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These represent a family of straight-line fringes making an angle of −θ and θ, respectively, with the x axis. For a collimated beam, the fringes run parallel to each other in both wedge plates. This, therefore, is a self-referencing method requiring no reference line or fiduciary line and also offers double the sensitivity.

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.

In a recent work, Rana et al.76 captured the self-images of a grating at the first and second Talbot planes and computed the statistical correlation coefficient. Correlation coefficient estimates the similarities between the two images; the value is 1 if the images are absolutely identical and 0 if they are uncorrelated. When the point source is at the focal point, the correlation coefficient is nearly one; otherwise, its value is less than one.

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]

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Confocal microscopyprinciple

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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.

It is assumed that the wedge edge is parallel to the x direction. Here n is the refractive index of the material of the wedge plate and Δx is the amount of shear along the x direction. This equation represents a family of straight lines that make an angle θ with the x axis where θ=tan−1(−Δx/2nαR). When the beam is collimated, i.e., R is infinite, θ=0 and the fringes run parallel to the x axis. For finite R, these fringes are inclined.

Silva48 demonstrated that Talbot interferometry is a good technique for collimation testing. He used two identical gratings: self-image of the first grating falls on the second grating, which is aligned in an infinite fringe mode. In this mode, the fringe width is directly proportional to the radius of curvature of the wave that illuminates the first grating. He also showed that the moiré fringes are formed when the second grating is rotated in its plane such that grating elements in the self-image of the first grating make an angle with the elements of the second grating. The moiré fringes rotate if the first grating is illuminated by a spherical wave. On collimation, the moiré fringes are parallel to a reference direction.

A technique for routine collimation testing should be simple, requiring a minimum of components, and should have good setting accuracy. It is suggested that a pair of well-designed wedge plates properly aligned or a pair of composite gratings enclosing an acute angle would be a good choice.

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]

Is confocal microscopylightmicroscopy

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]

A Fresnel bi-prism, usually used for conducting interference experiments, would be an ideal element when used in reflection along with a composite grating for collimation testing using moiré fringes.

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]

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]

Prasad et al.87 wrote a 4×4 lenslet array on LCTV and used it for collimating a beam by finding the centroids of the point spread functions of the lenslet array.

It may be noted that the angle of rotation of the fringe from the reference direction is directly proportional to the shear and inversely proportional to the angle of the wedge. Therefore, Shukla et al.17 suggested a set of wedge plates with different angles and nominal thicknesses for use at certain ranges of angle of incidence to achieve optimum sensitivity for beams of diameters ranging from 3 to 100 mm. Udupa et al.18 gave the design of a shear plate to be used with an unexpanded laser beam. It can be used both for collimation testing and to measure the radius of curvature of the beam.

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.

A point-like source is to be placed at the front focal point of the collimating lens to obtain a collimated beam. If it is not placed at this location, the beam may be either divergent or convergent, that is, it becomes a spherical wave. If Δf is the axial shift of the point-like source from the focal point, the radius of curvature R of the emergent beam is R=±f2Δf. If the axial shift is outward from the focal point, it is a convergent beam; if it is inward (toward the lens), it is a divergent beam. When Δf=0, R becomes infinite and the beam is then collimated. Many methods of collimation testing rely on this characteristic, that is, they check whether the beam is spherical or not.

A large number of techniques and methods to collimate a beam originating either from an incoherent or a coherent source have been investigated and demonstrated. These exhibit a range of setting accuracies. Table 1 is a compilation of data from various publications. Though there are a large number of techniques that have been mentioned in this paper, two of them, namely shear interferometry and Talbot interferometry, have been researched and used the most for collimation testing. Wedge plate shear interferometry provides better sensitivity as compared with PPP shear interferometry. It is also to be noted that the setting sensitivity in shear interferometry depends on (i) the angle of the wedge, (ii) F# of the collimating lens, and (iii) magnitude of the shear. Shear interferometry can be performed in a relatively small space. The setting sensitivity in Talbot interferometry depends on (i) the frequency of the grating, (ii) number of the Talbot plane, and (iii) F# of the lens. Talbot interferometry requires a relatively large space to perform. However, if a grating of very small period is used at its first Talbot plane, the space requirement is considerably reduced.

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]

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]

What is confocal microscopyused for

Another modification of the conventional shear plate interferometer is the quadruple-pass lateral shear interferometer in which a double prism, which acts as a shear device, is used as a beam splitter in a Michelson type interferometer and half of the beam is reflected back.14

The author would like to thank the Department of Physics of Alabama A&M University for providing an excellent work environment and support.

A wedge plate shear interferometer uses a wedge plate of a very small wedge angle α (α∼10  arc sec.). When placed normal to the beam, the beams reflected from the front and back surfaces are angularly sheared and there is a variable path difference between the two beams. When the incident beam is collimated, a straight-line fringe pattern with fringes running parallel to the edge of the wedge results. When this wedge plate is placed inclined to the beam, the reflected beams are laterally shifted, resulting in rotation of the fringes. The direction of rotation depends on whether the beam is convergent or divergent. Obviously, this is more sensitive than the PPP shear interferometer. Mathematically, we can describe the fringe formation by xΔxR+2nαy=mλ.

Confocal microscopyppt

If one of the mirrors of a Michelson interferometer is replaced by a phase-conjugate mirror, then one observes interference between the convergent and divergent beams when the beam is not collimated, giving curved fringes whose curvature changes on either side of the collimation condition.42,43 A straight-line fringe pattern is obtained only when the beam is collimated. Darlin et al. increased the sensitivity of this technique using a bi-mirror along with a phase conjugated mirror.44

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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]

A collimated optical beam is required in several applications such as metrology, optical processing, free space propagation of information, and laser-based instrumentation. Prior to the advent of the laser, the size of the aperture at the focal point of a collimating lens determined the degree of collimation based purely on geometrical optics consideration. Because the laser beam can be focused to a tiny size, effectively making it a point source, the degree of collimation is governed by diffraction at the aperture of a collimating lens. A large number of procedures have been developed to collimate the beam. We examine the development of these techniques from the historical perspective.

Wang et al.11 used a PPP as a shear plate for collimating the beam from a laser diode. First, the beam was corrected using a combination of aspheric lens and cylindrical lenses and then collimated by obtaining a fringe-free field.

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]

When we wish to expand and collimate a laser beam from a laser oscillating in TEM00 mode, we assume that a planar wavefront of beam waist 2ω0 lies at the front focal plane of the lens of focal length f. The spot size 2ω(f) at the lens aperture is 2ω(f)=2ω0[1+(fz0)2]12, where z0=πω02λ and λ is the wavelength of laser radiation. The beam waist 2ωc lies at a distance f from the lens; ωc is nearly the same as ω(f). If the spot size 2ω(f) is equal to the diameter D of the lens, 86.5% of the incident power is transmitted by the lens. The irradiance across the lens aperture is not uniform, being maximum at the optical axis and about 14% at the periphery of the lens aperture. If a more uniform beam is required, it is to be over-expanded at the expense of available power.

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This gives a family of straight-line fringes running parallel to the y direction. The fringe width x¯ is given as x¯=λ/(2nα+ΔxR).

Anand and Narayanamurthy89 recorded an interference pattern on a bismuth silicone oxide (Bi12SiO20) crystal. The path difference between the recoding beams is large but within the coherence length. It is shown that the diffraction efficiency is maximum when the recording beams are collimated. Later, Anand et al.90 also showed that the irradiance of the beam passing through a birefringent material (quartz) placed between crossed polarizers varies with departure from collimation: it is minimum when the beam is collimated.

Bass and Whittier86 used a small corner-cube retroreflector that is translated a small distance perpendicular to the beam axis and measured the translation of the retroreflected beam. If the displacement of the retroreflected beam is equal to the displacement of the corner-cube retroreflector, the beam is collimated. This method of collimation is based purely on geometrical optics.

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]

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]

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]

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The use of spiral and evolute gratings in Talbot interferometry does not yield good sensitivity. The Dammann grating appears to be promising, but its cost may be prohibitive to be routinely used for collimation testing.

The beam from the collimating lens is incident on a PPP that is placed inclined to the beam. The beams reflected from the front and back surfaces of the plate are laterally sheared, and the interference is observed in the overlap region. The magnitude of shear depends on the angle of incidence, and for smaller angles shear is linearly related to the angle of incidence. When the point source is axially shifted by Δf from the focal point, the beam from the collimating lens can be described by a path function W(x,y), which is given by W(x,y)=x2+y22R,where x and y are Cartesian coordinates on a plane perpendicular to the optical axis and R is the radius of curvature of the beam. Taking this as the path function of the beam reflected from the front surface of the PPP, the path function for the beam reflected from the back surface can be described by the path function W(x−Δx,y), which is given by W(x−Δx,y)=(x−Δx)2+y22R′,where Δx is the shear, assumed to be along the x direction, and R′ is the radius of curvature, being different from R due to additional travel distance through the glass plate. Ignoring the phase change on reflection at the front surface, the condition for bright fringes in the interference pattern can be expressed as W(x,y)−W(x−Δx,y)=x2+y22ΔRR2+xΔxR=mλ,where m is the fringe order, λ is the wavelength, and ΔR=R′−R. Further, the term containing (Δx)2 is dropped. When near collimation condition, Δf≪f and hence R′∼R, the quadratic term can also be dropped. The interference pattern contains straight-line fringes: the fringe width x¯ is given by x¯=λRΔx=λf2ΔxΔf.

Avudainayagam and Chitralekha79 observed that Lau fringes, for small grating separations, rotate when the first grating is illuminated by a nearly collimated beam and the second grating is rotated in its own plane. The fringes rotate in the same direction as the second grating when the illumination is by a diverging wave and in the opposite direction when it is by a converging wave. When the first grating is illuminated with a collimated beam, the Lau fringes stagger and then vanish.

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].

For over a century, telescopes have been aligned and tested for their performance using the star test.1 This is a visual but quite useful test. A telescope then can be used for setting up a collimator. The telescope receives the beam from the collimator, and the position of the pinhole (aperture) near the front focal point of the collimating lens is axially adjusted until its sharp image is seen in the telescope. The beam exiting the collimating lens is now collimated. It should be noted that this is the method used to set up a collimator for doing an experiment either with a prism or a grating on a spectrometer.

It is obvious that both of these techniques require a composite wedge plate in which two identical wedge plates are aligned antiparallel. To avoid this cumbersome alignment, a single wedge plate is fixed on a mount that could be rotated exactly by 180 deg to obtain the second interferogram.27–29 Essentially, the technique provides double the sensitivity and possesses self-referencing. Zhang et al. used a wedge plate in which the resulting interference pattern appearing on a ground glass plate is grabbed and stored. The wedge plate is rotated by 180 deg, and the interference pattern is again grabbed. These two patterns are digitally subtracted to obtain the moiré fringe pattern.30 At collimation, a fringe-free field is obtained; otherwise, straight-line moiré fringes are observed. Li et al.31 proposed a method to obtain two sets of interference fringes from a single wedge plate. The wedge plate acts as a beam splitter in a Michelson interferometer. The mirrors are tilted such that two sets of interference fringes appear side by side. This technique was devised to overcome the problem of aligning two wedge plates8,23 or the requirement for an accurate rotator.27–29 However, obtaining these two sets of fringes side by side is not easy. This problem was, however, eliminated by Lee et al. by masking the lower half of one mirror and the upper half of the other mirror of a Michelson interferometer.32 The two patterns thus formed are one above the other. This arrangement has been demonstrated for both configurations: rotation of fringes and change of fringe width. It is shown that both configurations provide the same degree of collimation. In another method, a right-angle prism in retroreflector mode is used.33 Half of the diagonal surface of the prism is coated. Two sets of fringes, one on the reflection from the mirrored surface and the other on the retroreflection from the other half-surface, are formed when the wedge plate is used as a beam splitter. An interesting arrangement to generate two fringe patterns from a single wedge plate uses a combination of a wedge plate and a diagonal coated cube.34 This reversal interferometer has been demonstrated in two different configurations. It is interesting to note that two US patents were granted on the techniques outlined above.35,36

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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]

Rajpal S. Sirohi is a fellow of the Indian National Academy of Engineering, National Academy of Sciences, Optical Society of America, Optical Society of India, SPIE, and Instrument Society of India and is an honorary fellow of Indian Society for Technical Education and Metrology Society of India. He is a senior editor for Optical Engineering. He has published 248 papers in archival journals and written several books. He is the recipient of many awards, notable are the Gabor and Vikram awards from SPIE and Padmashri from the Government of India.

Confocal microscopyPDF

The wedge plate shear interferometer is quite sensitive, but it requires a reference line—a fiduciary line—to which the fringes are parallel when the beam is collimated. Sirohi and Kothiyal proposed a double wedge plate shear interferometer using two identical wedges with their apex opposite to each other and wedge edges parallel to each other.8 Equations for fringe formation in both wedge plates are xΔxR+2nαy=mλ,xΔxR−2nαy=m′λ.

Zhao and Chung92 used a one-order binary phase circular Dammann grating for collimation testing. The grating generates two uniform-intensity impulse rings in the far field. The separation of the rings is minimum when the beam is collimated.

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.

The fringe width becomes infinite when Δf=0, that is, when the point source is right at the focal point. On either side of this position, straight line fringes running parallel to the y axis are formed.

It is known that interference between two laterally shifted spherical waves produces a straight-line fringe pattern whereas interference between two laterally shifted plane waves produces a uniform illumination. With the advent of lasers, it became possible to observe interference patterns even when waves with large path difference were superposed. The first application of shear interferometry for collimation testing using a plane parallel plate (PPP) for shearing was reported by Murty.5 Subsequently, a wedge plate and several of its variants were used for collimation testing.6–8

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It has been pointed out that the PPP shear interferometer has its limitations when used for testing collimation because we cannot distinguish between a fringe of infinite width and a fringe of width equal to the aperture of the PPP. Further, large diameter beams cannot be tested for collimation because PPP samples only a portion of the beam. It is suggested that a shear interferometer with a tilt orthogonal to the shear direction would produce straight line fringes when the beam is collimated, and the fringes would rotate when the beam departs from collimation.15 It is much easier to detect a change in the direction of fringes than to identify the total absence of fringes. Therefore, it is better to use a wedge plate than a PPP as a shear device.

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]

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.

In a method proposed by Zhang et al.,91 a small circular aperture samples a beam that needs to be collimated. The aperture is placed abutting a high-quality collecting lens. Behind this lens, two planes that display identical diffraction patterns of the aperture are found. The separation between these planes is related to the wavefront error of the beam.

A shear interferometer with a large shear was realized by Shukla et al.9 by utilizing two plates whose separation could be varied to change the amount of shear. They claim this to be more sensitive for collimation testing. When the collimation of a narrow beam is to be tested, a PPP does not serve the purpose as the two beams from the front and back surfaces do not overlap. A shear plate with a small shear can be realized either using two plates placed parallel but close to each other or placing the diagonal surfaces of two right angle prisms (double prism) parallel to each other but with a small gap.10

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]

A Michelson interferometer can also be adapted for shear interferometry. One of the mirrors of the interferometer is replaced by a right-angle prism, which folds the incident wavefront. Shear is introduced by the shift of the prism in its plane. The right-angle prism can be replaced by a pair of mirrors enclosing an angle of (90  deg−α):α being very small. This provides self-referencing and doubles the sensitivity. Such an interferometer can also be used to check collimation of nonvisible beams provided the beam splitter is appropriately chosen. Chen et al.45 used such an interferometer to test the collimation of a beam from a CO2 laser.

Disadvantages ofconfocal microscopy

These techniques use both a wedge plate and a grating. Darlin et al.37 used a single wedge plate along with a composite grating to produce a moiré pattern. This hybrid arrangement provides double the sensitivity due to the composite grating. The techniques described so far use wedge plates of very small angles (∼10  arc sec.), resulting in a few fringes in the field of view for visual observation. By contrast, one can also use a wedge plate with a large angle (∼80  arc sec.) such that the fringes formed when the beam is collimated have a higher spatial frequency (∼2.5 lines per mm). A pair of such wedge plates of slightly different wedge angles is placed in the beam at two locations.38 These are adjusted to produce identical fringe patterns, which on superposition produce a moiré pattern. This moiré pattern is used for collimation testing. Disawal et al.39 used a single wedge plate of about 50 arc sec wedge angle and a linear grating to produce a moiré pattern and calculated the phase of moiré fringes using a temporal phase-shifting technique. At collimation, the phase is constant across all pixels along the shear direction.

Instead of using a shear plate, the beam is sheared with a pair of sandwiches of holo-lenses.12 The holo-lenses are recorded with a collimated beam as a reference wave. This experimental arrangement is long and cumbersome. Rajkumar et al.13 used holo-elements in close proximity. The holo-elements are recorded by interference between two collimated beams and hence are essentially sinusoidal gratings.

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.

Several other researchers demonstrated the application of Talbot interferometry for collimation testing.49–52 Instead of using two gratings, a single grating with its self-image formed by reflection from a plane mirror on itself has been used for collimation testing.49 The arrangement is relatively compact, but departure from collimation is detected by the change in fringe width. A modification of this uses a right-angle prism (porro prism) instead of a plane mirror for folding the path so that the self-image of the grating is formed on itself.53 The porro prism is placed such that its edge is vertical and the grating elements make a small angle θ with the horizontal. Due to retroreflection, the grating image is folded about the vertical; thus, its elements make an angle −θ with the horizontal. The position of a porro prism is adjusted so that the retroreflected self-image falls on the grating, thereby forming a moiré pattern in which the fringes run parallel to the edge of the prism for a collimated beam. If the incident beam is spherical, the moiré fringes rotate. The method is self-referencing and easy to perform.

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]

Interestingly, the wedge plate can also be used in another orientation—the wedge edge is perpendicular to the lateral shear direction. In this situation, the condition for fringe formation is xΔxR+2nαx=mλ.

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]

Henning and Carlsten46 proposed a cyclic interferometer, in which shear is introduced by the shift of one of the mirrors, for measuring the radius of curvature of a short-coherence length laser beam and collimating the beam. By replacing a mirror of the cyclic shear interferometer by a bi-mirror, self-referencing can be built.47 This self-referencing cyclic shear interferometer is demonstrated for both fringe rotation and fringe width mismatch modes of operation.

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]

Instead of the two gratings being separated by a finite distance, Patorski80 suggested an arrangement in which the separation between the two gratings is infinite. This is achieved by placing the first grating illuminated by an incoherent source at the front focal plane of a lens and the second grating after the lens. In such an arrangement, second grating self-images at certain planes where a third grating is placed to observe moiré fringes, essentially making this equivalent to Lau interferometry. Such an arrangement with three gratings with elements aligned parallel to each other has been used by several researchers for collimation testing.81–84 At collimation, a fringe-free field is obtained, whereas there are straight-line fringes if the beam is not collimated. In another variant, the second and third gratings are rotated in their plane oppositely by a very small amount resulting in the formation of fringes. The fringes run parallel to the x axis when the beam is collimated and rotate in opposite directions when the beam is convergent or divergent.82,83 The phase of these fringes is measured to automate the system using the phase-shifting technique84 and the Fourier fringe analysis technique.85

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]

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]

If the path difference between the two arms of a Michelson interferometer is very large, the fringes in the interference pattern will be circular when the incident beam is a spherical wave. A fringe-free field is obtained only for a collimated beam.41

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]

For checking collimation of beams with diameters much larger than the diameter of the wedge plate, the beam is sampled at several locations. If a single wedge plate is used to check the collimation of beams of various sizes up to wedge plate diameter, it is designed so that the number of fringes observed with the smallest beam size is about two. This may ensure the same sensitivity for other beam sizes.

A collimated optical beam is required in several applications, such as metrology, optical processing, free space propagation of information, and laser-based instrumentation. Errors in measurement can occur if the beam is not properly collimated. Due to its importance, this topic has been extensively researched. Many diverse methods and techniques have been explored. Some of these are simple and practical, while many of them are of academic interest. Main techniques for collimation testing are shear interferometry and Talbot interferometry. Classical interferometry along with a phase conjugated mirror or a bi-mirror has also been used for collimation testing. There are also some papers using techniques that do not fall into either in shear interferometry or Talbot interferometry. This paper attempts to describe these techniques.

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]

If this pair of wedge plates is rotated by 90 deg in their plane, it converts to an interferometer in which the widths of the fringes are the same in both wedge plates when the beam is collimated. Otherwise, the fringe widths are different in both wedge plates.8,23,24 Vernier acuity now plays a part in ascertaining the collimation. Unlike a single wedge plate shear interferometer, the double wedge plate shear interferometer can be used, in fringe mode, for collimation testing with good setting accuracy.

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]

When a beam from an incoherent source is to be collimated, an aperture of diameter 2a is placed at the front focal point of the collimating lens of focal length f. The semidivergence angle θ of the beam is now given by θ=tan−1 af.

If a point source is placed at the front focal point of a positive lens, the beam leaving the lens is a parallel beam under geometrical optics consideration. Note that a parallel beam is an academic idealization and does not exist in practice. The spherical wave from the point source is diffracted at the lens aperture, and the exiting beam is now called a collimated beam. This beam, if captured by another positive lens of a larger aperture, will not focus to a point but to a region with an irradiance distribution called an Airy pattern, which occurs due to diffraction at the first lens. By contrast, if the beam is captured by a lens of smaller aperture, the Airy pattern would be due to diffraction at the second lens. The central disc of this pattern contains about 85% of the energy of the beam, and its size is taken as an effective source size, which then defines the degree of collimation. The degree of collimation is half of the angle that the source subtends at the center of the collimating lens, which is given by α=0.61λ/D, where D is the diameter (aperture) of the lens. Obviously, this angle will be smaller for a larger aperture lens. Therefore, a point source can be replaced by a source of finite size.

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.

Because the phase of the moiré fringes varies linearly when the beam departs from collimation, several methods that determine the phase when the point source is translated through from an out-of-focus position to an in-focus position by small amount have been investigated. Methods used for determining the phase information include the phase-shifting method,67 Fourier transform,68 and windowed Fourier transform.69 Patorski et al.70 used circular and linear gratings along with wavelet transform to determine the phase in the moiré pattern. Sanchez-Brea et al.71 proposed a two-grating arrangement in which the second grating has two-halves shifted by a quarter of the period. A pair of photodetectors is placed behind each half. When the first grating is translated with a uniform velocity, the phase difference between the heterodyne moiré signals depends on the degree of collimation of the beam. These signals can also generate Lissajous figures; the ellipticity of the figure depends on the degree of collimation. When the beam is collimated, the Lissajous figure is a circle. In a modification to this technique, the second grating consists of a mask of many Ronchi gratings appropriately shifted. Signals from photodetectors behind each Ronchi grating in the mask are used to generate Lissajous figures without the translation of the first grating.72 It may be remarked that phase estimation techniques are accurate, precise, automated, and relatively easy to implement.

Laser scanningconfocal microscopy

In another classical method of beam collimation, the beam from the collimating lens is retroreflected and the location of the pinhole is axially adjusted until a sharp image of the pinhole is formed on itself.

When the beam is collimated, the fringe width is x¯=λ/2nα. For a beam departing from collimation, the fringe width will be either more or less than that for a collimated beam. This configuration is generally not used as it is not as sensitive as the one that detects the change in inclination of the fringes. Riley and Gusinow6 presented the theory of wedge plate shear interferometer and demonstrated its application for collimation testing. Later, Grindel7 applied wedge plate shear interferometry for both collimation testing and for measuring the radius of curvature of a concave mirror. Dhanotia and Prakash,16 instead of obtaining a collimated beam by visually observing the fringe pattern obtained with wedge plate shear interferometry, used the Fourier fringe analysis technique to calculate the phase of the wavefront, thereby achieving a much better setting accuracy.

For coherent sources, diffraction limits the degree to which a beam can be collimated. The diffraction limited divergence is given by α=0.61λD, where D is the diameter of the collimating lens. Obviously, a large aperture collimator provides a well collimated beam. For a collimating lens with an aperture of 10 cm, the diffraction limited divergence is on the order of 4  μrad when red radiation from a He–Ne laser is used.

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]

For an incoherent source, the divergence is controlled both by the diameter of the aperture at the front focal point and the focal length of the collimating lens. A ratio in the range of 1/250 to 1/1000 is not uncommon and is decided by the available light throughput.

It may also be noticed that when fringe processing techniques are used in both of these techniques, the setting accuracy is considerably improved. This, however, requires additional hardware such as CCD camera(s) and fringe processing software.

The Lau effect is an interference phenomenon involving a pair of coarse gratings in tandem with the grating elements parallel to each other and back illuminated by a polychromatic extended source. Colored fringes are observed at infinity for all separations of the gratings. High-contrast fringes are observed for distances na2/2λ between the gratings when they are illuminated by narrowband light, where n is an integer, a is the grating period, and λ is the mean wavelength.

Recently, collimation testing based on determining the period of self-images using a variogram function has also been reported.73–75 The technique uses a single grating. In one technique, the period of self-image of the grating is compared with the period of the grating.73 In a variation of this technique, the periods of the self-images at two different Talbot planes are compared.74 The periods of the grating and its self-images are the same when the grating is illuminated by a collimated beam. With spherical beam illumination, the periods are different. This technique is also applied to collimate a beam from a white LED.75

Confocal microscopydiagram

Langenbeck proposed an improved collimation technique in which one of the mirrors of a Michelson interferometer is replaced by a pair of corner cube reflectors (CCRs), making it an inverting interferometer.40 Displacing the CCRs perpendicular to the optical axis introduces shear. The orientation and spacing of the fringes are controlled by the plane mirror. On collimation, the fringes will be parallel in both retroreflected beams, whereas they will be oppositely rotated when the beam departs from collimation. Langenbeck even proposed a compact arrangement consisting of several small CCRs on a glass plate that has a transmissive reference coating: interference between the beam reflected from the reference coating and retroreflected beams from the CCRs generate several interference patterns. Fringes in all CCR patterns will be parallel to each other for a collimated beam.

The Fresnel diffraction pattern of a slit imaged on a CMOS sensor has been used to collimate a beam.93 It is shown that the separation between the primary peak point and the secondary peak point in the diffraction pattern is related to the defocusing.

A composite grating consists of two identical gratings that either enclose an acute angle or an obtuse angle. A pair of such gratings is used in Talbot interferometry for collimation testing.23,60,77,78 This approach provides self-referencing and doubles the sensitivity. Another variant of the composite grating consists of two gratings of slightly different periods.26,65 A pair of these can be used in Talbot interferometry. At collimation, the fringe width in both halves is the same. For a convergent beam, the fringe width in one half is greater than in the other half. This changes when the beam is divergent, thereby providing self-referencing.

What exactly is magnification? A basic definition of magnification is the ratio of the size of a specific feature of an object or sample as seen in an image ...

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.

A grating illuminated with a plane wave self-images itself periodically. The separation between the self-images is zT=2a2/λ, where a is the period of the grating and λ is the wavelength. The distance zT is known as Talbot distance. When the grating is illuminated by a spherical wave, the self-images are not equally spaced, and the period of the self-image is different than that of the grating. Talbot phenomenon, when used in conjunction with moiré phenomenon, yields a technique known as Talbot interferometry. When two linear gratings with the grating vectors making an angle of +θ and −θ with the x axis are in close proximity to each other, a moiré pattern with fringes running along x axis is formed: the fringe spacing is y¯=a/2 sin θ. If the period of one of the gratings changes, the moiré fringes rotates, i.e., they make an angle with the x axis. When the two gratings are identical and aligned such that their grating elements are parallel to each other, the moiré pattern will be of uniform illumination (infinite fringe mode). Gratings with slightly different periods but with grating elements parallel to each other and along the x axis produce a moiré pattern in which the period of moiré fringes is y¯=ab/|a−b|, where a and b are the periods of the gratings. The moiré fringes are parallel to the x axis.

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]

Multiple reflections produce narrower (sharper) fringes. Sirohi et al.19 used a coated wedge plate in transmission for collimation testing and for some other applications such as lens testing. Senthilkumaran et al.20 used a coated wedge plate both in transmission and in reflection for collimation testing and showed that the reflection mode provides better setting accuracy. Matsuda et al. designed a wedge plate such that there is only one fringe running parallel to the base when the beam is collimated.21 Such a plate can be used to check collimation of any size beam. Further, it is much easier to sense the inclination of the narrow fringe; therefore, a coated wedge plate shear interferometer has better sensitivity compared with its uncoated counterpart. It has also been shown that it could be used to collimate a beam even when the collimating lens suffers from aberrations.22

It is also possible to align the collimator by measuring the modulation transfer function (MTF) of the collimator. This is feasible because there are methods available that can be used to measure the MTF in almost real time.2

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]

However, the simplest method is to observe the size of the beam at several planes along its direction of propagation, and the position of the pinhole is adjusted until the sizes at several planes are nearly the same. The beam from a CO2 laser was collimated by observing the shape and size of the beam taken on a thermal paper.3 The knife edge method is also used to get the beam spot size at various locations along the direction of propagation. Many classical methods for obtaining collimated beam are discussed by Van Heel.4

Several modifications/new configurations of double wedge plate shear interferometer have been proposed and demonstrated.25,26

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]

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.

In the method proposed by Senthilkumaran,88 the test beam is an input beam to a Michelson interferometer with a large path difference. A spiral phase plate is introduced in one of the arms (longer arm) of the interferometer, and the interference pattern is observed. On collimation, radial fringes are observed; otherwise, the fringe shape is spiral.

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

Dec 27, 2022 — Diffraction is the process by which a beam of light is spread out as a result of passing through a narrow aperture or across an edge. In ...

Talbot interferometry has also been performed using a single grating, which is kept in the beam, and a CCD is located at the Talbot plane.54,55 The self-image captured is subtracted from a previously stored image of the grating. Departure from collimation produces a self-image with a slightly different period, which on subtraction, produces moiré fringes. The correct position of the point source is determined by observing identical moiré fringes on either side of the collimation condition.55 It is known that divergence or convergence can be altered by changing the beam size. This idea is utilized by inserting a two-lens system after the collimating lens to increase the divergence/convergence and then using Talbot interferometry for collimation testing.56,57 Apart from a linear grating, other grating types such as circular gratings,58 spiral gratings,59–61 spiral gratings with wavelet transform,62 arc gratings,63 triangular gratings,64 and evolute gratings60 have been employed for collimation testing by many researchers. In another arrangement, instead of two identical gratings, a linear grating and a differential grating (composite grating containing linear gratings of different periods in each half) are used: the differential grating is placed at the Talbot plane of the linear grating.65 At collimation, both halves have the same pattern, whereas with a noncollimated beam, the fringes in the two halves have different widths. Instead of keeping both of the gratings stationary and observing the moiré fringes, the first grating is translated with a constant velocity in the direction of the grating vector and the phase of the heterodyne moiré signal is calculated.66 At collimation, the phase slope is zero.