The tissue culture cell method facilitates direct observation of living cells and is an outstanding superiority of tissue culture techniques. It allows the experimenter to observe and record the process of changes in living cells at different stages of cell culture. The most commonly used methods of observation and recording include phase contrast microscope observation and videography, microfilm and closed-circuit television observation and video recording. Source of content: Tissue Culture and Molecular Cytology Techniques. (Beijing Publishing House)
Operation method
Phase contrast observation and photographic methods
Principle
Cultured live cells in the general microscope observation, the cell is transparent, the contrast is very small, it is difficult to observe the clear structure of the cell, only the application of the application of the microscope with a phase contrast device, in order to make the object and the back of the bottom of the contrast enhancement, to be able to see clearly the outline of the cell and some of the microscopic structures, such as the mitochondria, nucleolus, chromatin, and so on.
Materials and Instruments
Cells Move I. Use of phase contrast device Caveat I. The use of phase difference device1. Requirements for culture bottles and dishesPhase contrast microscope observation requires the substrate to be flat, uniform texture, good light transmission.The general glass bottle is not suitable for phase contrast observation and photography, and the plastic culture bottle with uniform texture is the best. 2. PreparationWipe the surface of the culture bottle clean before observation and photography, do not let the residual stains affect the light transmission. 3. DimmingAdjust the illuminationAdjust the illumination degree and optical axis, so that the field of view is uniformly illuminated. Correcting the optical axis is an important condition for micrography, and it is not easy to get the ideal effect when it is neglected.The result is not easy to get the ideal effect when ignored. 4. in the absence of self-control camera device for the first time, should be magnification, lighting intensity and exposure conditions recorded one by one; do a set of tests with different exposure times, and make a set of different exposure times.To; do a group of different exposure time test, after developing the best combination of selection, as a standard for future re-photography.Cells1. When observing the liquid evaporates continuously, nutrient solution should be added from the side of the specimen at any time; however, it should not be too much to prevent the liquid from spreading over the large cover sheet above.However, it should not be too much in order to prevent the liquid from spreading over the large cover sheet above, which will affect the oil immersion observation and photography. 2. This method is only for short time observation of cells; it is better to use the heated carrier table to maintain the cell function, but it can be observed for about 2 hours, and if the observation time is too long, the cell morphology and functional status will change with the evaporation of the culture medium and the change of pH.If the observation time is too long, with the evaporation of the culture medium and the change of pH, the morphology and functional status of the cells will be changed. For more product details, please visit Aladdin Scientific website.
Phase-contrast concentrator Phase-contrast objective lens
A phase contrast device or microscope consists of two main parts: a phase contrast concentrator and a phase contrast objective lens.
In each phase-contrast objective lens, there is a halo lens in the optical system; in the concentrator there are several convertible phase plates, a certain magnification The use of a certain magnification of the receiver needs to be consistent with the corresponding phase plate. Phase contrast observation on the strict requirements of the illumination, the optical axis should be aligned, the field of view should be uniform illumination luminosity. The optical axis should be aligned and the luminosity of the field illumination should be uniform.
1. Working steps
(1) Adjust the phase plate first, so that the spotter phase plate number and the magnification of the receiving eyepiece (phase plate) are consistent;
(2) Then pull out the receiving eyepiece, and then change the auxiliary telescope, move the auxiliary lens barrel and adjust the concentrator phase plate, so that the visual field of the two halos of the same size. field of two apertures of the same size (if different means that the magnification is not consistent) must match each other;
(3) and then re-exchange the original connection eyepiece, that is, into the phase difference image; when replacing the different multiples of the objective lens, need to be according to the above procedures When replacing the objective lens with a different magnification, it is necessary to re-adjust according to the above procedure. Cells
Cultured cells grow on the bottom of the bottle, the most suitable for inverted light phase contrast microscope observation, and need to be accompanied by a long focal length collector, in order to observe the thicker culture dishes. It is most suitable for inverted optical phase contrast microscope observation.
The wall thickness of culture flasks is >20 μm, which is limited to the 40× objective lens, if the cells are observed with an oil immersion lens, the cells are more delicate. To observe the finer structures of the cells with an oil immersion microscope, a support culture method is required; the cells are cultured on long cover slips.
When observing To make a temporary specimen from a bottle, proceed as follows
1. take a large carrier sheet, another small piece of high quality filter paper cut into a long square window shape, placed on the carrier sheet;
2. Use a pipette to put a few drops of culture solution into the paper frame, so that it fills the space inside the frame and soaks the paper frame;
3. Place a cover sheet with cells on it (with the cells facing upwards) and cover it with a large cover sheet.
