Proper Use of Inverted Fluorescence Microscope
Inverted fluorescence microscope is a multifunctional microscope used in the field of high-end biological science research. Its structure is complex and expensive. Therefore, in order for medical graduate students to correctly grasp the operation and use of inverted fluorescence microscope, teachers must first demonstrate the actual operation of inverted fluorescence microscope. Before, a course was set up to learn the basic knowledge of inverted fluorescence microscopes, introducing the structure, principle, function and basic usage methods of inverted fluorescence microscopes, and then in the specific operation, for different specimens, demonstrate different functions, from shallow to deep, gradually To standardize the use of inverted fluorescence microscopes by medical graduate students.
1. Correct use of bright field observation. One of the functions of the inverted fluorescence microscope is bright field observation. The basic principle is that after the specimen is irradiated by light, the light absorption of each part is different to realize imaging in a bright background. It is mainly suitable for stained or colored cells, Observation of specimens such as tissue sections.
2. Correct use of phase contrast observation. The second function of the inverted fluorescence microscope is phase contrast observation. Its basic principle is to convert the optical path difference of different parts of the sample into amplitude difference. Observation is realized through a phase contrast objective lens with a phase plate and a condenser lens with a ring diaphragm. It is mainly applicable to For the observation of living cells or unstained tissue sections, in the process of experimental teaching, in addition to correctly guiding medical graduate students to observe the basic operation methods of phase contrast observation, it is more important to guide them to understand and master the specimens used for phase contrast observation. For them, microscopic observation of cultured cells is the most commonly used in future scientific research. First of all, for the selection of cell equipment, it is generally recommended to use glass and plastic culture dishes, culture plates and culture bottles. Most of them are colorless and transparent, and can be observed directly on an inverted fluorescent microscope without pouring off the cell fluid and other treatments. Secondly, during microscopic observation, do not open the cover of the equipment at will, so as not to contaminate the cells. Finally, for operators, they need to wear gloves, masks and white coats, which can not only protect their own health but also prevent cell contamination.
3. Correct use of fluorescence observation. The third function of the inverted fluorescence microscope is fluorescence observation. The basic principle is to use excitation light to excite the sample to generate fluorescence, so as to realize the observation of the shape and position of the sample. It is mainly suitable for the observation of fluorescently labeled samples. Fluorescence observation is an important function of an inverted fluorescence microscope, and its accessories are expensive and special to use. In order for medical graduate students to correctly grasp the operation and use of fluorescence observation, teachers should not only teach them the basic operation methods, but more importantly, teach them to master its particularity. First of all, in the preparation of specimens, the slide glass and cover glass should have a smooth surface and appropriate thickness (the slide glass should be between 0.8mm and 1.2mm, and the cover glass should be about 0.17mm); the specimen should have an appropriate thickness and avoid cells Overlapping or masking by impurities; the mounting medium should use an equal volume mixture of glycerol and carbonate buffer (0.5mol/L pH9.0-9.5). Secondly, before fluorescent observation, the electric light in the room and the white light of the inverted fluorescent microscope should be turned off to ensure the environment in a dark room. Finally, in the fluorescence observation, the observation time should be strictly controlled, generally 1-2 hours/time is appropriate, otherwise the fluorescence will attenuate and disappear; when adjusting the light source, wear protective glasses to avoid the damage of ultraviolet rays to the glasses.
4. Correct use of image capture. In scientific research, it is not only required that the inverted fluorescence microscope can realize various observations, but also requires that the inverted fluorescence microscope can realize image capture. The inverted fluorescence microscope system combines an optical microscope with a computer through photoelectric conversion, not only for microscopic observation on the eyepiece, but also for real-time dynamic image observation, editing and saving on the computer.
