How to choose between inverted microscope and fluorescence microscope?
Microscope is an important instrument in cell culture and related derivative experiments. At present, there are various types of microscopes on the market, and choosing a microscope that meets the needs and is suitable is a challenge. Below, we will introduce the principles of inverted microscopes and fluorescence microscopes for everyone to choose from.
The inverted microscope, like a regular microscope, mainly consists of three parts: mechanical part, lighting part, and optical part. The composition of an inverted microscope is the same as that of a regular upright microscope, except that the objective lens and lighting system are reversed, with the former under the stage and the latter above the stage. This structure significantly expands the effective distance between the lighting spotlight system and the stage, making it easier to place thicker observation tools such as culture dishes and cell culture bottles (of course, glass slides can also be used), while the working distance between the objective lens and the material does not need to be very large. Inverted microscope is used by medical and health institutions, universities, and research institutes for observing microorganisms, cells, bacteria, tissue cultures, suspensions, sediments, etc. It can continuously observe the process of cell and bacterial proliferation and division in culture medium, and can capture any form of this process. Widely used in fields such as cytology, parasitology, oncology, immunology, genetic engineering, industrial microbiology, and botany.
Fluorescence microscopy is used to study the absorption, transportation, distribution, and localization of substances within cells. For the tested object, there are two ways to generate fluorescence: spontaneous fluorescence, which is directly emitted by ultraviolet irradiation; Secondary fluorescence occurs when the observed object is treated with fluorescent dyes and exposed to ultraviolet light before emitting fluorescence. Some substances in cells, such as chlorophyll, produce spontaneous fluorescence after being exposed to ultraviolet radiation; Some substances themselves may not emit fluorescence, but if stained with fluorescent dyes or fluorescent antibodies, they can also emit secondary fluorescence under ultraviolet radiation. A fluorescence microscope uses a high luminous efficiency point light source to emit a certain wavelength of light (UV 365nm or UV blue 420nm) through a color filtering system as excitation light, which excites the fluorescent substances in the sample to emit various colors of fluorescence. After that, it is observed through magnification of the objective lens and eyepiece. In this way, even with weak fluorescence, it is easily recognizable and highly sensitive under strong contrasting backgrounds. It is mainly used for studying cell structure, function, and chemical composition.
Fluorescence microscopy can be divided into two types: transmission type and falling type. The former is more primitive, while the latter is more advanced. The basic construction of the two types of fluorescence microscopes is similar, with the main difference being that the transmitted excitation light passes through the specimen, producing fluorescence as a whole. The fluorescence then enters the objective lens, and the higher the magnification, the weaker the fluorescence; The falling excitation light projects onto the surface of the specimen, producing fluorescence that enters the objective lens. The higher the magnification, the stronger the fluorescence, making it suitable for high-power observation. The main components of a fluorescence microscope include a mercury lamp light source, an excitation filter plate, a spectrophotometer (drop type), a suppression filter plate, and a dark field condenser (transmission type). In addition, due to the severe heating of mercury lamps, most of them are also equipped with heat absorbing filters. Some fluorescence microscopes also have a phase contrast objective and a circular aperture, allowing for phase contrast observation. Some fluorescence microscopes adopt an inverted structure, which is also an inverted microscope, and so on.
In addition, the above-mentioned microscopes can be assembled into digital microscopes by installing CCD, which converts the physical images seen by the microscope into digital analog images and images them on a computer. As a result, we can shift our research in the micro field from traditional binocular observation to reproduction through displays, thereby improving work efficiency.
