Confocal microscopy

From EyeWiki


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

Characteristic Features of IVCM Images:

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,sequence scan. A section scan is a single image. Volume scan acquires multiple images from a user-selected starting depth- 40 images over max. 80 microns depth. 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] 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]


IVCM Application In Different Ocular Surface Conditions
  1. Normal Central Cornea (ageing, contact lens wear-related changes)
2. Dry Eye Disease
3. Corneal ectatic disorders, dystrophies, degenerations
4. ICE syndromes
5. Keratitis (microbial, fungal, parasitic, viral)
6. Post-surgical evaluation of the cornea ( cataract surgery, LASIK, LASEK , PRK, keratoplasties )
7. Corneal deposits ( pseudoexfoliation syndrome, drug-induced )
8. Systemic diseases ( DM, Parkinson's disease, SLE, RA )
9. Evaluation of the conjunctiva ( conjunctivitis, neoplasia )
10. Limbal stem cell deficiency ( chemical or thermal injury, Stevens-Johnson syndrome )
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 screening tool for patients undergoing treatment.
Description of Corneal Epithelium

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 smaller diameter 8-10 μm with dark cytoplasm and bright border. 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]

Hyper-reflective dendritic structures have been documented at the level of 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] This dendrtitic cells represent Langerhans' antigen presenting cells, that play a role in immune response. Langerhans cells (LCs) are professional antigen presenting cells of the ocular surface and can be detected in the normal uninflamed cornea.[16] Increased density of LCs have been detected in the central cornea of patients with keratoconjunctivitis, keratitis.[17][18]

Description of Bowman's layer:

It is an amorphous 10 μm thick membrane posterior to 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.

Description of Corneal Stroma:

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 oval in the posterior. Myelinated nerve fibers can be visualized in the anterior stroma, but the orientation and size are variable which makes the 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]

Description of Descemet's membrane:

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

Description of Endothelium:

This is a layer of endothelial cells which are 4-6 μm thick and 20 μm in diameter with a hexagonal or polygonal shape. On IVCM 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]

Corneal Nerves:

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.

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 and due to this feature physicians can explore corneal innervation after keratoplasty, PRK, LASIK Aas well as in dry eye, diabetic neuropathy.[31] Another study highlighted the importance of exprienced observers in manual assessment of corneal nerve parameters.[32] Sub-basal corneal nerve recovery after DMEK to its preoperative values occur 4-10 months after surgery.

Description of Conjunctiva:

Normal bulbar conjunctiva epithelium is comprised of superficial, intermediate and basal cells. Also large hypo reflective oval-shaped cells are seen throughout the epithelium. Those cells represent 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 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.


Heidelberg Retinal Tomograph 3 with Rostock Cornea Module (HRT3-RCM) application in the clinic

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 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 and 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.

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.

IVCM Findings in Contact Lens Wearers

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 showed that contact lens wear cause 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 scanned by tandem scanning confocal microscope. Decreased corneal sensitivity in contact lens wearers is not associated with decreased nerve fiber bundle density either.[38] Various studies showed 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. 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 lens can be displaced and impinge further onto the bulbar conjunctiva. Both the cornea and conjunctiva are susceptible to physical irritation form the lens.The observation in one study suggest that contact lens wear induce conjunctival epithelial thinning, accelerated formation of microcysts, increased epithelial cell density,but no impact on Langerhans or goblet cell density.[41] Also in contact lens wearers LC density in central and peripheral parts of cornea was twofold higher than in normal controls, implying mechanical irritation of the corneal surface from contact lens wear.[42] The mechanical irritation from contact lens wear either soft or rigid may promote inflammatory mediatos (cytokines, growth factors) release and keratocyte redistribution in corneal stroma.[43] In summary, confocal microscopy is an imaging modality that can be utilized to further investigate changes in cornea and conjunctiva related to contact lens wear.

IVCM and Limbal Stem Cell Deficiency

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 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 case 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 ( large oval hyperreflective cells). Large number of dendritic cells are present in the conjunctival epithelium. Corneal epithelium is metaplastic with dendritic cells present in the subepithelial region in limbal stem cell deficiency.The Bowman layer is not identified and the stroma has a fewer keratocytes when compared with a normal cornea .[45] Corneal basal cell density along with subbasal nerve plexus density decrease in patients with limbal stem cell deficiency, basal epithelial cells become severly metaplastic.[46][47] Analysis of the limbal stroma showed replacement of hyperreflecive niche structures by bright fibrotic structures.[46]

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 more reliable than IC analysis in evaluation of outcomes after limbal stem cell transplantation in the long term as well.[48] IVCM has been successfully utilized to document the clinical and morphological corneal findings in the early stages of aniridic keratopathy showing focal opacities in basal epithelium, altered sub-basal nerves, tear film instability and degradation of limbal palisade microstructure.[50]

Keratoconus by Confocal Microscopy

Keratoconus is a progressive noninflammatory corneal ectasia in which the cornea assumes a conical shape. It is characterized by stromal thinning and that may be the result of a loss of keratocytes, extracellulat matrix or both.[51] Investigators showed good repeatability and reproducibility for keratocyte count by LSCM.[52] In subjects with keratoconus ICVM showed significant reduction in keratocyte cell count and that decline was correlated with indices of disease severity. Anterior keratocyte density was significantly lower in contact lens-wearing keratoconic subjects.[53] Also morphologic alterations to the epithelium and Bowman's layer have been described. Images obtained by Tomey Confoscan P4 revealed 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.[54]

Keratoconus is progressive in 20% of cases and can be treated by lamellar or penetrating keratoplasty. The technique of corneal collagen cross-linking is a conservative approach to slow or arrest progression of the disease and 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.[55] 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.[56] [57]

Confocal microscopy evaluates microstructural changes of the cornea 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 manifestation of topographic signs. Some KCN relatives with normal corneal topography may have keratoconus diagnoses or subclinical KCN in the future. For this patient category detection of early forms of KCN is essential before keratorefractive surgery to avoid subsequent complications.[58]

Confocal Microscopy in Corneal Dystrophy

Corneal dystrophies are a group of inherited disorders that are usually bilateral, symmetric, slowly progressive and not related to environmental or systemic factors.[59] Disorders in this group can affect various corneal layers resulting in certain microstructural as well as gross morphological changes. Confocal microscopy can visualize the changes non-invasively at a cellular level and it has been applied to delineate those 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.[60]

Confocal microscopy performed in eyes with Reis–Buckler dystrophy, granular dystrophy and lattice type-I dystrophy demonstrated the diversity of the deposit pattern among these three autosomal-dominant corneal dystrophies.[61] 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.[61] 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 and it should not be misdiagnosed as fungal hyphae.[62] In rare dystrophies such as Fleck and pre-Descemet's membrane corneal dystrophy, confocal microscopy visualized intracellular deposits throughout the stroma and intacellular as well as extracellulat deposits immediately anterior to Descemet's membrane, respectively.[63]

It has also been shown that anterior corneal cellular and structural abnormalities begin early in the course of Fuchs’dystrophy, before the onset of clinically evident edema. IVCM demonstrated depletion of anterior stromal cells and high extracellular reflectivity even in 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 back scatter and irregular anterior corneal surface.[64] This subclinical changes are relevant for postoperative visual outcomes after introduction of newer surgical techniques like DSEK.[65]

IVCM in Systemic Diseases

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.[66] 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.[67]

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, In contrast, the MGUS immunoprotein deposits associated with IgA-gammopathy involved the anterior and midstroma with sparing of the epithelium and endothelial layers. Endothelium has not been involved in this case series.[68]

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.[69]

In Vivo Confocal Microscopy After Ocular Surgery

IVCM in Refractive Surgery:

Laser in situ keratomileusis (LASIK) is a leading technique in refractive surgical correction of ametropias. IVCM helps clinicians to examine flap-related complications after refractive surgeries, describe changes in corneal nerves and sublayers.[70] This technique images small particles at the flap interface as well as Bowman layer's microfolds.[70]

IVCM as a high resolution imaging modality can assess and compare corneal modifications caused by different types of lasers used to create LASIK flap.[71] Some studies showed that LASIK with IntraLase provides more reproducible flap thickness and fewer interface particles.[71][72] 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.[73][74]

Studies of in vivo confocal microscopy after LASIK have shown that the number of subbasal and stromal nerve fiber bundles decreased by more than 90% 1 week after LASIK and increased from 3 months to 1 year after surgery.[70][75][76]

After photorefractive keratectomy (PRK), IVCM shows the regeneration of the epithelium covering the wound. It has also been observed that activated keratocytes were responsible for the clinically visible haze which could be evaluated objectively with this technique.[77] Another study described corneal wound healing after myopic PRK and showed epithelial thickness increased 21% by 12 months after PRK 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.[78]

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.[79]

In Vivo Confocal Microscopy in Management of Microbial Keratitis

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, fungal and Acanthamoeba keratitis (AK) in particular. Delayed diagnosis of these infections is typical due to the time delay of corneal cultures and slow-growing organisms such as fungi and Acanthamoeba. A fast and reliable diagnosis of Acanthamoeba and fungal keratitis is essential to ensure an optimal outcome.

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.[80] 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.[80]

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 [81] or have "single file arrangement"[82]. Interestingly, the arrangement of Acanthamoeba cysts in clusters or chains was associated with a worse outcome of AK [81]. IVCM had a sensitivity of 90% and specificity of 100% in case of both clinical and objective evidence of AK.[83] Numerous studies have demonstrated usefulness of IVCM for the diagnosis of AK.

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.

Fusarium hyphae and Candida pseudo filaments were imaged by HRTII‐RCM [84] . 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[85].

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