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Images of Descemet's membrane have a hazy appearance. It is 6-10  μm thick with cellular structures not identifiable on IVCM images. Normal Descemet's membrane is not visible when imaged by IVCM in young people, but becomes more visible with aging.[23]

Hyper-reflective dendritic structures have been documented at the level of the basal epithelium and Bowman's membrane in 12-30% of normal volunteers with a mean density of 34 ± 3 cell/mm² in the central cornea and 98 ± 8 cell/mm² in the periphery.[15] Langerhans cells (LCs) are professional antigen presenting cells of the ocular surface and can be detected in the normal, un-inflamed cornea.[16] Increased density of LCs have been detected in the central cornea of patients with keratoconjunctivitis.[17][18]

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Anterior corneal cellular and structural abnormalities begin early in the course of Fuch's dystrophy, before the onset of clinically evident edema. IVCM demonstrated depletion of anterior stromal cells and high extracellular reflectivity even in patients with mild cases of Fuch's dystrophy. IVCM images visualized reticular networks of probably fibroblasts located deep to the basal epithelial layer that were highly reflective, hence contributing to corneal backscatter and irregular anterior corneal surface.[63] This subclinical changes are relevant for postoperative visual outcomes after introduction of newer surgical techniques like DSEK.[64]

In vivo confocal microscopy (IVCM) is an emerging noninvasive imaging and diagnostic tool, which enables morphological and quantitative analysis of ocular surface microstructure. The principal of confocal microscopy was patented in 1957 by Marvin Minsky .[1]The key elements of Minsky's confocal microscope design included the pinhole apertures, point-by- point illumination of the specimen and rejection of out-of-focus light.[2] Tandem-scanning microscopes were the first commercialized confocal microscopes introduced in the 1960s in Czechoslovakia. In 1969, the first laser scanning confocal microscopes were developed at Yale University.[3]

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Fabry disease is an X-linked genetic disorder determined by the deficient activity of α galactosidase A, a lysosomal enzyme, that causes an error in glycosphingolipid metabolism. Cornea verticillata and stromal haze are the most characteristic and frequent ocular findings of this disorder. Confocal microscopy has been utilized to describe microscopic corneal and conjunctival findings in patients with Fabry disease (FD) related keratopathy. Structural alterations were found throughout the entire ocular surface epithelium. IVCM could be a a useful technique in facilitating the diagnosis of FD-related ocular surface manifestation and to detect variations while monitoring the effect of enzyme replacement therapy in the future.[68]

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Heidelberg Retina Tomograph Rostock Cornea Module- HRT III RCM has advantage of imaging corneal structures with much higher resolution. Its magnification (800 ×) is high enough to visualize fungal hyphae and yeast in the cornea. The high resolution allows visualization of yeasts, which has never been described with previous confocal microscopes.

IVCM can also be used to measure the depth of cysts invasion and to monitor the progression and response to the antimicrobial therapy. Numerous studies described cyst morphology in AK, and a few analyzed the arrangement of these structures. Cysts can be organized in chains and/or clusters [80] or have "single file arrangement"[81]. Interestingly, the arrangement of Acanthamoeba cysts in clusters or chains was associated with a worse outcome of AK [80]. IVCM had a sensitivity of 90% and specificity of 100% in case of both clinical and objective evidence of AK.[82] Numerous studies have demonstrated usefulness of IVCM for the diagnosis of AK.

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The superficial layer is difficult to image by IVCM. It is comprised of superficial epithelial cells, wing cells and basal epithelial cells. The superficial cells are usually 40-50 μm in diameter with hyper-reflective cytoplasm and a bright nuclei of 10 μm. [11] The wing cells are 20-30 μm in diameter with very thin borders and the average wing cell density is 5000 cell/mm².[12] Basal epithelial cells have a smaller diameter of 8-10 μm with dark cytoplasm and bright borders. They show honeycomb pattern and variable cell densities among studies-from 3600 to 8996 cell/mm².[12][13] IVCM has demonstrated a decrease in basal epithelial cell density in diabetic patient population.[14]

ICVM showed that increase in corneal epithelial thickness after myopic LASIK persists for at least 7 years and that the central corneal thickness increases during the first year after PRK and remains stable thereafter up to 7 years.[78]

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After photorefractive keratectomy (PRK), IVCM shows the regeneration of the epithelium covering the wound. Activated keratocytes that are responsible for the clinically visible haze could be evaluated objectively with this technique.[76] Another study described corneal wound healing after myopic PRK and showed that epithelial thickness increased 21% by 12 months and remained unchanged to 36 months after PRK, but there was no change in stromal thickness between 1 and 36 months after PRK. Activated keratocytes and corneal haze peaked at 3 months after myopic PRK.[77]

The corneal limbus is a semitransparent, vascularized transition zone between cornea and sclera. The palisades of Vogt are believed to harbor corneal epithelial stem cells and are distinctive normal features of the human corneoscleral limbus. The palisades of Vogt were first clinically described in 1914 by Streiff who called them radial stripes. They are a series of radially oriented fibrovascular ridges and in between them there is a thickened conjunctival epithelium or so-called interpalisades. In vivo confocal microscopy is useful in observing limbal microstructures. Clusters of hyperreflective structures were observed in the anterior limbal stroma , but not in the corneal stroma.[44] Stem cells of the corneal epithelium reside in the basal limbal part of the corneoscleral junction. Deficiency of these epithelial stem cells can be either inherited (aniridia, multiple endocrine deficiency, epidermal dysplasia) or acquired (burns, cicatricial pemphigoid, Stevens-Johnson syndrome, contact lens-associated, neurotrophic keratopathy, multiple surgeries, chronic limbitis, severe microbial infections extending to limbus). Live imaging of corneolimbal epithelial architecture became possible with the advent of in vivo confocal microscopy. In cases of limbal stem cell deficiency, the limbal epithelium is replaced by conjunctival epithelium characterized by cells with or without evident nuclei, along with goblet cells (which are characterized by large oval hyper-reflective cells). Large numbers of dendritic cells are also present in the conjunctival epithelium, mostly in the subepithelial region. The Bowman layer can not be identified and the stroma has fewer keratocytes when compared with a normal cornea .[45] Corneal basal cell density along with subbasal nerve plexus density decreases in patients with limbal stem cell deficiency and basal epithelial cells become severely metaplastic.[46][47] Analysis of the limbal stroma showed replacement of hyper-reflecive niche structures by bright fibrotic structures.[46]

Microbial keratitis is a major blinding eye disease in the world and is more common in contact lens wearers. IVCM proved to be a useful tool in the early diagnosis of microbial keratitis, and particularly in fungal and Acanthamoeba keratitis (AK). Delayed diagnosis of these infections is common due to the time delay of corneal cultures and slow-growing fungi and Acanthamoeba. A fast and reliable diagnosis of Acanthamoeba and fungal keratitis is essential to ensure an optimal outcome.

Patient examination in the clinic is conducted over anesthetized cornea and may last from 5-15 min with patient rarely experiencing discomfort. The system consists of 670 nm diode laser and horizontally-mounted optics upon which a disposable plastic sterile cap is placed (Tomo-Cap; Heidelberg Engineering GmbH). The cap comes in touch with the corneal surface through a refractive index-matching ophthalmic gel (Comfort Gel; Bausch & Lomb GmbH, Berlin, Germany). For confocal imaging, the ophthalmic gel is placed on the tip of objective lens to serve as a cushion and to eliminate bright reflections. Scans are collected from epithelium to endothelium while using the (HRT3-RCM) streaming software function. The acquisition rate for sequence scans can be set to 30 frames/second and up to 100 images can be stored. Image acquisition of 8 frames/sec is typical for patients without nystagmus. In order to obtain good images, the patient's corneal surface has to be in good contact with the objective lens. This often requires manual manipulation of head and eyelids. Access to cornea layers further from the apex is achieved by manually placing a fixation target into which the patient is instructed to view.[35] A digital camera mounted on a side provides a lateral view of the eye and objective lens, which helps monitor the position of the lens on the ocular surface during the examination.

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The HRT Rostock Cornea Module is a contact lens system attached to the Heidelberg Retina Tomograph (II) and features three unique imaging modes for maximum versatility: section scan, volume scan, and a sequence scan. A section scan is a single image. Volume scan acquires multiple images from a user-selected starting depth. A sequence scan is a dynamic movie of 1-30 frames of variable depth.[7] Images acquired via LSCM (laser scanning confocal microscopy) are en-face, i.e. parallel to the surface of the cornea, have a field of view of either 300×300 μm or 400×400 μm (depending on the internal lens power) with the lateral resolution of 1-2 μm and the axial resolution of 4 μm. Images taken with slit-scanning microscope (Confoscan Series) have the following parameters: 1-2 μm for lateral and 25-27 μm for axial resolution.[8] The LSCM operator can manually select the depth of interest and adjust the image brightness. The IVCM has the advantage of imaging through moderately opaque tissues ( scarring or edema of the cornea) and also observes the dynamic process in the cornea, i.e. inflammatory reaction monitoring in infectious keratitis, wound healing after refractive surgery. [9][10]

Confocal microscopy enables evaluation of corneal microstructural changes in patients with manifest keratoconus (KCN), subclinical KCN and in topographically normal KCN relatives. This technique is useful for the determination of early corneal changes before the manifestation of topographic signs.[57]

Stromal nerves can be divided into 2 groups: sub-Bowman's and mid-stromal nerves. Sub-Bowman zone appear as hyper-reflective linear 1-5μm thick structure. The thicker mid-stromal nerves run straight and show dichotomous bifurcation. Corneal nerves can be analyzed qualitatively and quantitatively by IVCM. Physicians can thus explore corneal innervation after keratoplasty, PRK, LASIK, dry eye, and diabetic neuropathy.[31] However, one study highlighted the importance of experienced observers in the manual assessment of corneal nerve parameters.[32]

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Stroma is composed of collagen fibers, keratocytes and interstitial substance. Keratocyte nuclei are 5-30 μm in diameter, have a bean-like shape in the anterior stroma and are oval posteriorly. Myelinated nerve fibers can be visualized in the anterior stroma, but the orientation and size are variable which makes quantification difficult. A study showed that stromal thickness imaged by IVCM was significantly higher in diabetic patients.[19] Keratocyte density is the highest in the anterior stroma, 50-100 μm posterior to the Bowman's membrane.[20] Confocal microscopy has been used to show keratocyte density in normal cornea, contact lens wearers, keratoconus etc.[21][22]

The use of IVCM in the scientific research has been expanding rapidly over the past years. It has also been implemented for the clinical diagnosis of different ocular surface conditions as well as a screening tool for patients undergoing treatment.

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IVCM as a high resolution imaging modality can assess and compare corneal modifications caused by different types of lasers used to create LASIK flap.[70] Some studies showed that LASIK with IntraLase provides more reproducible flap thickness and fewer interface particles.[70][71] Interface particle could be either metal or plastic particles from the microkeratome blade or lipid products, migrated corneal epithelial cells, synthetic material such as sponge particles, powder from surgical gloves, or inflammatory cells.[72][73]

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Acathamoeba is a ubiquitous protozoan found in water, soil, air and has a 2-stage life cycle: trophozoites and cysts. Both of these forms are identifiable on IVCM exam along with corneal nerves and inflammatory cells.[79] Acanthamoeba cysts are hyper-reflective 15-28 micrometers in size with a double wall. They are usually spherical, but may sometimes appear ovoid. The trophozites are usually 25-40 micrometers in diameter, also hyper-reflective with surrounding hypo-reflective edema.[79]

Potential complications of this procedure include corneal abrasion and infection. Careful disinfection of the lens surface is important to minimize the risk. The risk of corneal abrasions may be higher in patients with epithelial defects, ulcers and corneal epithelial or basement membrane dystrophies.

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It is a valuable technique to visualize immunoprotein deposits as well as to determine the extent of corneal involvement in gammopathies. The immunoprotein crystals related to IgG-kappa gammopathy could be found in the epithelium. In contrast, the MGUS immunoprotein deposits associated with IgA-gammopathy involved the anterior and mid-stroma with sparing of the epithelium and endothelial layers. Endothelium has not been involved in this case series.[67]

Fusarium hyphae and Candida pseudo filaments were imaged by HRTII‐RCM [83] . The Fusarium solani patients' corneas revealed numerous high‐contrast lines 200–300 μm in length and 3–5 μm in width with branches at 90° angles. Candida albicans-infected corneas revealed numerous high‐contrast elongated particles measuring 10–40 μm in length and 5–10 μm in width, resembling pseudo filaments. In all fungal keratitis cases inflammatory cells were present at the epithelial layer. IVCM is a valuable tool in diagnosing filamentous fungal keratitis and clinically applicable grading scale could facilitate the interpretation of the IVCM images[84].

This is a layer of cells that are 4-6 μm thick and 20 μm in diameter with a hexagonal or polygonal shape. On IVCM, they are identified as bright cell bodies with dark cell borders. The nuclei are rarely recognizable. Increasing age cases a reduction in endothelial cell density by approximately 0.6% per year and increase in cell variation.[24] In studies using (HRT-II)/Rostock Cornea Module (RCM) (Heidelberg Engineering GmbH, Heidelberg,Germany) the average endothelial cell density varied between 2550 and 2720 cell/mm².[25] Diabetic patients show an increase in endothelial cell damage and polymegathism with increasing duration of diabetes.[26][27]

The most used IVCM used today in clinical practice and reported in the published literature are Confoscan Series (Nidek Co. Ltd., Gamagori, Japan) and the Heidelberg Retina Tomograph II (HRT-II)/Rostock Cornea Module (RCM) (Heidelberg Engineering GmbH, Heidelberg,Germany). The Confoscan 4 confocal microscope uses a slit scanning design. The slit scanning microscope has the advantage of scanning many points in parallel, but the disadvantage is that this microscope is truly confocal only in the axis perpendicular to the slit. [4] Confoscan 4 has 20× lens that works 12 mm from the corneal apex for endothelial cell count, allowing a fully no-contact exam. Its z-adapter agrees with mean corneal thickness measured with the Tandem Scanning confocal microscope when both instruments are correctly calibrated.[5] HRT-II/Rostock Cornea Module has 63× lens and optional 10 × to see deeper (lens, zonules), uses a 670 nm diode laser and provides 1 micron resolution. HRT-II/Rostock Cornea Module does applanate cornea and the use of PMMA disposabel cap and gel is mandatory for the exam.[6]

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Nerves enter the cornea from a peripheral, mid-stromal depth and proceed anteriorly terminating between corneal epithelial cells. These nerves loose their myelin sheath within 1 mm of the limbus and are subsequently surrounded by Schwann cell sheaths.[28] Nerves bundles from anterior stroma penetrate through Bowman's membrane to form sub-basal nerve plexus that runs parallel to the ocular surface.[4] Corneal nerves are easily identified by IVCM. Sub-basal corneal nerves are hyper-reflective linear structures. The sub-basal nerve diameter can vary from 0.52 μm to 4.6 μm.[29] Sub-basal nerve density values vary between studies mainly due to different types of confocal microscopes used for imaging. Nerve density values reported from studies using HRTII RCM are higher than values reported from studies using slit scanning confocal microscopes: 21.6 mm/mm² [8] via HRTII RCM vs. 15.18 mm/mm² via slit scanning microscope. [30] Nerve fiber bundles are arranged radially in cornea and converge toward 1-2 mm inferior to the central cornea to form whorl or vortex pattern.

Corneal dystrophies are a group of inherited disorders that are usually bilateral, symmetric, slowly progressive and not related to environmental or systemic factors.[58] Disorders in this group can affect various corneal layers resulting in certain micro-structural as well as gross morphological changes. Confocal microscopy can visualize the changes non-invasively at a cellular level. This technique has been used to delineate changes in posterior polymorphous corneal dystrophy (PPCD), Fuchs' endothelial dystrophy, bowman and stromal corneal dystrophies. In a case series of patients with PPCD, confocal microscopy showed reduced endothelial cell density, hyperreflectivity at the level of Descemet's membrane surrounding the endothelial lesions, which have been classified into 3 main forms: vesicular, band and diffuse.[59]

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The diagnosis of LSCD is mostly clinical with definitive changes observed by slit-lamp biomicroscopy, but there is some limitation with this technique associated with subtle changes, particularly in partial LSCD. Superficial corneal neovascularization, conjunctivalization and ocular surface inflammation are often subtle and nonspecific in partial LSCD. Impression cytology (IC) analysis can also provide objective evidence of LSCD, but it does not offer analysis on deeper corneal layers. IVCM is more reliable diagnostic technique in patients with the suspected diagnosis of LSCD and it showed a substantial degree of concordance with IC analysis.[48][49] IVCM is a useful tool and may be more reliable than IC analysis in evaluation of outcomes after limbal stem cell transplantation in the long term as well.[48]

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It is an amorphous 10 μm thick membrane posterior to the basal epithelium, featureless and grey on confocal microscopic images with discrete nerves bundles in the field of view. Some keratocytes may be seen in the background.

Corneal cross linking has been shown to reduce the progression of keratoconus and potentially avoid the need for keratoplasty. Reduction in anterior and intermediate keratocytes followed by gradual repopulation has been  described by HRT II-RCM confocal microscopy after riboflavin-UVA-induced corneal collagen cross-linking.[54] IVCM has also been successfully used to characterize keratoconic cornea after deep anterior lamellar keratoplasty (DALK) and penetrating keratoplasty (PKP). Images obtained by Confoscan P4 ( Nidek Technology, Padova, Italy) confirmed that keratocyte density was significantly lower in the DALK group than in the PKP group throughout the stroma. One of the possible mechanisms of such cellular changes of the donor tissue could be the mechanical trauma to the donor tissue during the surgery.[55] [56]

The availability of confocal microscopy in the clinic provides an opportunity to study cornea changes after contact lens wear. Confocal microscopy can be performed over the contact lens to observe changes in corneal cellular morphology. Studies have shown that contact lens wear causes stromal acidosis and hypoxia.[36][37] However, long-term contact lens wear and its associated acidosis and hypoxia have no significant effect on keratocyte density. Decreased corneal sensitivity in contact lens wearers is not associated with decreased nerve fiber bundle density, either.[38] Various studies have shown that extended contact lens wear does cause a loss of keratocytes.[39][40] The effects of contact lens wear on the bulbar conjunctiva can be investigated by LSCM as well. When a soft contact lens is worn, it completely covers the cornea and impinges 2 mm onto the bulbar conjunctiva. During eye movement or blinking, contact lenses can be displaced and impinge further onto the bulbar conjunctiva. Both the cornea and conjunctiva are susceptible to physical irritation from the lens. The observation in one study suggests that contact lens wear induces conjunctival epithelial thinning, accelerates formation of microcysts, increases epithelial cell density, but has no impact on Langerhans or goblet cell density.[41] Also in contact lens wearers LC density in central and peripheral parts of cornea has been reported to be twofold higher than in normal controls, implying mechanical irritation of the corneal surface .[42] The mechanical irritation from contact lens wear either soft or rigid may promote inflammatory mediators (cytokines, growth factors) release and keratocyte redistribution in corneal stroma.[43]

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Studies of in vivo confocal microscopy after LASIK have shown that the number of sub-basal and stromal nerve fiber bundles decreased by more than 90% 1 week after LASIK and increased from 3 months to 1 year after surgery.[69][74][75]

Normal bulbar conjunctival epithelium is comprised of superficial, intermediate and basal cells. Large, hypo reflective oval-shaped cells are also seen throughout the epithelium. Those cells represent the goblet cell population in the conjunctival epithelium. Goblet cells constitute approximately 10% of the conjunctival epithelial cell population and they are scattered in the conjunctival epithelium.[33] Conjunctival epithelium also contains LC, which function as tissue macrophages. Epithelial cells in the palpebral conjunctiva are much smaller than those of the bulbar conjunctival epithelium. At the corneal limbus, the conjunctival epithelium and the stroma form palisades of Vogt. IVCM has been also used to study the inflamed conjunctiva in different types of conjunctivitis. The common feature is that the inflammatory cells are significantly increased in epithelium and the stromal collagen meshwork contains hyper-reflective debris as well.[34] The episclera and sclera are too deep to be visualized by IVCM.

IVCM may help to identify suspicious conjunctival lesions, but it does not replace histology for the diagnosis of the tumors.

Confocal microscopy performed in eyes with Reis–Buckler dystrophy, granular dystrophy and lattice type-I dystrophy demonstrated a diversity of a deposit pattern among these three autosomal-dominant corneal dystrophies.[60] In this case series epithelium was involved in 30% of the eyes and stroma was involved in all eyes. Some of the confocal findings near the Bowman membrane were common for all three dystrophies.[60] This technique may be used in addition to slit-lamp biomicroscopy in atypical corneal dystrophies, as histopathologic studies cannot be obtained systematically for all patients affected. In lattice corneal dystrophy linear and branching structures with changing reflectivity were visualized in the stroma.[61] In Fleck and pre-Descemet's membrane corneal dystrophy, IVCM found intracellular deposits throughout the stroma.[62]

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Laser in situ keratomileusis (LASIK) is a leading technique in refractive vision correction. IVCM helps clinicians to examine flap-related complications after refractive surgeries and describe changes in corneal nerves and sublayers.[69] This technique images small particles at the flap interface as well as Bowman layer's microfolds.[69]

Keratoconus is a progressive noninflammatory corneal ectasia in which the cornea assumes a conical shape. It is characterized by stromal thinning that may be the result of a loss of keratocytes, extracellular matrix, or both.[50] Investigators showed good repeatability and reproducibility for keratocyte count by LSCM.[51] In subjects with keratoconus ICVM showed a significant reduction in keratocyte cell count, a decline that was correlated with indices of disease severity. Anterior keratocyte density was significantly lower in contact lens-wearing keratoconic subjects.[52] Morphologic alterations to the epithelium and Bowman's layer have also been described, such as disruption of Bowman's layer and the occasional presence of epithelial cells and keratocytes. Most of the changes observed with CM have been correlated with the findings obtained by light microscopy.[53]

IVCM is a non-invasive method that has been proposed to diagnose as well as to assess the progression of diabetic neuropathy. It enables the study of corneal nerve alterations in various ocular diseases, after corneal surgery and in systemic diseases. The correlation between reduced corneal nerve bundles with loss of corneal sensation and severity of somatic neuropathy has been shown in patients with type 1 diabetes.[65] IVCM detects early peripheral neuropathy and also shows that corneal nerves recover with improved glycemic control withing 6 months after pancreatic transplantation in diabetic patients.[66]

A polarized 3D system uses polarization glasses to create the illusion of three-dimensional images by restricting the light that reaches each eye (an example of stereoscopy). To present stereoscopic images and films, two images are projected superimposed onto the same screen or display through different polarizing filters. The viewer wears low-cost eyeglasses with a polarizing filter for each eye. The left and right filters have different polarizations, so each eye receives only the image with the matching polarization. This is used to produce a three-dimensional effect by projecting the same scene into both eyes, but depicted from slightly different perspectives with different polarizations. Multiple people can view the stereoscopic images at the same time.