope can be used to carry the sample for observation.
3. Selection of mounting medium
If the sample only needs to be observed once and the fluorescence is not easily quenched, you can choose a certain concentration of glycerol mixture to seal the slide. If the sample needs to be placed for a period of time and taken multiple times, an anti-fluorescence quenching mounting medium should be selected to reduce the loss of fluorescent signals.
