Aplication of in Vivo Confocal Microscopy in Ophtalmology—Overview

Confocal microscopy is a method which has been increasingly used over the last decade in the study of the anterior ocular surface. The method allows testing and in vivo high resolution imaging of the structures of the anterior eye segment, at a cellular level, which is close to the histological examination of tissues. The data provided by this method allow for a better understanding of both the functional and pathological processes occurring in the anterior ocular surface not only for scientific purposes but also in clinical practice. The aim of the present work is to summarize the current knowledge and applications of confocal microscopy of the anterior ocular surface.


Introduction
In vivo confocal microscopy is a non-invasive method, which provides high resolution tissue images at a cellular level in real time, permitting the quantitative, qualitative and morphological analyses of a living tissue These capabilities can be employed to image structures of ocular surface in the eye, in vivo, both for research and for the diagnosis and treatment of human disease [1]- [7]. The ocular surface is composed of cornea, eyelid and the bulbous conjunctiva, as well as the eyelids [8]. The tear film plays an important role in maintaining the homeostasis of this system.
The method provides information and an opportunity for a better understanding of the ongoing processes in these structures, in depth.
The principle of confocal imaging, patented in 1957 by Marvin Minsky [9] [10], was designed to overcome certain limitations of conventional light microscopes, where the whole specimen is evenly illuminated by the light source, due to which the quality of images is distorted by the background unfocused part of the specimen.
In contrast, with confocal microscopes the light beam emanates from a single point, the aperture being located in front of the detector, eliminating the signal outside the focus-"confocal" derives from this configuration. Since it detects only light obtained from fluorescence close to the focal plane, the optical resolution of the image, especially in the depth of the specimen, is considerably better than that of conventional light microscopes.
One of the most widely used laser scanning confocal microscopes was designed by White, Amos, Durbin and Fordham [11] to image specific macromolecules in embryos [9]. This and some other microscopes developed at the time were the forerunners of modern microscopes, which are available to biomedical researchers.
Over the last decade, the method has become increasingly popular and widely applicable in many fields of medicine and biology. The advantages of confocal microscopy are hidden in the extremely high quality images, which provide information in detail about the microstructure of the examined specimen. In addition, the method has the potential to be used in many areas of biomedicine.
Master and Thaer described the application of a slit scanning confocal microscope, which performs a unique real-time video image of a live human cornea [8] [12] [13].
There are three types of CFM according to the type of illumination used: 1) Tandem Scanning Confocal Microscopy (TSCM), which is based on a modified technology with a Nipkow disc-a metal disc with multiple holes with a size of 30 microns, which rotates at a high speed. It was first introduced by Cysts with hyperreflective cells are sometimes found in the stroma. Relatively large cells (about 25 μm) are detected, supposed to be goblet cells. Fine capillaries up to 20 μm in size with blood cell elements in the lumen of the vessel-small hyperreflective structures-are found in the stromal layer. The vessels close to the eyelash margin have a larger diameter, and the vessel wall can be observed in an image in the oblique diameter.
The eyelash margin is represented by a layer of epithelial cells and the secretory ducts of the Meibomian glands, in which lipid accumulations can be detected [20].
The bulbar conjunctiva is thinner than the palpebral conjunctiva. It is characterized by smaller epithelial cells (about 10 μm) with hyperreflective inclusions (lymphocytes and plasma cells), as well as fewer goblet cells, compared to the palpebral and fornix conjunctiva. Тhe palisades of Vogt are clearly visible in the limbal area.

Cornea
Confocal microscopy allows in vivo layer-by-layer images of the cornea, which are similar to a histological section ( Figure 1).
Epithelial layer (Corneal epithelium): it is composed of 5 -6 layers of epithelial cells, functionally and morphologically divided into 3 zones: Superficial cells: they have a polygonal shape, 40   Wing cells: they have a polygonal shape and well-defined borders with a mosaic appearance (Figure 1(a)). They range in size: the more superficially positioned ones are 50 μm in diameter, whereas the deeper ones are 20 μm. The density varies from 5000 cells/sq•mm in the central part of the cornea up to 5500 cells/sq•mm. in the periphery [8].
Basal cells: they have an elongated shape, a small diameter of 8 -10 μm and a height of 20 μm. They are characterized by clearly distinguishable borders and a nucleus hard to differentiate (Figure 1(b)). The density in the central cornea varies from 6000 cells/sq•mm up to 9000 cells/sq•mm, and 10,000 cells./sq.mm in the periphery [2] [8] [20] [23] [24].
Bowman's membrane (anterior limiting membrane): an amorphous membrane located just below the basal epithelial cells, with a thickness of approximately 10 μm. It is composed of collagen fibrils and contains unmyelinated nerve fibers from the subepithelial nerve plexus [20] [21].
Corneal nerves. The cornea is the structure of highest sensitivity in the human body. This is due to the large number of nerve fibers running through it. This extensive innervation affects the integrity of the corneal epithelium and the healing of corneal injuries [20] [25]. The visualization and examination of these nerve fibers is possible with confocal microscopy. Nerve fibers are sensory, deriving from the ophthalmic nerve and the terminal branches of the trigeminal nerve. They have no myelin sheaths, their thickness varying from 2 μm to 10 μm. Nerve fibers enter from the peripheral part of the cornea into the anterior and middle parts of the stroma. They run parallel to the corneal surface, moving along radially before making a sharp turn at 90 degrees towards Bowman's membrane [20] [26].
The nerve fibers reach the corneal apex mainly along the meridians at 6 -12 h, 5 -11 h or 7 -1 h and keep a course predominantly in an upward-downward direction (12 -6 h); in the other areas, they are oriented in a nasal-temporal direction [26].
In the anterior parts of the stroma, just before Bowman's membrane, the nerve fibers divide in three directions. Some of them branch without penetrating Bowman's membrane, forming the Subepithelial Plexus (SEP) (Figure 1(c)). Thin nerve fibers of the SEP are characterized by fibers connected with anastomoses. The profile of these fibers is hyperreflective, and their tickness usually ranges from 4 to 8 μm [25] [26] [27] [28]. Others penetrate Bowman's membrane directly, following a perpendicular or slightly oblique course or form fine branches just before piercing the membrane. After passing through Bowman's membrane, they change their course to 90 degrees and run between the basal epithelial layer and the membrane towards the center of the cornea, forming the Basal Epithelial Plexus (BEP). They then form multiple branches in all directions along the corneal surface, where they end loosely or in the direction of the center of the cornea [20] [26] [27].
The nerve fibers from the basal epithelial plexus usually run parallel to each other, forming Y or T-branches. Open Journal of Ophthalmology Confocal microscopy images show the stromal nerve fibers as thick [8], almost always taut, highly reflective structures, often located near keratocytes, the deep stroma lacking nerve fibers.
The nerve fibers of the basal epithelial plexus are characterized by lower reflectivity, a predominantly granular structure and tortuous course. The thicker fibers branch out, reuniting after a certain distance to form a fiber of the same size. There are also many small nerve branches which form connections between long nerve fibers [20] [26] [27].
Corneal stroma: makes up about 90% of the volume of the cornea. It consists of three main components: cellular-keratocytes, amorphous substance (glycoproteins, glucose-aminoglycans-keratin sulfate and chondroitin sulfate) and nerve fibers. The nuclei of the keratocytes are highly reflective, in the shape of a "bean" in the anterior parts of the stroma (Figure 1(d)), and "egg-shaped" in the deep layers ( Figure 1(e)). The other cell structures are not visible -they appear black. The density of keratocytes is higher in the anterior parts of the stroma. The nerve fibers in the stroma are larger in diameter than the subepithelial ones [8] and keep a rectilinear course.
Descemet's membrane: Posterior limiting lamina. This membrane is the basal membrane of endothelial cells. Its thickness varies from 6 μm to 10 μm. In a confocal image it is seen as a homogeneous acellular layer. Normally it cannot be imaged in young individuals. With the advancement in age, however, it is easier to image (Hollingsworth et al. 2001) [8] [21].
Corneal endothelium: a single layer of endothelial cells with a hexagonal or polygonal shape with dimensions: 4 -6 μm thick and about 20 μm in diameter. In a CFM image they resemble a "honeycomb" mosaic with clear dark borders and hyperreflective cytoplasm, without polymegatism (variations in cell size) and pleomorphism (variations in the shape of the cell) ( Figure 1(f)). The nucleus is rarely distinguished [8] [21]. The total number of endothelial cells in a healthy individual is approximately 500,000 cells. The density in the central cornea is 2500 -3000 cells/sq•mm [8]. Limbal region: it is the boundary zone between the cornea and conjunctiva and the underlying sclera, which plays an important role in maintaining the integrity of the cornea. A border is formed between the corneal epithelium and the conjunctival epithelium, which consists of approximately 10 -12 layers. Here the palisades of Vogt are found too [20]. It is assumed that it is in the palisades of Vogt that limbal stem cells are synthesized for the regeneration of the corneal epithelium [29] [30]. When CFM imaged, the epithelial cells of the conjunctiva appear more highly reflective, with unclear borders and smaller in size; they have larger and brighter nuclei. In the border zone, epithelial cells are characterized by inhomogeneous reflectivity and variations in shape and size [20]. The palisades of Vogt are visualized as trabecular extensions in the conjunctival epithelium, located radially to the limbus. Their edges are well demarcated, especially in the deeper layers, at the end of which concentrations of hyporeflective cells are differentiated, supposed to secrete stem cells [20].
In the subepithelial space, near the limbus, there are blood vessels-limbal plexus.

In Vivo Laser Scanning Confocal Microscopy (LSCM) Characteristics in Anterior Ocular Surface Diseases
The ability of CFM to produce rapid in vivo layer-by-layer images of tissues, even with decreased corneal transparency, makes it an extremely valuable method in the diagnosis, follow-up and treatment of many pathological conditions of the anterior segment of the eye. It gives us a new understanding of the ongoing pathophysiological processes at a cellular level, enabling us to monitor and evaluate treatment.

Corneal Dystrophies
The most common classification of corneal dystrophies is anatomically based (IC3D classification of Corneal Dystrophies). The dystrophies are divided into groups, depending on the corneal layer affected.
In 2015, the IC3D classification of Corneal Dystrophies-Edition 2, was revised and published, the templates being updated, as a result of which the dystrophies are now divided into four major groups: epithelial and subepithelial, epithelial-stromal, stromal and endothelial [31].
Until recently, the clinical evaluation, diagnosis and differentiation of corneal dystrophies were based on biomicroscopy, but it was unable to provide information at a cellular level. It necessitated the performance of a biopsy, as well as genetic and histological examination, which are invasive and time-consuming methods [21].
On the other hand, CFM is a rapid non-invasive method which provides information at a cellular level in vivo, quickly finding a place in the diagnosis of corneal dystrophies [32].
The most common among them is Epithelial Basement Membrane Dystrophy and a lack of halo [43].
With RBCD, fine fusiform deposits can be observed in the anterior and rarely in the posterior parts of the stroma [31]. Lattice corneal dystrophy (LCD) is caused by the mutation of Arg124Cys and Leu527Arg in the TGFBI gene [45]. Through biomicroscopy, a multitude of fine fibers and radial particles in the stroma, corresponding to amyloid deposits, are Open Journal of Ophthalmology observed. Kaufman et al. were the first to describe a case of primary corneal amyloidosis, demonstrating the high hyperreflectivity of the amyloid deposits with CFM [46]. Consequently, many studies established the presence of hyperreflective extracellular punctate deposits in the basal epithelium layer [44] [47]. Linear filaments with different reflectivity and unclear borders, with thickness of up to 50 μm and a dimeter of 80 -100 μm, are found in the stroma [44] [47] [48], (Figure 3). А reduction of the long nerve fibers is detected in the subbasal nerve plexus, which results in decreased corneal sensitivity, described in the case of lattice type 2 (familial amyloidosis) [32] [49].
Granular corneal dystrophy (GCD) is with autosomal dominant inheritance, caused by a mutation in the TGFBI gene. Hyaline deposits, involving the central corneal zone, are found in the corneal stroma and Bowman's membrane, leading to reduced corneal transparency [45]. CFM images show hyperreflective "snowball" or trapezoid-like deposits in the posterior epithelial layers and Bowman's membrane. The deposits in the stroma vary from 50 μm to 500 μm in diameter, the size decreasing in the deeper layers. The lesions are dense with well-defined borders against the surrounding normal stroma [32] [44].
Stromal corneal dystrophies Macular Corneal Dystrophy (MCD) is characterized by the appearance of whitish opacities with unclear borders in the central area of the cornea and limbus, located in the stroma, hence its name. The epithelium appears grayish and defective, often developing erosions. The cornea is reduced in thickness. The condition is caused by a mutation in the Carbohydrate Sulfotransferaze 6 gene-CHST6. Kobayashi et al. conducted a CFM examination of two patients with macular dystrophy. They established highly reflective deposits in the basal epithelium, as well as in the anterior and middle sections of the stroma, some being dark striated. Normal keratocytes were not visualized [31] [32] [47].
Schnyder crystalline corneal dystrophy (SCCD) is an autosomal dominant disease. When biomicroscopy is performed, cholesterol deposits are observed in the stroma and Bowman's membrane, with diffuse grayish opacification of the surrounding cornea. The degree of alterations progresses with age from the central to the peripheral part of the cornea, in some cases with unilateral involvement. Thanks to CFM, highly reflective deposits in the form of elongated Open Journal of Ophthalmology crystals are differentiated in the anterior sections of the stroma. Decreased keratocyte density is also observed [50] [51] [52]. Fine crystalline deposits are found in the posterior parts of the stroma, decreasing in brightness and number further deep [51] [53]. Over the course of time, the cornea loses its normal architecture; extensive intra-and extracellular crystalline deposits, as well as the accumulation of extracellular matrix, are observed. This all leads to a loss of corneal transparency and disintegration in the subbasal nerve plexus. Nerve fibers exhibit pronounced tortuosity [32] [50] [52].
Congenital stromal Corneal Dystrophy (CSCD) shows a slow to no progression, and has autosomal dominant inheritance. Through biomicroscopy, opacities in all layers of the stroma and bilaterally in all areas of the cornea could be seen. No vascularization or surface erosions are detected. Pachymetry shows increased corneal thickness. The cells in the epithelial layer manifest no changes in the CFM image. Increased reflectivity is established in the anterior parts of the stroma [31].
Fleck Corneal Dystrophy (FCD) is a rare, autosomal dominant dystrophy with bilateral involvement. Small punctate gray-whitish opacities have been biomicroscopically detected in all layers of the stroma. Between the opacities, spreading from the center towards the periphery, the cornea appears normal. Intracellular hyperreflective punctate deposits of various shape are differentiated in the stroma by CFM. They consist mainly of small spherical materials with a size of 1 -18 μm, which are sometimes encapsulated in cystic structures with dimensions of 50 -110 μm in diameter, in the middle and posterior sections of the stroma [32] [54] [55]. Reflective inclusions are detected in the layer of the subbasal nerve plexus, the number and density of nerve fibers being reduced, which contributes to the reduced corneal sensitivity in Fleck dystrophy [56].
Posterior amorphous corneal dystrophy (PACD) is characterized by the presence of grayish-white, sheet-like opacities in all layers of the stroma, with a high concentration in the posterior stroma. The pachymetric map shows corneal thinning of up to below 380 μm, and the topography reveals flattening of the corneal curvature of up to 41D with hypermetropia. Protrusion of Schwalbe's line, iridocorneal adhesions, corectopia, pseudopolycoria and anterior synechiae have been reported. Erdem et al. described a case of PACD in which CFM reveals micro-folds and diffusely distributed sheet-like deposits extracellularly in the posterior stroma, just before the endothelial layer [57].
Central Cloudy Dystrophy of Francois (CCDF) is a bilateral, symmetrical dystrophy. Numerous dense polygonal grayish opacities, occupying the central area of the cornea, resembling crocodile shagreen, are established. Visual acuity is normal. Kobayashi et al. reported two cases of CCDF, where small highly reflective granules and deposits in the anterior stroma were differentiated by CFM. No changes were detected in the epithelial layer, the middle sections of the stroma or the endothelial layer, but fine low-reflective striae of highly variable appearance were visualized in the posterior parts of the stroma [32] [58].
Pre-Descemet Corneal Dystrophy (PDCD) usually occurs after the age of 30. Polymorphic opacities (similar to Cornea farinata and Fleck) are biomicroscopically established in the corneal stroma [32]. CFM reveals extra-and intracellular polymorphic hyperreflective inclusions, varying in size, right in front of the Descemet's membrane [59]. It is assumed that intracellular inclusions are actually modified keratocytes, and the small extracellular particles are lysosomes, containing lipofuscin [32] [60].
Endothelial dystrophies Fuchs' Endothelial Corneal Dystrophy (FECD) is a bilateral slowly progressing corneal dystrophy, which results in dysfunction of the endothelial cell layer. It is clinically manifested with pronounced changes in the endothelial layercornea guttata, pigment deposits, thickening of the Descemet's membrane, edema of the stroma and epithelium with bullae, subepithelial fibrosis [32]. A number of rounded hyporeflective zones with a centrally illuminated hyperreflective peak (point) in the endothelial cell layer [61]- [66], as well as pronounced pleomorphism, polymegatism and a reduced number of endothelial cells [31], are visualized on the CFM (Figure 4). Some blurring of the collagen fibrils and a sparse population of keratocytes in the anterior sections [58] were established in the stroma. In all the cases described, a thickening of the Descemet's membrane was detected [67] [68]. A lack of subbasal nerve plexus is also revealed [68].
Grupcheva et al. used CFM for differential diagnosis of corneal diseases, occurring with reduced corneal transparency and edema, which demonstrates the ability of CFM to differentiate the presence of guttae in cornea guttata, as well as Fuchs' dystrophy, emphasizing the capabilities of CFM to produce images of endothelial cells in the presence of corneal edema, unlike specular microscopy [69].
Posterior polymorphous corneal dystrophy (PPCD) is frequently taken into consideration with regards to Fuchs' dystrophy, when a differential diagnosis is made. It is often asymmetric. Opacities are detected both in the Descemet's membrane and the endothelial layer: grayish opacities, single or grouped vesicular lesions, grayish-white linear opacities, which may also affect the stroma. Vesicular lesions are visualized on CFM: rounded hyporeflective areas with demarcated single cellular elements in the middle, bearing a resemblance to a "doughnut". There is pronounced polymegatism of the endothelial cells [31].
There are no published data from CFM studies on Congenital hereditary endothelial dystrophy (CHED) or X-linked endothelial corneal dystrophy (XECD).

Langerhans cells (LCs)
CFM allows in vivo imaging and evaluation of Langerhans Cells (LCs) in the human cornea, clarifying their morphology and distribution. LCs are corpuscular particles with projections (dendrites) and a diameter of up to 15 μm. Their distribution increases from the center to the periphery of the cornea. Morphologically there are three types of LCs: with missing dendrites, with small dendrites and such with long dendrites, forming a network.
LCs with long dendritic projections are located in the corneal periphery, whereas in the central parts there are LCs without any projections, which probably accounts for their immaturity. Immature forms are capable of capturing antigens, while mature forms are able to sensitize T-cells, through MHC (Major histocompatibility complex) molecules, thus being part of the immune system [8] [20] [70].
Zhivov et al. reported a significant increase in the density of LCs when contact lenses are worn: 78 ± 25 cells/mm 2 in the center up to 210 ± 24 cells/mm 2 in the periphery, the gradient of distribution from the center to the periphery being maintained. In the center of the cornea, the density decreases significantly the longer the period of wearing contact lenses is [8] [71].
All of this suggests that LCs are involved in the immune and inflammatory responses, thus determining cell-mediated immunity [20].  [73]. Leukocyte infiltration was differentiated as hyperreflective cells in the layer of wing epithelial cells. During an inflammatory process, the mature forms of LCs predominate, localized in the layer of basal epithelial cells.
Bacterial keratitis Corneal infiltrates are identified as massive leukocyte infiltration at the level of wing and basal epithelial cells (Figure 5), while in corneal ulcers, in addition to the tissue lesion, there is edema of the surrounding tissue and infiltration of leukocytes and mature Langerhans cells [73] (Figure 6).
Viral keratitis Viral keratitis can be easily and accurately diagnosed with CFM. A so-called "wire network" is detected, consisting of nerve fibers from the subepithelial nerve plexus and dendritic cells [8]     Acanthamoeba keratitis Acanthamoeba keratitis often remains undiagnosed due to non-specific clinical manifestations. Microbiological tests require time, leading to delays in specific treatment and frequent complications. The fast and reliable identification of Acanthamoeba is one of the most valuable applications of CFM in clinical practice. The differentiation of cysts and trophozoites secures a quick diagnosis and timely treatment [8] [82].
The first identification of Acanthamoeba with CFM was reported in 1992 [83], and shortly thereafter the American Academy of Ophthalmology adopted CFM as an accompanying diagnostic method [84].
The cystic form is characterized by a double wall, hyperreflective structure, a spherical or ovoid shape with dimensions of 15 -28 μm in diameter in the epithelium and stroma (Figure 8(a) and Figure 8(b)). The double wall is not always visible, which complicates the differentiation from leukocytes or epithelial cell nuclei. Trophozoites are 25 -40 μm in diameter, having a hyperreflective structure and ovoid shape [85].
Numerous different clinical studies attribute 80% -100% reliability to the CFM diagnostic data on Acanthamoeba, while the data from slit-lamp biomicroscopy have been found unconvincing [12] [85].
Mycotic keratitis Mycotic keratitis is a primary cause of blindness, especially in the tropics. It often remains undiagnosed due to the high variability of the clinical finds. There is often poor susceptibility to treatment, leading to the development of endophthalmitis and blindness.
The diagnostic gold standard is microbiological testing, but it is time-consuming and the cultures frequently remain negative.
CFM provides quick and accurate diagnostics by imaging the hyphae and filaments with high reliability -78% -94%, according to various studies [8] [85].
Aspergillus hyphae are hyperreflective and have dichotomous branches below 45˚ with dimensions of 5 -10 μm in diameter [86]. Conversely, Fusarium demonstrates branches below 90˚. Paecilomyces show varying thin branches, forming rings [87]. The hyperreflective structures must be differentiated from nerve fibers, which have a more regular architecture, those in the stroma having Open Journal of Ophthalmology

Dry Eye
Dry eye syndrome is one of the most common diseases in ophthalmology. It is characterized not only by disorders in the tear film but also by a violation of homeostasis and the structure of the anterior ocular surface.
With its capabilities, CFM allows us to see changes in the corneal epithelium, immune and inflammatory cells, nerve fibers, keratocytes and Meibomian glands at a structural level.
The alterations in the epithelial layer are characterized by decreased superficial cell density and increased basal cell density [89] [90] [91] [92].
In the layer of superficial epithelium, some variations in size are detected, as well as an increased number of hyperreflective cells (probably desquamable) with clearly demarcated nuclei [8].
During the more advanced stages of the disease, alterations in the deeper layers of the cornea are reported too. In the basal epithelial layer, there is an increased number of LCs in the central and peripheral cornea. The amount reported does not determine the severity of the disease, but can be used to monitor treatment [8] [93] [94] [95].
The nerve fibers of the subbasal nerve plexus demonstrate a pronounced tortuosity with a granular structure of the fibers and abnormal branches [85].
In their study, Castillo et al. reported decreased nerve fiber density in the subbasal nerve plexus [96].
Kheirkhah et al. conducted a study on 45 patients (90 eyes) with moderate to severe dry syndrome, using CFM to examine the density of endothelial cells, subbasal nerve fibers and dendritic cells. The control group comprised 15 healthy patients (30 eyes), of corresponding age and sex. The results demonstrated a significant decrease in endothelial cells and subbasal nerve fibers, as well as a sig-nificant increase in dendritic cells in the dry syndrome group, compared to the control group [97].
CFM can also provide imaging and evaluation of the Meibomian glands. A qualitative study of the structure is performed -diameter, density, diameter of the orifice, as well as the periglandular density of the inflammatory cells [97] [98] [99] [100].
Furthermore, the method permits a semi-quantitative assessment of the glandular secretion, taking into account the reflectivity, as well as the structure of the duct wall [99] [100] [101].
When there is a dysfunction of the Meibomian glands, their size is increased but the density is decreased, hyperreflective secretion being found in the ducts. In the case of dry eye syndrome, the Meibomian glands are reduced in volume, the inflammatory cells demonstrate an increased density, and the periglandular tissue is homogeneous [85].
A number of CFM studies have been conducted on the conjunctiva of patients suffering from dry syndrome. Hong et al. [102] established the formation of epithelial cysts, as well as a reduced density of conjunctival epithelial cells and goblet cells.
In CFM images, goblet cells figure as oval hyperreflective cells of large size and relatively homogeneous brightness. The density of goblet cells on CFM is 332 ± 137 cells/ mm 2 compared to impression cytology, where the reported density is 200 ± 141 cells/mm 2 in patients with Sjogren's syndrome, a significantly positive correlation being established [101].
Wakamatsu et al. [103] reported that patients with Sjogren's syndrome demonstrated a reduced density of conjunctival epithelial cells, in comparison to the group of healthy individuals. The same find was established in patients with dry syndrome. The group of patients with Sjogren's syndrome showed a significant increase in the density of conjunctival epithelial microcysts, compared to the group of healthy individuals, while the dry syndrome group demonstrated no significant difference.
The CFM studies performed on patients with dry eye syndrome reveal a decrease in goblet cell density and an increase in the density of inflammatory cells [101].

Contact Lenses
CFM considerably contributes to our understanding of the processes occurring in the anterior ocular surface when contact lenses are worn, at a cellular level. In contrast, there was no such possibility to produce an image of a live eye in the past.
There have been several clinical trials with CFM performed by different au-thors on patients wearing contact lenses [103]- [109]. In the epithelial layer, a larger size of the cells has been established, especially in cases where hard contact lenses are worn. During the process of normal epithelial desquamation, the lenses with high oxygen permeability have the smallest impact [85] [110]. An increased number of LCs has also been reported, in both the center and the periphery of the cornea, within the layer of subbasal nerve plexus. This suggests that the prolonged use of contact lenses may lead to a change in the immune status of the cornea [111].
A number of studies have ascertained a loss of keratocytes with the prolonged use of contact lenses, although there is disagreement about the extent of this effect. Mechanical irritation of the ocular surface results in the subsequent release of inflammatory mediators, which can lead to keratocyte apoptosis [104] [105] [112]- [118].
Corneal sensitivity is also affected by the long-term use of lenses. CFM visualizes the reduction and distribution of nerve fibers, leading to a reduced corneal sensitivity [103] [104].
Chronic morphological changes are also found in the endothelial cell layer, in response to chronic irritation from the prolonged use of contact lenses, increased polymegathism of the cells being reported too [119].

Corneal Surgery
In addition to diagnostics and monitoring of the anterior segment diseases, CFM gives us the opportunity to evaluate, monitor, and observe the correlation between the morphological and functional processes which take place in the cornea at a microstructural level, after a surgery has been performed. CFM reveals that the structure and architecture of corneal innervation is not static but dynamic. The recovery process of the nerve fibers is dependent on many factors, such as post-operative period, age, diagnosis and the surgical procedure itself.

Corneal transplantation
After Penetrating Keratoplasty (PK), severely reduced innervation is observed due to the nerve fibers being cut at all levels both in the donor and the recipient corneas. The reinnervation process is slow, and the structure of the nerve fibers is abnormal. After being monitored for more than 3 years, none of the patients showed a full morphological and functional recovery of the corneal innervation. It has been established that the reinnervation of the central cornea with subbasal nerve fibers takes approximately two years, and the stromal nerve fibers about seven months postoperatively [120].
Lamellar keratoplasty has gained wide popularity over the last decade in the treatment of diseases engaging the anterior and posterior corneal surface. Although there is no rupture of nerve fibers from the subbasal nerve plexus, except for the area of incision, there are significant changes in the subbasal and stromal nerve fibers before and after endothelial Keratoplasty (EK) has been performed on patients with Fuchs' dystrophy. The stromal nerve fibers have a pronounced tortuosity and are closely connected with the keratocytes, which suggests an in-teraction between them. The subbasal nerve fibers show an abnormal structure and a reduced number, which is also associated with decreased corneal sensitivity [85] [121].

Refractive surgery
Laser in situ Keratomileusis (LASIK) and Photorefractive Keratectomy (PRK) are the most widely used refractive corneal procedures. In both types of surgery, corneal tissue is removed through excimer photoablation, a flap being formed on the corneal surface with LASIK.
In PRK, subbasal nerve fibers cannot be identified in the treated area. Full recovery of the nerve fiber density in this area is reported after about 2 years [122], and morphological changes are detected after more than 5 years postoperatively [113] [115]. Despite the anatomical structural changes, which are visualized with CFM, the recovery of corneal sensitivity begins to be clinically evident 4 -6 weeks after the procedure, complete recovery being achieved after 6 -12 months [123] [124] [125].
In the first month after LASIK has been performed, a 90% decrease in the density of subbasal and stromal nerve fibers is observed, compared to the preoperative period [126]. A number of studies have ascertained that after the first 6 months, reinnervation in the center of the cornea is visualized [127], and complete regeneration is achieved within 2 -5 years, according to various authors, after the procedure [122] [128].
A correlation is found between corneal sensitivity, the morphology and density of the nerve fibers in the postoperative period [128] [129] [130].
A study comparing corneal healing and reinnervation, conducted by Sonigo et al., reported that no difference could be found between the flap made by a Femto Laser and that made by a mechanical microkeratome [131].
Corneal sensitivity after Laser-Assisted Sub-Epithelial Keratectomy (LASEK) is fully restored 3 months after the procedure, although the density of nerve fibers in the subbasal nerve plexus is decreased by half, 6 months after the procedure Darwish et al. [132] reported in their study.
Lee et al. also reported significantly reduced nerve fiber density after LASEK and incomplete recovery after a 6-month postoperative period [127].
With both LASIK and PRK, a reduced density of keratocytes was observed over a 5-year postoperative period [133] [134].
Small Incision Lenticule Extraction (SMILE) is an innovative method of correcting refractive errors, where through a minimal incision, an intrastromal lenticle previously formed by a Femto Laser is removed. The method has become widespread in ophthalmic surgery due to its high efficiency, minimal invasiveness, safety and predictability [135] [136] [137] [138]. The minimal invasiveness of the method implies a minimal reduction in the density of nerve fibers. Meiyan et al. [135] reported in their study that SMILE causes a considerably less reduced density of nerve fibers in the subbasal nerve plexus 1 week, 1 month and 3 months after the procedure, in comparison to Femto LASIK, density being in correlation with corneal sensitivity. Open Journal of Ophthalmology Corneal Collagen Cross-linking (CXL) and Keratoconus CFM applied to corneas with keratoconus reveals changes in all the layers of the cornea. Elongated superficial epithelial cells and enlarged basal epithelial cells are visualized. The significant decrease in the density of basal epithelial cells and keratocytes corresponds to the severity of the disease. Vogt's striae are depicted as hyporeflective lines in the stroma. There is also marked polymorphism and polymegatism, as well as significantly reduced ECD. In the layer of the subbasal nerve plexus, an abnormal architecture and reduced density of nerve fibers are visualized [139].
CXL is a minimally invasive surgical procedure aimed at strengthening the tissue in corneal ectasia and stopping the progression of keratoconus [140] [141] [142].
The action of CLX is based on a photochemical reaction between Riboflavin (Vit B 12) and Ultraviolet rays (UVA).
The processes and effect after the application of CXL at a microstructural level can be visualized with CFM. [148] [149]. Messmer et al. [148] reported a significant correlation between the studied parameters and the function of blebs. Filtering blebs with optically empty epithelial microcysts, stroma with extensive non-encapsulated hyporeflective zones, low degrees of vascularization and tortuosity all correlate with good function.   In functioning blebs, normal epithelium is established; loose hyporeflective subepithelial tissue with numerous optically empty spaces is detected too. In non-functioning blebs, an absence or a reduced number of epithelial microcysts, dense hyperreflective subepithelial tissue and a large number of blood vessels are found [146] [147]. Caglar et al. reported a significant correlation between the morphological CFM appearance of FB and the function of FB. The presence of epithelial microcysts, capsule-free stromal cysts, minimal vascularization and decreased vascular tortuosity are associated with good FB function, and conversely, hyperreflective dense stroma is associated with poor FB function [150].

Conclusion
In vivo confocal microscopy is a relatively new method, which provides detailed information about ocular structures at a cellular level. In vivo laser scanning confocal microscopy is a non-invasive examination, allowing layer-by-layer high-resolution imaging at a microstructural level of the tissues of the central and peripheral cornea, eyelid and bulb conjunctiva, eyelids and tear film. It reveals new possibilities to clinicians for evaluation and better understanding of the processes taking place in the structures of the normal anterior ocular surface in abnormal states, as well as after anterior eye segment surgery.