Confocal microscopy

Confocal microscopy is currently considered one of the most promising methods for in vivo skin imaging, which is why interest in it is unusually high among both clinicians and representatives of research institutes.

[ 1 ], [ 2 ], [ 3 ], [ 4 ]

Confocal Microscopy Capabilities

In dermatology, confocal laser microscopy is used for:

• study of the penetration of compounds into the skin (paths of penetration, kinetics, distribution in the skin);
• observation of the functioning of the glands (determination of the active and passive state);
• studies of the microcirculatory bed (including in real time);
• diagnostics of neoplasms.

Without discussing the advantages and disadvantages of the above-mentioned types of confocal microscopy, we note that in recent years, fluorescence laser confocal microscopy has become increasingly popular.

[ 5 ], [ 6 ], [ 7 ]

Confocal microscopy for skin examination

The confocal microscope provides two invaluable opportunities - the study of tissues at the cellular level in a state of physiological vital activity and the demonstration of the results of the study (i.e. cellular activity) in four dimensions - height, width, depth and time. For the quality of the image and the depth of the study, the most important role is played by the ability of the tissue to transmit light, in other words, its transparency. The confocal microscopy method is contactless, the light beam does not cause any harm or discomfort to the patient being examined or the experimental animal.

Confocal scanning laser microscopy (CSLM) is used to examine the skin. The method allows one to see the epidermis and the papillary layer of the dermis with a resolution close to histological. All examination results are displayed on the monitor and saved as a package of image files (as a microfilm (in dynamics) or microphotographs).

There are two types of the method:

• reflective (reflectance CSLM) - based on the fact that various intracellular and intercellular structures have different refractive indices of light, which allows obtaining a contrast image.
• fluorescence (fluorescence CSLM) - uses laser light that penetrates the skin and excites exo- or endochromophores in it, which in response begin to emit photons (i.e. fluoresce).

Lateral resolution is the minimum distance between points located on a horizontal plane, i.e. a plane parallel to the skin surface. Axial resolution is the minimum distance between points located on a plane perpendicular to the skin surface.

[ 8 ], [ 9 ], [ 10 ], [ 11 ], [ 12 ], [ 13 ], [ 14 ]

History of confocal microscopy

The idea of creating a microscope capable of showing a section of living tissue at the cellular level was actively developed 130 years ago. The main element of modern microscopes was designed at the end of the 19th century and was a rotating disk with tiny holes arranged in a spiral. This disk was invented in 1883 by a German student Paul Nipkow, after whom it was named - the Nipkow disk (or Nipkow disk). The invention was based on the ability of light, passing through tiny holes in the disk and a magnifying lens, to penetrate deep into the tissue and illuminate a cell fragment at a distance from the surface. When the disk rotates quickly, the fragments form a single picture. By moving the structure away from or closer to the object, it is possible to vary the depth of the optical section of the tissue being studied.

It was only with the advent of video recorders in the 1980s and computers capable of processing images in the early 1990s that it became possible to create and effectively use the modern microscopes that are used today.