Fluorescence microscopy: study features

The term "microscopy" has Greek roots. In translation, it means the study of objects using high-precision instruments. . Lately, luminescent and electron microscopy have become increasingly popular.

Resolution

Under it, as a rule, understand the minimum distance, on which can be clearly distinguishable objects. The resolution of the equipment will determine the degree of penetration into the microscopic world, the possibility of considering the size of the element under investigation. With a large increase in the boundaries of objects can merge. Accordingly, there are certain limits beyond which the approximation of elements is meaningless.

Method of luminescence microscopy: mechanism

When the energy that is absorbed by the substance is converted, a visible light is produced in the visible radiation . It is called luminescence. This phenomenon is due to the fact that some substances under the influence of light begin to emit rays with another (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 can not be seen in the ultraviolet light can radiate a bright shine if it is 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, fluorescence microscopy is effective in a darkened room. When using this kind of research, the object is viewed in the light, which he himself emits. The chemical composition of tissues, cells will affect the quality of the study. Luminescence microscopy is considered to a certain extent a histochemical study.

Classification

может быть первичной или вторичной. Luminescent microscopy can be primary or secondary. In the latter case, the object is processed by special connections giving a glow. основывается на собственной способности исследуемого элемента излучать свет. Primary luminescence microscopy is based on the intrinsic ability of the element under investigation to emit light.

Equipment

Luminescent microscopy is performed using a variety of devices. The illuminator serves as their main element. 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 the 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 trapped only under one condition. The surplus of the rays emerging from the source must be cut off by 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 coming from the light source.

What does the installation consist of in visible radiation? It has a bright light source and a biological microscope. Between the mirror of the device and the lamp, a blue-violet filter is installed. It can be FS-1, UFS-3 and so on. The yellow filter is put on the eyepiece of the microscope. With their help, a blue-violet light falls on the object. He 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. The lighting is set according to Kohler's method, with one exception. The diaphragm of the condenser must be fully opened. In the study it is important to use non-fluorescing immersion oil. To reduce its own luminescence, nitrobenzene (2-10 drops / 1 g) is added to it.

Fluorescence microscopy: application in microbiology

The advantages of this type of research are:

1. Possibility of obtaining color images.
2. High contrast level of self-emitting elements on a black background.
3. Detection and localization of specific types of viruses and microbes.
4. Possibility of studying transparent and opaque living organisms.
5. 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.