The correct use of inverted fluorescence microscopy
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 master the operation and usage standards of inverted fluorescence microscope, teachers need to first teach the basic knowledge of inverted fluorescence microscope before demonstrating the actual operation of inverted fluorescence microscope. They should introduce the structure, principle, function, and basic usage methods of inverted fluorescence microscope, and then demonstrate different functions for different specimens in specific usage operations, gradually making the operation and usage of inverted fluorescence microscope standardized for medical graduate students.
1. Correct use of visual observation. One of the functions of an inverted fluorescence microscope is to observe in a bright field of view. Its basic principle is to achieve imaging in a bright background based on the different light absorption of each part of the specimen after being illuminated by light. It is mainly suitable for observing stained or colored cells, tissue slices, and other specimens.
2. Correct use of differential observation. The second function of inverted fluorescence microscope is phase difference observation. Its basic principle is to convert the optical path difference of different parts of the sample into amplitude difference, and observe through a phase difference objective with a phase plate and a spotlight with a circular aperture. It is mainly suitable for observing live cells or unstained tissue slices. In the process of experimental teaching, in addition to correctly guiding medical graduate students to follow the basic operating methods of phase difference observation, it is more important to guide them to understand and master the specimens using phase difference observation. For them, the most commonly used method in future scientific research is microscopic observation of cultured cells. Firstly, for the selection of cell equipment, it is generally recommended to use culture dishes, plates, and bottles made of glass and plastic materials. They are mostly colorless and transparent, and can be directly observed under an inverted fluorescence microscope without the need to pour out cell fluid or undergo other treatments. Secondly, during microscopic observation, do not open the lid of the equipment casually to avoid contaminating the cells. Finally, for operators, gloves, masks, and white coats should be worn to not only protect their own health but also prevent cell contamination.
3. Correct use of fluorescence observation. The third function of an inverted fluorescence microscope is fluorescence observation. Its basic principle is to use excitation light to excite the sample to produce fluorescence, thereby achieving the shape and positioning observation of the sample. It is mainly suitable for observing fluorescence labeled samples. Fluorescence observation is an important function of inverted fluorescence microscopy, with expensive accessories and special usage. In order for medical graduate students to correctly master the operating norms of fluorescence observation, teachers not only teach them the basic operating methods, but more importantly, teach them the uniqueness of it. Firstly, in specimen preparation, the surface of the slide and cover glass should be smooth and the thickness should be appropriate (the slide should be between 0.8mm and 1.2mm, and the cover glass should be around 0.17mm); The specimen should have an appropriate thickness while avoiding cell overlap or impurity masking; The sealing agent should use an equal mixture of glycerol and carbonate buffer (0.5mol/L pH 9.0-9.5). Secondly, before fluorescence observation, the lights in the room and the white light of the inverted fluorescence microscope should be turned off to ensure that they are in a dark room environment. Finally, in fluorescence observation, the observation time should be strictly controlled, generally 1-2 hours per time, otherwise fluorescence will decay and disappear; When adjusting the light source, protective glasses should be worn to avoid UV damage to the glasses.
4. Correct use of image capture. In scientific research, it is not only required that inverted fluorescence microscopy can achieve multiple observations, but also that inverted fluorescence microscopy can achieve image capture. The inverted fluorescence microscope system combines an optical microscope with a computer through photoelectric conversion, allowing for not only microscopic observation on the eyepiece, but also real-time dynamic image observation, editing, and storage on the computer.
