Standard Model of particle physics

A healthy human eye is able to distinguish two points that are 0.2 mm apart. To observe finer details, it is necessary to increase the viewing angle. With the use of microscopic techniques, we can distinguish individual points between which there is a smaller distance, and thus obtain more detailed information about the structure of the observed sample.

The first light microscope was constructed more than 400 years ago in the Netherlands by Zacharias Janssen. And in the course of the following years, the construction of microscopes underwent further development. In 1830, it was possible to eliminate the chromatic defect of the objective, and later the astigmatism of light microscope lenses was eliminated. In the 1930s, an electron microscope was constructed, in which a stream of accelerated electrons is used instead of light radiation. Thanks to the use of a stream of electrons as corpuscular radiation with a significantly lower wavelength than light radiation, the electron microscope is able to distinguish details in the range of 0.2 nm. Meanwhile, in light microscopy, fluorescent dyes (fluorochromes) that emit visible light upon absorption of excitation radiation were discovered, and fluorescence microscopy became widespread. Towards the end of the 20th century, confocal microscopes appear. With the use of some modern microscopic techniques, we are able to obtain a three-dimensional image of an object, distinguish structures at the atomic level or selectively detect individual molecules.

Resolution of microscopic techniques
The resolving power of the microscope is determined by the relationship, where dmin is the minimum distance between two points that can be distinguished using the microscope. R = 1/dmin

If we consider the sample as an optical grating perpendicular to the optical axis, according to Abbe's theory, interference maxima and minima arise in the image plane after the bending of a uniformly passing beam of rays on the optical grating. n∙d∙sinα = k∙λ

n – refractive index

d - distance between two scratches

α - aperture angle (angle of deviation of the rays from the optical axis)

λ - wavelength of light in a vacuum

To resolve the real grating image, there must be a zeroth and first order maximum in the image plane. For an aperture angle α that still passes through the lens, the smallest resolution limit is dmin:

dmin = λ/(n∙sinα) Where n ∙sinα = A - numerical aperture

To achieve the highest resolution of the microscope, it is necessary to ensure the maximum values ​​of the numerical aperture:

a) Appropriate lens construction

b) Immersion layer between the specimen and the objective – a transparent substance with a higher refractive index (e.g. cedar oil)

Microscopic techniques according to the arrangement of the system
Incident Light

Light hits the surface of the sample from the observation side. It is used in the imaging of opaque samples and in the study of surfaces.

Passing Light

The preparation is partially transparent, it is illuminated by light, and the passing rays enter the objective. When working with tissue cultures, a so-called inverted microscope is used, where the light source is located above the sample and the objective below.

Bright field method – a light cone passes through the specimen and enters the objective.

Dark field method – the light cone is deflected and only rays enter the objective due to scattering, reflection or bending on the structure of the specimen. The rays then fall into the lens, where they display the outlines of the specimen's structures on a dark background.

Objects for transmitted light microscopy must have sufficient contrast. Therefore, preparations for light microscopy are enhanced by staining. When the radiation passes through the specimen, its amplitude will decrease.

Light emission

Fluorescence microscopes work on the principle of luminescence. In the preparation, a luminescent component is either present, or a fluorescent substance (fluorochrome) is applied to it. When a quantum of radiation is absorbed by a luminescent substance, a quantum of a different, mostly longer wavelength is emitted.

Light Microscopy
Image quality and resolution depends on the quality of the optical system and the light source. A classic light microscope uses radiation in the visible light region. But there are also other methods of light microscopy using monochromatic light, polarized light or electromagnetic radiation in wavelengths other than the visible range of light.

Ultraviolet Microscopy

Using ultraviolet radiation increases the resolving power of the microscope. The image is recorded photographically or using a special CCD camera. Ultraviolet radiation is also used to display objects containing UV-absorbing substances.

Infrared Microscopy

Some objects penetrate infrared radiation more easily than visible light and relatively strong preparations can be imaged. Infrared radiation with wavelengths of 750 – 1100 nm is used. The image is recorded on special photomaterials sensitive to IR.

Polarization Microscopy

Polarizing Microscopy combines a light microscope and polarimeter. A polarizer is included in the lighting system and an analyzer is behind the lens. In this way, information can be obtained about the optical activity of the observed preparation. This type of microscope is mainly used in mineralogy, in biology it is suitable for observing some anisotropic systems, e.g. striated muscle.

Laser Confocal Scanning Microscopy

The source of radiation in a confocal microscopeie is a laser. The laser beam passes through the first confocal diaphragm onto a dichroic mirror and is focused by the objective to a specific point on the specimen. The light reflected by the sample passes back through the objective, the dichroic mirror and the second confocal diaphragm in the back focal plane. This aperture will prevent the passage of radiation from places outside the optical plane. The light then falls on the detector (photomultiplier). The information is transmitted to the scanning device together with the coordinates of the given point, in which the resulting image is processed from the information from the individual scanned points. A confocal microscope makes it possible to guide a series of optical sections through a sample. It is characterized by a higher resolution compared to other methods of light microscopy.

Electron Microscopy
The function of light rays is replaced by a stream of electrons emitted in a vacuum by an electron nozzle. And the optical lenses of the light microscope are replaced by a system of electromagnetic lenses whose fields act on passing electrons.

According to the imaging method, we distinguish between transmission (TEM) and scanning electron microscopy (REM).

In a transmission electron microscope, a stream of electrons passes through the object and optics and forms an image on a luminescent screen. It is a system of direct observation and image capture.

In the ''scanning electron microscope ', the object is streaked by an accelerated beam of electrons, and the released electrons from the sample are registered by the appropriate detector. Subsequently, the resulting image is processed. It is a system of indirect observation and image capture.

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