Confocal microscopy

Confocal microscopy is now considered to be one of the most promising methods of skin imaging in vivo, and therefore interest in it both among clinicians and among representatives of scientific research institutes is unusually high.

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

Confocal microscopy capabilities

In dermatology, confocal laser microscopy is used for:

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

Without discussing the merits and demerits of the above varieties of confocal microscopy, we note that in recent years, fluorescence laser confocal microscopy has been gaining in popularity.

[5], [6], [7],

Confocal microscopy for skin examination

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

To study the skin, confocal scanning laser microscopy (CSLM) is used. The method allows you to see the epidermis and papillary dermis layer with a resolution close to the histological. All survey results are displayed on the monitor and saved as a package of image files (in the form of a microfilm (in dynamics) or microphotographs).

There are two types of method:

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

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

[8], [9], [10], [11], [12], [13], [14], [15]

The history of confocal microscopy

The idea of creating a microscope capable at the cellular level to show an intravital cut of living tissue was actively developed 130 years ago. The main element of modern microscopes was designed at the end of the XIX century and was a rotating disk with the smallest holes spirally located. This disc was invented in 1883 by a German student Paul Nipkov, in honor of which he received his name - the Nipkov disk (or nipkov disk). The invention was based on the ability of light, passing through the smallest holes in the disk and the magnifying lens, to penetrate into the depth of the tissue and illuminate the cell fragment at a distance from the surface. When the disc rotates quickly, the fragments are added to the overall picture. By removing or approximating the structure to the object, the depth of the optical section of the tissue being examined can be varied.

Only with the advent of VTRs in the 1980s and computers capable of processing images, in the early 1990s there was a real opportunity to create and effectively apply those modern microscopes that are used in our time.