The term "microscopy" has Greek roots. In translation, it means the study of objects using high-precision instruments. Recently, fluorescence and electron microscopy have become increasingly popular.
Resolution
Under it, as a rule, they understand the minimum distance at which clearly distinguishable objects can be located. The degree of penetration into the microscopic world, the ability to consider the size of the element under study will depend on the resolution of the equipment. At high magnification, the boundaries of objects can merge. Accordingly, there are certain limits beyond which the approximation of elements is meaningless.
Luminescence microscopy method: mechanism
When the energy absorbed by a substance is converted into visible radiation, a glow occurs. It is called luminescence. This phenomenon is due to the fact that some substances, under the influence of light, begin to emit rays with a different (usually large) wavelength. In addition, some objects that, under normal lighting, have a certaincolor, under the influence of ultraviolet change their color.
Specifics
An object that cannot be seen under ultraviolet light can emit a bright luster if it is treated with a special substance. In it, the elements glow in different colors in the dark. The strength of the radiation is different, but, as a rule, it is small. In this regard, fluorescence microscopy is effective in a darkened room. When using this type of study, the object is viewed in the light that it itself emits. The chemical composition of tissues, cells will affect the quality of the study. Fluorescence microscopy is considered to be a histochemical study to a certain extent.
Classification
Fluorescence microscopy can be primary or secondary. In the latter case, the object is processed with special compounds that give a glow. Primary fluorescent microscopy is based on the element's own ability to emit light.
Equipment
Fluorescence microscopy is performed using a variety of devices. Their main element is the illuminator. It is equipped with a UV lamp. In addition, the devices use a set of filters. In some devices, there are quite a lot of different configurations. Depending on which color is used to excite luminescence - ultraviolet or blue, an appropriate filter is placed between the light source and the object under study. Since the glow of a microscopic element is energetically weaker than the exciting light, it will be captured only under one condition. Excess rays from the source must be cut off by a yellow-green filter. It is located on the eyepiece of the device. The most pronounced effect of luminescence will be when the filter completely cuts off the rays emanating from the light source.
What does a visible light installation consist of? It contains a bright light source and a biological microscope. A blue-violet filter is placed between the mirror of the device and the lamp. It can be FS-1, UFS-3 and so on. The yellow filter is put on the eyepiece of the microscope. With their help, blue-violet light falls on the object. It excites luminescence. But this light can interfere with seeing the glow. Therefore, on the way to the eye, it is cut off by a yellow filter. Lighting is set according to the Koehler method, with one exception. The condenser diaphragm must be fully opened. When examining, it is important to use a non-fluorescent immersion oil. To reduce its own glow, nitrobenzene is added to it (2-10 drops / 1 g).
Luminescence microscopy: applications in microbiology
The benefits of this type of study are:
- Colour imaging capability.
- High contrast self-emitting elements on a black background.
- Detection and localization of certain types of viruses and microbes.
- The ability to study transparent and opaqueliving organisms.
- Study of life processes in their dynamics.
It should also be said that fluorescent microscopy contributes to the development of the finest methods of histo- and cytochemistry, express diagnostics.
