Confocal intravital microscopy of the cornea

Confocal microscopy of the cornea is one of the modern methods of investigation; allows to conduct intravital monitoring of the cornea state with visualization of tissues at the cellular and microstructural level.

This method, due to the original design of the microscope and its high resolution, makes it possible to visualize living tissues of the cornea, to measure the thickness of each of its layers, and to assess the degree of morphological disturbances.

The purpose of confocal microscopy of the cornea

Characterize the morphological changes in the cornea, arising from various inflammatory and dystrophic diseases, as well as from surgical interventions and the effects of CR.

Morphological data are needed to assess the severity of the pathological process, the effectiveness of treatment and determine the tactics of patient management.

Indications for the procedure

• Inflammatory diseases of the cornea ( keratitis ).
• Dystrophic diseases of the cornea ( keratoconus, Fuchs's dystrophy, etc.).
• Syndrome of "dry eye".
• Conditions after surgical interventions on the cornea (through corneal transplantation, keratorefractive operations).
• Conditions associated with wearing contact lenses.

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

Preparation

This study is possible without the use of anesthetics. A drop of immersion liquid is placed on the lens of the confocal microscope lens. This eliminates the direct contact of the lens with the cornea and minimizes the risk of epithelial damage.

[5], [6], [7], [8], [9],

Technique of the confocal microscopy of the cornea

The study is performed on a confocal microscope ConfoScan 4 (Nider) with an increase of 500 times. The device allows you to examine the cornea over its entire thickness.

The size of the investigated zone is 440 × 330 μm, the thickness of the scanning layer is 5 μm. A lens with a drop of gel is brought to the cornea to touch and set so that the thickness of the immersion liquid layer is 2 mm. The device design allows you to examine the cornea in the central zone and its paracentral areas.

Contraindications to the procedure

Relative contraindication marked irritation of the eye against the background of an acute inflammatory process.

[10], [11]

Normal performance

Normal morphological picture of the cornea

The anterior epithelium consists of 5-6 layers of cells. The average thickness of the entire epithelium is approximately 50 μm. According to the morphological structure, the following layers are distinguished (from the inside outward): basal, subulate and superficial.

• The innermost (basal) layer is represented by small dense cylindrical cells with no visible nucleus. The basal cell borders are clear, bright.
• The middle layer consists of 2-3 layers of spine-shaped (winged) cells with deep invaginations, into which outgrowths of neighboring cells are built. Microscopically, cell boundaries are fairly well distinguishable, and the nuclei may not be defined or be fuzzy.
• The surface layer of the epithelium is represented by one or two layers of polygonal cells with clear boundaries and a homogeneous density. The nuclei are usually brighter than the cytoplasm, in which one can also distinguish a near-nucleus dark ring.

Among the cells of the surface layer distinguish between dark and light. The increased reflectivity of epithelial cells indicates a decrease in the level of metabolism in them and the beginning of their desquamation.

The Bowman membrane is a transparent structure that does not reflect light, so it is impossible to visualize it when confocal microscopy is performed.

Subbasic nerve plexus is located under the Bowman membrane. Normally, the nerve fibers look like bright strips running in parallel on a dark background, contacting each other. Reflectivity (reflectivity) can be uneven in fiber length.

The stroma of the cornea occupies 80 to 90% of the thickness of the cornea and consists of a cellular and extracellular component. The basic cellular elements of the stroma are keratocytes; constitute approximately 5% of the volume.

A typical microscopic pattern of the stroma includes several bright irregular oval-shaped bodies (keratocyte nuclei) that lie in the thickness of a transparent dark gray or black matrix. Normally, visualization of extracellular structures is impossible because of their transparency. The stroma can be conditionally divided into sublayers: anterior (located directly under the Bowman membrane and constitutes 10% of the thickness of the stroma), anterior, middle and posterior.

The average density of keratocytes is higher in the anterior stroma, gradually their number decreases towards the posterior layers. The density of the cells of the anterior stroma is almost twice that of the cells of the posterior stroma (if the density of the cells of the anterior stroma is taken as 100%, then the density of the cells of the posterior stroma is about 53.7%). In the anterior stroma, the nuclei of keratocytes have a round bean-shaped shape, and in the posterior oval and more elongated.

The nuclei of keratocytes can differ in brightness. The different ability to reflect light depends on their metabolic state. Brighter cells are considered to be activated keratocytes ("stress cells"), whose activities are aimed at maintaining internal corneal homeostasis. In the norm and field of view, there are single activated cells.

Nerve fibers in the anterior stroma of the cornea are visualized as bright homogeneous bands, often forming bifurcations.

The descemet membrane is normally transparent and is not visualized by confocal microscopy.

The posterior epithelium is a monolayer of hexagonal or polygonal flat cells with a uniformly light surface against a background of distinct dark intercellular boundaries.

The device provides the possibility of manual or automatic calculation of cell density, their area and coefficient of variability.

Pathological changes in the structure of the cornea

Keratoconus is characterized by significant changes in the anterior epithelium and stroma of the cornea.

[12], [13], [14], [15], [16], [17], [18], [19], [20]