The term "microscopy" has Greek roots. In translation, it means the study of objects using high-precision instruments. Recently, luminescent and electron microscopy have become increasingly popular.
Resolution
Under it, as a rule, understand the minimum distance at which clearly distinguishable objects can be. 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. With a large increase, the boundaries of objects can merge. Accordingly, there are certain limits beyond which the approximation of elements is pointless.
Fluorescence Microscopy Method: Mechanism
When the energy that a substance absorbs 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 have a certain color under normal lighting, under the influence of ultraviolet light, change their color.
Specificity
An object that cannot be seen in ultraviolet light can emit a bright shine if treated with a special substance. In it, the elements glow in different colors in the dark. The intensity of the radiation is different, but, as a rule, it is small. In this regard, luminescent microscopy is effective in a darkened room. When using this type of research, the object is viewed in the light that it itself emits. The chemical composition of tissues and cells will affect the quality of the study. Luminescent microscopy is considered to some extent a histochemical study.
Classification
Luminescent microscopy may be primary or secondary. In the latter case, the object is processed with special compounds that give a glow. Primary fluorescence microscopy is based on the intrinsic ability of the element under study to emit light.
Equipment
Luminescent microscopy is performed using a variety of devices. The main element is the illuminator. It is equipped with an ultraviolet lamp. In addition, the devices use a set of filters. In some devices there are quite a lot of different configurations. Depending on what 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 luminescence of the microscopic element is energetically weaker than the exciting light, it will be captured only under one condition. Excess rays emanating from the source should be cut off with a yellow-green filter. It is placed 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 the installation in visible radiation consist of? It has a bright light source and a biological microscope. A blue-violet filter is installed between the mirror of the device and the lamp. It can be FS-1, UFS-3, and so on. A yellow filter is attached to the eyepiece of the microscope. With their help, a 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 with a yellow filter. Lighting is installed using the Koehler method, with one exception. The condenser diaphragm must be fully open. When researching it is important to use non-fluorescent immersion oil. To reduce its own glow, nitrobenzene (2-10 drops / 1 g) is added to it.
Luminescent microscopy: application in microbiology
The advantages of this type of research are:
- The ability to obtain color images.
- High contrast of self-emitting elements on a black background.
- Detection and determination of localization of certain types of viruses and microbes.
- The ability to study transparent and opaque living organisms.
- The study of life processes in their dynamics.
It must also be said that luminescent microscopy contributes to the development of the finest methods of histo- and cytochemistry, rapid diagnostics.