In Vivo Confocal Microscopy in Eye Disease 1447175166, 9781447175162

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In Vivo Confocal Microscopy in Eye Disease Golshan Latifi Scott Hau

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In Vivo Confocal Microscopy in Eye Disease

Golshan Latifi • Scott Hau

In Vivo Confocal Microscopy in Eye Disease

Golshan Latifi Department of Cornea and External Disease Farabi Eye Hospital, Tehran University of Medical Sciences Tehran, Iran

Scott Hau Department of Cornea and External Disease, National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust University College of London, Institute of Ophthalmology London, UK

The author(s) has/have asserted their right(s) to be identified as the author(s) of this work in accordance with the Copyright, Designs and Patents Act 1988. ISBN 978-1-4471-7516-2    ISBN 978-1-4471-7517-9 (eBook) https://doi.org/10.1007/978-1-4471-7517-9 © Springer-Verlag London Ltd., part of Springer Nature 2022 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer-Verlag London Ltd. part of Springer Nature. The registered company address is: The Campus, 4 Crinan Street, London, N1 9XW, United Kingdom

Contents

 Principles of In Vivo Confocal Microscopy����������������������������������������������������   1 Parisa Abdi and Mehrnaz Atighehchian Normal Anatomy����������������������������������������������������������������������������������������������   13 Scott Hau Inflammation and Keratitis����������������������������������������������������������������������������   29 Scott Hau Corneal Dystrophies����������������������������������������������������������������������������������������   61 Golshan Latifi Conjunctiva and Limbus ��������������������������������������������������������������������������������   91 Golshan Latifi and Victor H. Hu Corneal Nerves ������������������������������������������������������������������������������������������������  125 Ioannis N. Petropoulos and Rayaz A. Malik Other Anterior Segment Applications of In Vivo Confocal Microscopy and Future Developments����������������������������������������������������������  153 Parisa Abdi, Mehrnaz Atighehchian, and Scott Hau Index������������������������������������������������������������������������������������������������������������������  171

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Principles of In Vivo Confocal Microscopy

Optical Principles Detailed three-dimensional visualization of biological tissues has been a challenge over the years due to the fact that images detected by light microscopic devices are often obscured by interfering signals from out-of-focus lights. As a result, the resolution in slit lamp biomicroscope is often limited to a resolution of 20 μm or a 40× magnification [1]. A number of modern digital optical processing technologies have emerged to address this issue. In vivo confocal microscopy (IVCM) is one of these non-invasive and rapid optical imaging techniques that has been developed and it provides real-­ time sectional biopsies with microscopic details of various tissue layers that have helped in improving our understanding of the cellular structures in normal and pathological states. Enhanced visualization of live specimens and observable intracellular details are major advantages with this method as well as improved diagnostic accuracy for pathologies which helps to reduce the number of unnecessary biopsies and cut sections [2]. The principle of IVCM is based on the principle of confocality of the examined object with the light source and the detector plane. This produces a point source of light and rejects out-of-focus lights to provide high resolution three dimensional images and reconstruct optical sections from tissues and cells [3]. This allows for magnification of up to 600 times, lateral resolution of up to 1–2 μm and axial resolution of up to 5–10 μm [1]. The principle of “confocality”, which means observing only a focal point at any moment, was first proposed by Marvin Minsky in the 1950s. He described how to exclude unwanted lights reflected from sources outside the focal point [4]. In this imaging method, illumination light and detection lenses are focused on the same spot on tissue so light interference (reflection, diffusion and diffraction) are reduced and the axial and lateral resolution of the image are significantly increased. However, this improvement in resolution is at the cost of a substantial decrease in the field of view [3]. The key point in this imaging technique is the ability to observe and © Springer-Verlag London Ltd., part of Springer Nature 2022 G. Latifi, S. Hau, In Vivo Confocal Microscopy in Eye Disease, https://doi.org/10.1007/978-1-4471-7517-9_1

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Principles of In Vivo Confocal Microscopy

capture adjacent points and reconstruct the whole image which has now become possible by high performance digital imaging techniques [5]. In IVCM, a specimen is illuminated with a focused light spot. The light beam passes through an aperture and is focused by an objective lens into a small area (a point) of a specimen. A mixture of emitted and reflected lights are then recollected by the objective lens from the illuminated specimen. A beam splitter, separates the mixed light and reflects them into a second pinhole diaphragm where after passing through the pinhole the light is detected by a photosensitive detector device. This aperture (the pinhole) which is in front of the detector, obstructs the out of focus lights that do not come from the focal point to make a sharp image and decrease interfering noises [3]. To build up a two-dimensional (2D) image perpendicular to the optical axis of the device, the light beam scans the tissue point by point using two oscillating mirrors. Deep layer imaging can also be achieved by optical movement of the focal plane [3] (Fig. 1). The basic components of IVCM are pin holes, objective lenses, detectors, fast scanning mirrors, wavelength filter selection and laser illumination. Although diode and solid-state lasers are more common, gas lasers are still used in some cases [3, 6].

Detector In Focus Light Emission Pinhole Illuminating Aperture

Out of Focus Light

Point Source Dichroic Mirror Objective Lens

Focal Plane Specimen

Fig. 1  Schematic principle of confocal microscopy. Light is passed through a dichromatic mirror and reflected to the objective lens, which focuses the light beam on a point in the sample. Scanning mirrors sweep the excitation beam over the sample point by point to make the image. Emitted beam passes back through the objective lens and the dichromatic mirror and is terminally detected by a detector. A pinhole is placed parallel to the image plane to eliminate out-of-focus light

Types of Confocal Microscopes

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Different pinhole size change the amount of light collected by the detector which determines the thickness of the optical section [3, 7]. There are three different imaging modes in IVCM: 1. The section mode enables saving a single image at a time. For instance, the cornea may be scanned manually in the X–Y–Z axes and images captured at the desired points. Manual scanning in the Z-axis may be performed in a wide depth range of 0 to 1500 μm. 2. The volume mode allows the observer to view 40 images in successive focal planes that are 2 μm apart. Therefore, 80 μm of tissue depth can be scanned in one axis. 3. The temporal mode can acquire a series of 100 images at a rate between 1 and 30 frames per second, and the result is saved as a 3–100 s movie [7–10].

Types of Confocal Microscopes The type of confocal microscope can be determined by its method of scanning and how the images are constructed. In the original confocal microscopes the illuminating light was held fixed and the stage could be moved in x, y to scan the sample and build the image. In modern confocal microscopes, the basic components (pinholes and objective lenses) are the same, but they also include fast-scanning mirrors and laser illumination [3]. The optical principles involve the illumination beam moving across the stationary sample and is controlled with a filter to turn the beam on and off, so the illumination output power is decreased and more controlled. This scanning technique varies among the main types of confocal microscopes for example, laser scanning confocal microscope (LSCM) uses a single point for scanning, while Tandem scanning confocal microscope (TSCM) uses a spinning disk containing a large number of holes arranged in a spiral fashion whereas Slit-scanning confocal microscope uses a very thin slit of light instead of a point [3, 5, 11, 12].

Tandem Scanning Confocal Microscope Tandem scanning confocal microscopes (TSCM) uses a miniaturized Nipkow spinning disk which contains a large number of (14,000) small holes (40–80 μm) arranged in a spiral fashion. Each hole has a conjugate pair with the same diameter and the same radial distance from the center of rotation, but on the exact opposite side. This ensures that when a hole is aligned with the illumination source the conjugate will be situated under the objective lens. The illumination source hole in tandem with the objective lens hole limit the illumination and detection lights in the plane of focus respectively, and this is necessary to achieve confocality [3, 13, 14]. During disk rotation, light excitation is delivered to the Nipkow disc that transmits approximately 1% of beam lights. The holes allow single spot white lights to come through the disc adjacent to one another, and project on to an array of

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Principles of In Vivo Confocal Microscopy Direction of rotation

Spread light source

Dichromatic mirror Camera

Nipkow discs

Lens

Pinhole array

Objective lens

Specimen

Fig. 2  Schematic principle of spinning disc confocal microscopy. Hundreds of pinholes are arranged in spirals on the Nipkow discs. In the first Nipkow disc each hole has a microlens that concentrates the light on the second disc which contains the pinholes. The light reaches the sample through the objective lens. Emitted light is collected by the objective lens and passes back through the pinhole disc. A dichroic beam splitter placed between the two Nipkow discs is responsible for reflecting the sample image to the detector

objective micro lenses and form image of these spots on the objective plane, thereby scanning the whole specimen [3, 5, 13, 14] (Fig. 2). If one follows the path of a light ray from the illumination source, they will pass through a pinhole before reaching the specimen. Then the light ray, after being reflected several times from internal mirrors, passes through the conjugate pinhole on the opposite side of the Nipkow disc to ensure out of focus rays in both illumination source and detection plane are filtered out. A very high portion of the reflected light passes to the observation side of the disc to form an image. However, scattering light from above or below the object plane will not be focused on the opposite side of the disc and will be filtered. The single spot visible through the Nipkow disc will

Types of Confocal Microscopes

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scan a line while the disc rotates. The spiral arrangement of the pinholes on the Nipkow disc is designed so that when the first pinhole scans the line completely and exits the observation plane the next pinhole comes into view to scan the adjacent parallel line, hence making a complete planar image [13–15]. There are limitations with this technique including the disc filters out most of the light, the resolution is defined by the size of the pinholes and the disc itself, and to get a single image the disc has to make a full mechanical rotation [15]. It is noteworthy that the spinning disk approach, employs multiple illumination and detection sites and sweeps across the samples whereas the galvanometer scans in laser scanning confocal microscopy are performed in a point by point manner. Even though TSCM has low intensity of illumination for image formation [3, 15], the spinning disk has the advantage of scanning a wide field without loss of resolution in thick tissues, provides greater detail and more information about the number and shape of cells, and has a faster speed than LSCM. Clinical versions of this type of IVCM are no longer available [7, 16].

Slit-Scanning Confocal Microscope An intermediate approach between single and multi-point scanning confocal microscopy is the slit-scanning confocal microscopy, which uses a different mechanism for scanning images. This technique can detect confocal image sections with an optical resolution of approximately 5–10 μm. A very thin vertical rectangular slit aperture (180 μm wide) is used to eliminate out-of-focus beam lights and projects a slit light instead of a point source that scans the whole specimen. All points in the axis of the slit will be scanned at the same time and the image is viewed through a conjugate slit of the same size. Slit-scanning systems cover more of the sample in one field of view and increase the imaging speed as well as reducing scanning time. The slit height can be adjusted, which allows a change in thickness of the optical section, and the slit width adjustment controls the amount of light that reaches the tissue and increases field brightness compared to the field luminosity in the Tandem Scanning microscope [3, 5, 17, 18]. Three-dimensional image distortion in this method is usually associated with eye movements. The detrimental effect of linear eye movement along the z-axis can be reduced by faster image acquisition. However, more rapid movement along the optical axis (z-scan) may lead to lower image resolutions [11]. Commercially available slit scanning CMs include: Tomey Corporation (Cambridge, MA, USA), Nidek Technologies (Gamagori, Japan) and Helmut Hund (Wetzlar, Germany) [7].

Swept-Field Confocal Microscope Another hybrid approach is the swept-field confocal microscopy (SFC).

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The SFC can be used in conjunction with both the pinhole or slit scanning techniques. Swept-field confocal microscopy uses a positionable aperture plate containing a variety of pinhole columns and slit apertures instead of pinholes embedded on a spinning disk. The emitted beams are directed through pinholes or slits before reaching the camera. The major advantages of this approach are the high speed scanning, high resolution and less artifacts that are created by moving apertures in a spinning disk system [19].

Laser Scanning Confocal Microscope (LSCM) Further improvement in IVCM technology was made by Webb in the 1980s, who used a coherent laser as a high intensity light source and a set of galvanometer scanning mirrors for rapid scanning of the ocular fundus. Using laser diodes has the advantage of providing high radiant flux with a very small spot size that allows for more efficient illumination and detection. With this spot size illumination, the need for illumination pinhole is eliminated and the resolution characteristics improves [8, 20]. In LSCM a coherent Helium Neon diode laser with 670 nm wavelength is used as a high intensity light source and the laser beam scans the specimen mediated by a pair of galvanometer scanning mirrors that provide fast scanning in along the x direction and slow scanning in y direction over the sample to produce an optical section. Galvanometer mirrors have a rotating mirror shaft which can control the position of the mirror and provide a very precise movement, while allowing the mirror to rotate at speeds up to 1 kHz. These mirrors tilt the beam in the x and y directions, respectively or in a raster fashion when combined. The reflected light from the specimen is refocused by the objective lens and passes through the galvanometer scanning mirrors again. After passing through the lens and pinhole, the ray reaches the photomultiplier tube detector (PMT) which consists of highly sensitive detectors that collect and convert the light signal into an electric one that can be recorded by the computer. Finally, all optical sections from top to bottom are collected to reconstruct a 3-dimensional (3D) image [3, 5, 7, 10] (Fig. 3). This type of confocal microscopy has the ability to adjust the pinhole size to change the optical section thickness that allows it to change the resolution of images. There are several geometric varieties for the pinhole that can potentially enhance image resolution. For instance, there are square or hexagonal pinholes for confocal aperture. However, the intensity of light that pass through the aperture is independent of the shape of pinholes [3, 5, 16]. The commercial well established IVCM of this type is the Heidelberg retina tomograph (HRT) (Heidelberg Engineering, Heidelberg, Germany). Its first generation was released in 1990, and the next generations, HRT II and HRT3 came into the market in 1998 and 2005, respectively. HRT uses a 670 μm diode laser to assess the optic nerve head [11, 14] (Fig. 4). In 2002 Heidelberg Engineering collaborated with Rostock Eye Clinic (University of Rostock, Germany) to design an optical accessory to the objective lens, the Rostock Cornea Module (RCM) which allowed microscopic imaging of the cornea.

Types of Confocal Microscopes

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Detector

Pinhole

Dichroic mirror Light source

Y galvanometer

X galvanometer

Objective lens

a

Specimen

b

Fig. 3 (a) Schematic principle of confocal laser scanning microscope. Excitation light, emitted from a laser source is passed through a dichroic mirror, and is scanned across the specimen by a pair of galvanometers, which scan in the x and y axes. Emitted light is reflected back via the galvanometers to the dichroic mirror where it is reflected through the pinhole to a PMT detector. (b) The raster scanning pass

This HRT II-RCM was released in 2004, producing high resolution imaging of the cornea, limbus, conjunctiva and sclera [11, 14]. The scanning and detection methods are the same in HRT as well as in HRT II/3-­ RCM. The module is combined with a manual scan in the x and y axis, and in the z axis for depth imaging that allows the focal plane to move over any depth of the cornea for detecting various cell layers. The distance from the cornea to the laser confocal microscope is stabilized with a disposable sterile cap which is a contact device in a sterile package (TomoCap®). Thus, it permits exact depth data (pachymetry) evaluation. The TomoCap® is a thin cap with a planar contact surface which is made of polymethyl methacrylate (PMMA) and is optically coupled to the lens. Optical coupling of the specimen to the cornea with the imaging device is achieved via the tear film or protective gel. The aqueous gel also bridges the air gap and minimizes the reflection at interfaces which otherwise cause signal loss [11, 14] (Fig. 5). One of the determinants of a microscope resolution is the amount of available light which is defined by the Numerical Aperture (NA) given by the equation: NA = n sinα . where n is the index of refraction of the medium in which the lens is working, and α is the half-angle of the aperture.

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Principles of In Vivo Confocal Microscopy

Fig. 4  The Heidelberg Retina Tomograph (HRT3) which allows confocal microscopy in combination with the Rostock Cornea Module (RCM). Reprinted from ‘High Resolution Imaging in Microscopy and Ophthalmology’, Chap. 12, p 267, by Oliver Stachs, Rudolf F. Guthoff, 2019 [14]. copyright permission under the terms of Creative Commons CC BY

The core component of the RCM is a water immersion objective lens with a high numerical aperture. It provides a focus at a distance of a few millimeters in front of the apex of the lens. The cornea is positioned in the focal plane. The higher refraction index of the PMMA cap (more than 1.49) increases the numerical aperture of the system. By placing the PMMA cap with a higher refractive index than the cornea, the focal plane is shifted toward the contact point, due to refraction according to Snell’s law. Therefore the NA as well as the resolution are improved. A beam focus less than 1 μm in diameter is provided for visualization and magnification of all the microstructures in the cornea and anterior segment [11, 14, 21].

Types of Confocal Microscopes

9 Cornea TomoCap

RCM objective

aqueous gel

Fig. 5  Schematic drawing of the Rostock Corneal Module and the Tomocap

Additionally, in the immersion technique the refractive index of the objective lens and immersion fluid are similar, so the beam passes unbroken and a high intensity of the light is taken up by the lens. Also in this technique, reflections at interfaces are eliminated. Recently, novel special caps with small tips have been developed [11, 14, 21]. Depending on the microscope lens, an additional lens may be placed between the HRT II and the microscope lens, so that the field of view of the scanning system which is fixed at 15° in the HRT II is reduced to approximately 7.5° to allow for an increase in magnification. The size of the field of view (FOV) of the image in the contact technique depends on the microscope lens and by using additional lenses, the FOV can be 250 μm × 250 μm, 400 μm × 400 μm or 500 μm × 500 μm [11, 22]. During standard imaging, a drop of topical anaesthetic and a drop of Carbomer based eye gel are instilled in the eye before examination. The patient is then positioned in the headrest and the RCM objective is adjusted to approach the patient’s cornea. This is facilitated by a small live camera that is on the computer screen and observing the laser reflex on the cornea. As the TomoCap touches the cornea, cell structures become visible. The images are captured in transverse (coronal plane) sections with the manual Z-feed. The focal plane is shifted through the whole cornea allowing live imaging of the various cell layers. As the distance between the microscope and the cornea is fixed, pachymetry becomes possible [11]. Cellular imaging requires a microscope lens with a short focal length, which also limits the working distance (WD). The depth of the layer which can be accessed within the tissue is confined to that distance. A WD of 2.2 mm is sufficient to image

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Principles of In Vivo Confocal Microscopy

cornea, limbus and sclera but deeper tissues like lens epithelium, can only be imaged with a microscope lens of lower NA, which in turn reduces the resolution [11, 14].

Noncontact Confocal Laser Scanning Microscopy Non-contact high resolution laser scanning microscopy is particularly important for evaluating tear film and can be used in imaging the posterior cornea like keratocytes and endothelium but the resolution is not as clear as compared to contact IVCM. In noncontact microscopy, the water-immersion objective along with the contact cap are removed and replaced by a long focal length dry objective lens. This way, it becomes possible to visualize even intraocular structures like lens capsule and other anterior segment structures. Due to additional effect of the corneal refraction on image magnification, it is necessary to use dry microscope lenses such as the Nikon *20, 0.35 Plan, ELWD (14  mm) or Nikon *10, 0.21  L Plan, SLWD (17  mm) to image intraocular structures instead of the Nikon *50, 0.45 CF Plan, SLWD (17 mm) [11, 23]. In addition, it is necessary to reduce the laser power in noncontact microscopy when the tear film is evaluated, because it produces high reflectivity. As HRT II was not successfully modified in this regard, a dramatically effective approach was to place neutral glass filters on the dry objective [11, 23]. Since the imaging sequence is rapid with this device, dynamic processes such as tear film break-up in patients with dry eye or assessing the retention time of medications in the tear film can be recorded [11, 23]. A summary of the main features comparing contact with noncontact IVCM is shown in Table 1. As a general guide: • The noncontact model is of great value in patients who do not tolerate corneal contact and eye drops. • The numerical aperture in the contact type RCM is higher than the noncontact type and as the numerical aperture increases, the resolving power improves. • In contact mode, there is a parallel alignment of the cornea with the Tomocap. This facilitates full-field image acquisition of all layers of the cornea which result in transverse ‘en face’ images. But in noncontact mode, it is impossible to Table 1  The main features of contact and verus non contact in vivo confocal microscopy In patients who do not tolerate corneal contact and eye drops Numerical aperture Resolving power Alignment of device to cornea

Non contact IVCM Contact IVCM Great value Not valuable

Section of image acquisition

Less Less Not parallel alignment Tangential sections

Imaging the tear film

Possible

More Better Parallel alignment Transverse ‘en face’ images Not possible

References

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maintain the cornea in stable parallel alignment with the RCM, so images are often captured in tangential sections. Albeit, this has the advantage of allowing multiple layers of the cornea to be imaged cross-sectionally in a single frame [23]. • Imaging the tear film is a clear advantage of the non-contact mode, which is not possible with the contact type instrument, since the Tomocap touches the cornea and also the anaesthetic drops and viscous gel interfere with normal tear film structures. However, it is worth to note that getting full-field image of the tear film is not a simple task, as the RCM not often remains parallel to the corneal surface [23].

Acknowledgement  We wish to express our great appreciation to Maryam Kasiri for her assistance in writing this chapter.

Disclosures  None to declare.

References 1. Jalbert I, Stapleton F, Papas E, Sweeney DF, Coroneo M. In vivo confocal microscopy of the human cornea. Br J Ophthalmol. 2003;87(2):225–36. 2. Wells WA, Thrall M, Sorokina A, Fine J, Krishnamurthy S, Haroon A, Rao B, Shevchuk MM, Wolfsen HC, Tearney GJ, Hariri LP. In vivo and ex vivo microscopy: moving toward the integration of optical imaging technologies into pathology practice. Arch Pathol Lab Med. 2019;143(3):288–98. 3. Elliott AD.  Confocal microscopy: principles and modern practices. Curr Protoc Cytom. 2020;92(1):e68. 4. Minsky M.  Memoir on inventing the confocal scanning microscope. Scanning. 1988;10(4):128–38. 5. Patel DV, McGhee CN. Contemporary in vivo confocal microscopy of the living human cornea using white light and laser scanning techniques: a major review. Clin Exp Ophthalmol. 2007;35(1):71–88. 6. Girkin JM, Ferguson AI, Wokosin DL, Gurney AM.  Confocal microscopy using an InGaN violet laser diode at 406nm. Opt Express. 2000;7(10):336–41. 7. Guthoff RF, Zhivov A, Stachs O. In vivo confocal microscopy, an inner vision of the cornea–a major review. Clin Exp Ophthalmol. 2009;37(1):100–17. 8. Paddock SW. Principles and practices of laser scanning confocal microscopy. Mol Biotechnol. 2000;16(2):127–49. 9. De Silva ME, Zhang AC, Karahalios A, Chinnery HR, Downie LE. Laser scanning in vivo confocal microscopy (IVCM) for evaluating human corneal sub-basal nerve plexus parameters: protocol for a systematic review. BMJ Open. 2017;7(11):e018646. 10. Patel DV, Zhang J, McGhee CN. In vivo confocal microscopy of the inflamed anterior segment: a review of clinical and research applications. Clin Exp Ophthalmol. 2019;47(3):334–45. 11. Guthoff RF, Baudouin C, Stave J. Atlas of confocal laser scanning in-vivo microscopy in ophthalmology. Springer Science & Business Media; 2007. 12. Prydal JI, Dily PN. Advances in confocal microscopy of the cornea. Eye. 1998;12(3):331–2. 13. Petráň M, Hadravský M, Egger MD, Galambos RO. Tandem-scanning reflected-light microscope. JOSA. 1968;58(5):661–4.

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14. Stachs O, Guthoff RF, Aumann S. In vivo confocal scanning laser microscopy. High Resolu Imag Microsc Ophthalmol. 2019:263–84. 15. Toomre DK, Langhorst MF, Davidson MW. Introduction to spinning disk confocal microscopy. ZEISS Campus. http://zeisscampus.magnet.fsu.edu/articles/spinningdisk/introduction. html. Accessed 2019. 2012. 16. Bayguinov PO, Oakley DM, Shih CC, Geanon DJ, Joens MS, Fitzpatrick JA. Modern laser scanning confocal microscopy. Curr Protoc Cytom. 2018;85(1):e39. 17. Wiegand W, Thaer AA, Kroll P, Geyer OC, Garcia AJ. Optical sectioning of the cornea with a new confocal in vivo slit-scanning videomicroscope. Ophthalmology. 1995;102(4):568–75. 18. Masters BR, Thaer AA. Real-time scanning slit confocal microscopy of the in vivohuman cornea. Appl Opt. 1994;33(4):695–701. 19. Castellano-Muñoz M, Peng AW, Salles FT, Ricci AJ.  Swept field laser confocal microscopy for enhanced spatial and temporal resolution in live-cell imaging. Microsc Microanal. 2012;18(4):753–60. 20. Webb RH, Hughes GW, Delori FC.  Confocal scanning laser ophthalmoscope. Appl Opt. 1987;26(8):1492–9. 21. Zhivov A, Stachs O, Stave J, Guthoff RF. In vivo three-dimensional confocal laser scanning microscopy of corneal surface and epithelium. Br J Ophthalmol. 2009;93(5):667–72. 22. Stave J, Guthoff R.  Darstellung von Tränenfilm und In-vivo-Kornea: Erste Untersuchungsergebnisse mit einem modifizierten konfokalen Laser-Scanning-­ Ophthalmoskop. Ophthalmologe. 1998;95(2):104–9. 23. Pritchard N, Edwards K, Efron N.  Non-contact laser-scanning confocal microscopy of the human cornea in vivo. Cont Lens Anterior Eye. 2014;37(1):44–8.

Normal Anatomy

Introduction The in vivo confocal microscope (IVCM) is a powerful tool in providing detailed analysis of the human cornea and adnexa. It offers non-invasive, high resolution, images of ocular tissues that are comparable to conventional light microscopy in terms of magnification and resolution. However, unlike light microscopy where the image is degraded by light reflected from structures around the point of focus, scattered out of focus light is eliminated by the use of confocal optics. The principle of confocal microscopy was first described by Minsky in 1957 [1] who proposed the concept of combining the observation (objective) and illumination (condenser) system to a single focal point, when this is achieved, the principle of confocality is achieved (see chapter “Principles of In Vivo Confocal Microscopy”). This greatly enhances the quality and contrast of the image achieving a lateral (x, y) resolution and axial resolution (z) of the order of 1–2 μm and 5–10 μm, respectively [2]. This improvement in image quality greatly increases the resolution and enables high magnification of the cornea and conjunctiva to be obtained.

Precorneal Tear Film The tear film covers the anterior part of the cornea and it provides a clear refractive interface for the cornea. The thickness varies between 7 and 10 μm and is composed of an outer lipid layer derived from the meibomian glands, thick aqueous layer from the aqueous component of the lacrimal gland, an inner mucin layer produced by the conjunctival goblet cells, in addition to a large number of polypeptides, growth factors and electrolytes [3]. Imaging of the tear film with non-contact laser scanning IVCM has been reported with a prototype lens that could be inserted into the Rostock Corneal Module (RCM) indicating a series of alternating light and dark interference fringes, interspersed with black spots of various sizes, resembling air bubbles in the tear film (Fig. 1) [4, 5]. In addition, it has been shown that there is a © Springer-Verlag London Ltd., part of Springer Nature 2022 G. Latifi, S. Hau, In Vivo Confocal Microscopy in Eye Disease, https://doi.org/10.1007/978-1-4471-7517-9_2

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Normal Anatomy

Fig. 1  Tear film imaged using non contact laser scanning microscopy. Reference bar = 50  m (From Pritchard, Edwards and Efron 2013 with permission)

good correlation between the interference pattern created by the lipid surface with physiologic measurements using non-contact tandem scanning IVCM [6]. Images published to date of the human in vivo tear film have been mainly used non-contact IVCM, due the fact that contact -IVCM involves physical contact between the microscope objective and the cornea, therefore, obtaining good quality of the tear film is not possible with the contact method [4].

Corneal Anatomy The cornea is made up of five main layers: epithelium anchored to a basement membrane, Bowman’s layer, stroma, Descemet’s membrane and the corneal endothelium.

Epithelium The corneal epithelium is composed of a stratified, squamous nonkeratinized epithelium and can be subdivided into three layers on IVCM: superficial epithelial cells, intermediate wing cells and the basal epithelial cells. The superficial desquamating cells are flattened and nucleated and demonstrate a range of sizes and shape (Fig. 2a, b). The cell size range between 40 and 50 μm in

b

Fig. 2 (a) Superficial desquamating epithelium showing the variation in the polygonal shaped cells with hyper-reflective cytoplasm and central nucleus surrounded by darker perinuclear space visible in some cells. (b) Superficial desquamating cells surrounded by smaller inferior epithelial cells with their bright central nuclei

a

Epithelium 15

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Normal Anatomy

diameter, 5 μm in thickness, and of polygonal in shape [5, 7–9]. Characteristically, these cells demonstrate hyper-reflective cytoplasm with a bright central nucleus surrounded by darker perinuclear space visible, though the degree of hyper-reflectivity is related to the amount of back-scattered light that varies depending on cell maturity, [10] and the type of light source used to generate the images [11]. The mean central cell density in the superficial epithelium has been estimated to be between 840 and 1213 cells/mm2, [5, 12] but prior instillation of fluorescein can increase the hyper-reflectivity of the cells and therefore, increase the density estimation [13]. The wing cells form 2–3 layers of cells beneath the desquamating epithelium and characteristically show bright cell border, dark cytoplasm and lower degree of pleomorphism (Fig. 3a). The wing cell layer can be subdivided into upper and lower wing cells [7, 9] with the latter being smaller but on a practical level, this is not easily distinguishable. The cell nucleus is not easily discernable and the size of the cells is approximately 20 μm in diameter. The mean central and peripheral cell density have been reported to be 5070 cells/mm2 and 5582 cells/mm2, respectively [14]. The single layer of basal epithelial cells rests on a basement membrane (basal lamina), about 40–60 nm in thickness, is anchored to Bowman’s layer via a complex mesh of anchoring fibrils and anchoring plaques [15]. This layer of cells have the smallest diameter of cells (8–10 μm) compared to the superficial and wing cell layers, and the mean cell density ranges from 6000 to 9000 cells/mm2 in the central cornea to 10,000 cells/mm2 in the periphery [12, 14, 16]. Similar to the wing cell layer, the cell nuclei are not easily discernable but there is a higher variation in the degree of cytoplasmic hyper-reflectivity (Fig. 3b). Due to light scattering, the cell border of both the wing cell layer and the basal epithelial cells are hyper-reflective, and this is governed by the type of organelles, membranes, microvilli and microplicae. It has been suggested that the microdesmosomes in the epithelial layer are the reason why the cell membranes are hyper-reflective [7]. The ratio between superficial, intermediate and basal cell density has been quoted to be approximately 1:5:10 [14]. There is no obvious difference in basal cell density estimation between male and female, and also with age [12, 17].

Bowman’s Layer Bowman’s layer is an acellular layer that lies beneath the basement membrane and it is around 12 μm thick. It consists randomly arranged collagen fibrils measuring 20–30 nm in diameter and on IVCM it has a featureless, grey, amorphous appearance. Polymorphic structures composed of fibrillar materials called Kobayashi structures (K-structures), measuring 5–15 μm in diameter, beneath the Bowman’s layer, have been previously described [18, 19]. It is thought that these structures (Fig. 4a) correspond to condensed anterior stromal collagen fibers that merge into Bowman’s layer and they account for the formation of the anterior corneal mosaic [19]. The appearance of the K structures can look different with eye disease and during inflammation, where inflammatory cells are seen surrounding these structures (Fig. 4b).

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Fig. 3  Section image of the epithelium. (a) Wing cell layer: depth = 24 μm. (b) Basal epithelium: depth = 45 μm

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Fig. 4  Images of Bowman’s layer. (a) Shows the distribution of Kobayashi (K) structures (arrows) and subbasal nerve plexus (arrow heads) in a normal cornea: depth = 54 μm. (b) Inflammatory cells surrounding K—structures (arrows) in a patient with keratoconus: depth = 51 μm

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Dendritic Cells Normal dendritic (Langerhans) cells are present between Bowmans’s layer and the basal corneal epithelium [20]. These cells are characterized by their small cell body and small or absence of processes to cells possessing long dendritic processes reflecting the immature and mature phenotype, respectively (see chapter “Inflammation and Keratitis”) [20]. A number of studies have produced data on the density and distribution of dendritic cells in normal, healthy eyes and the range of mean cell density, using laser scanning confocal microscopy, are 21–34 cells/mm2 in the central cornea and 70–98 cells/mm2 in the peripheral cornea, respectively [20–28].

Stroma The cornea stroma forms the major thickness of the cornea (approximately 90%) and it consists predominantly of 2 μm thick, flattened collagenous lamellae, orientated in a parallel fashion to the surface of the cornea. Amongst the lamellae, there are numerous stellate shaped, flattened keratocytes, with thin cytoplasmic extensions emanating from their cell body. On IVCM, the keratocyte nuclei are visible as hyper-reflective, irregular, oval shaped cells distributed against a dark background. The dark background is due to the inherent optical properties of the collagen lamellae [29]. The keratocyte density in the anterior 10% of the stroma is the highest ranging between 24,320 to 28,838 cells/mm3 and this then reduces to 18,850 to 19,947 cells/mm3 immediately adjacent to Descement’s membrane [30, 31]. There is a general decline in keratocyte density with age: it reduces by 0.9% and 0.3% in the anterior and posterior stroma [17], respectively and overall, it reduces by approximately 0.45% per year [30]. In the anterior and mid-stroma, straight, thicker hyperreflective nerves are observed and they branch in a dichotomous pattern (Fig. 5a–h) [7].

Cornea Nerves The cornea nerves can be subdivided into three layers: subbasal nerve plexus, subepithelial nerve plexus and stromal nerves. The subbasal nerve plexus is located between the Bowman’s layer and the basal epithelium (Fig. 6a). When imaged by IVCM, the nerve fibers are linear, beaded and have a homogeneous reflectivity. They branch in a Y or H—shaped configuration [32, 33]. Detail mapping of the subbasal nerve plexus has identified a radiating pattern of nerve fiber bundles converging to a point 1–2  mm inferior of the cornea apex forming a whorl-like pattern (Fig. 6b). It has been found that the nerve fibers migrate in a clockwise, centripetal direction, at a rate of 26 μm per week, reflecting the dynamic nature of the subbasal nerve plexus [34]. The subbasal nerve plexus reduces by approximately 0.9% per year [17].

f

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Fig. 5 (a–h) Montage of in vivo confocal images from the anterior stroma just below Bowman’s layer to the posterior stroma just anterior to Descemet’s membrane. Depth measurements are shown on the bottom of each image. Stroma nerves (arrow) are shown in two of the images (c, f)

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Fig. 6  Corneal nerves. (a) Subbasal nerve plexus. (b) Subbasal nerve plexus corneal whorl. (c) Subepithelial plexus (arrows). (d) Stroma nerve

Quantitative evaluation of the subbasal nerve plexus density (SBND) produces a range of values due to the different methods of reporting and the type of machine used for image acquisition [33]. The SBND values have been reported to range from 8.4  mm/mm2 using tandem scanning confocal microscopy (TSCM) to 21.7  mm/ mm2 with laser scanning confocal microscopy (LSCM) [35, 36]. Compare to both slit scanning confocal microscopy (SSCM) and TSCM, the image contrast with LSCM is superior, which may explain the higher values found. Recent developments in both manual and automated software have allowed nerve morphology to be quantified into various parameters including corneal nerve branch density, corneal nerve fiber length, tortuosity coefficient [37], and more recently corneal nerve fractal dimension analysis (see chapter “Corneal Nerves Useful for Ophthalmology But Indispensable for Neurology”) [38]. Between Bowman’s layer and the anterior stroma lies the subepithelial nerve plexus. The nerves in this layer are more sparse, lower contrast, exhibit beads or

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varicosities, and more apparent in the mid-peripheral cornea compared to the center (Fig. 6c) [33]. Stromal nerves are present in the anterior and mid stroma but are not seen in the posterior stroma. Compared to the subbasal nerve plexus and subepithelial nerves, the stromal nerves are much thicker, more hyper-reflective, and branches in a dichotomous pattern (Fig. 6d). The diameter of stromal nerves has been reported to range from 5.5 μm [39] to 11.4 μm, [40] and stromal nerve density, using SSCM to range between 3.7 and 4.2  mm/mm2. However, due the way the nerves traverse obliquely in the cornea, the sparse nature of stromal nerves, and the difference in axial resolution between microscopes, the density estimation may not be accurate or repeatable [33].

Descemet’s Membrane Descemet’s membrane is the modified basement membrane of the endothelium and it is of 6–10 μm in thickness [7, 9]. Histologically, it is made up of two parts, the anterior banded third that is produced in the prenatal period and a homogenous, non-banded posterior two-thirds that is deposited by the endothelial cells during life. This non-banded layer increases around fivefold to approximately 10  μm at 80 years of age [41]. Due to the increase in thickness with age, it may explain why the Descemet’s membrane is more visible in elderly subjects [42] but in general, this layer is not normally visible on IVCM.

Endothelium The endothelium is posterior to Descemet’s membrane and it is made of regular hexagonal hyper-reflective cells, arranged in a honeycomb pattern, with hypo-­ reflective border with occasional nuclei seen (Fig. 7a). The cells are approximately 20 μm in diameter, normal cell density in a young healthy adult is between 2500 and 3000 cells/mm2 [7, 14]. The cell density decreases by approximately 0.6% per year, as determined by specular microscopy [43]. Unlike the cell membrane of epithelial cells, the cell boundary of endothelial cells are hypo-reflective. Comparative studies between LSCM and non-contact specular microscopy have found that IVCM tends to underestimate endothelial cell density in eyes with a reduced cell density (10 years) wearers, particularly rigid contact lenses [41]; and in subjects transitioning from low to high oxygen

Fig. 1  Representative images of the central human subbasal innervation from a healthy individual captured with a laser scanning CCM (Heidelberg Retina Tomograph III, Heidelberg Engineering GmbH, Germany). Subbasal nerve fiber and branch density appear dense with minimal variation across the images (Right to left: nasal to temporal apex)

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transmissibility lenses [42]. Another investigation found no effects of soft contact lens wear on the morphology and distribution of corneal nerves despite subclinical corneal edema in overnight wearers [43]. Upregulation of interleukin-6 and decreased expression of MUC5AC mRNA, a molecular marker of tear film stability has been reported in soft contact lens wearers, but without alterations in SNP morphology [44]. More recently, a small but significant decrease in corneal nerve density was found in contact lens wearers, but it did not correlate with elevated levels of tear film nerve growth factor [45]. The most significant changes in SNP morphology have been demonstrated in orthokeratology lens wearers. Subbasal nerves lose their characteristic curvilinear appearance and whorl pattern and are replaced by a network of highly tortuous nerves accompanied by topographic change and reduced corneal sensitivity [46, 47]. Long-term (>1 year) orthokeratology treatment results in reduced subbasal nerve density, which does not recover to pre-treatment levels up to 1 month after discontinuation [48]. In summary, it appears that regular contact lens wear is associated with altered corneal sensitivity and tear film quality but not corneal nerve density. The long-term effects of orthokeratology treatment on SNP morphology are presently unknown.

Corneal Nerve Alterations Following Corneal Surgery Refractive Surgery Refractive surgery is known to induce changes in SNP morphology and density amongst other corneal structures. Following photorefractive keratectomy (PRK), by week 8 the subepithelial nerves begin to reinnervate the wounded cornea from the periphery towards the center; and at month 3 non-branched nerve fibers are detectable at the ablation zone. Regeneration is complete at 6–8 months post-operatively albeit with an abnormal branching pattern compared to controls [49]. Corneal sensitivity follows a similar pattern with partial recovery at weeks 4–8 and complete restoration by month 12 [49]. Other studies have reported that subbasal nerve density remains ~60% decreased at month 12 after surgery and returns to preoperative levels by month 24 [50, 51] and remains unchanged until year 5 [51, 52]. However, in some individuals a complete recovery of the subbasal nerve density has not been observed even at 36 months after surgery [53]. Laser-assisted in situ keratomileusis (LASIK) results in rapid degeneration and complete loss of subbasal nerves in the flap area with a variable recovery time. Studies report regenerating nerves as early as 1–2 weeks [54], 1 month [55] or even 4 months [56]. Central corneal reinnervation occurs approximately 6 months after surgery [49, 54] with the nasal area being less affected than the temporal cornea [54]. Indeed, the hinge flap position may influence corneal sensitivity and nerve regrowth as a superior-hinge flap transects more nerves than a nasal-hinge flap [57]. The central subbasal nerve density has been reported to remain >50% reduced at 12 months [49]; 35% less at 24 months [51] and 34% less at 36 months [51, 58] while the inferior whorl is not visible for up to 24 months post-operatively [55].

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Corneal sensation is significantly reduced 1  week after surgery and recovers on average 6 months after surgery (range 3–16 months) [59–62]. Corneal nerve regeneration is ~10% faster at 3  months in flaps created with a One-Use Plus Sub-­ Bowman’s keratomileusis compared to femtosecond laser or a mechanical microkeratome [63]. The differences in recovery time of subbasal nerve morphology between PRK (up to 2 years) and LASIK (up to 5 years) may be attributed to ablation depth.

Penetrating Keratoplasty Corneal transplantation is the most frequent and successful form of transplantation in humans and indications include Fuchs’ dystrophy, keratoconus, infectious keratitis and corneal scarring [64]. Endothelial keratoplasty, deep anterior lamellar keratoplasty or penetrating keratoplasty are the available options depending on the degree of corneal involvement. Stromal nerves can be observed in 88% of the grafts, 4 days after keratoplasty but appear thinner compared to the healthy cornea [65]. In a serial investigation thin stromal nerves were present in some patients after 6  months and all patients within 12  months while subbasal nerves were undetectable throughout the study [66]. In clear penetrating grafts, keratocyte and subbasal nerve density are markedly lower compared to controls, 12.8 ± 8.7 years post-operatively with no subbasal nerves in 48% of clear grafts and regenerated nerves follow a random pattern as opposed to the curvilinear organization in the healthy cornea [67]. Irregular morphology of regenerating nerves has been confirmed histologically, characterized by a tortuous network of nerves in the stroma. Subbasal nerves lose their whorl configuration, are highly tortuous and reduced in number and are mainly located in the periphery with only a few nerves crossing the graft-host junction [68]. However, corneal sensitivity improves 12  months after transplantation, despite the absence of central subbasal nerves [69], which may be attributed to the lower resolution of earlier CCM models and contribution of peripheral nerves [68]. Indeed, central reinnervation of the basal epithelium was found 2  years after grafting, particularly in young patients, and correlated with the highest level of sensitivity [70]. A comparative study of deep anterior lamellar and penetrating keratoplasty showed a near complete recovery of corneal sensitivity 2 years after surgery in 91% of cases, independent of the procedure of choice [71].

Corneal Collagen Cross-Linking Corneal collagen cross-linking is a minimally invasive procedure to strengthen the collagen fibrils to slow down the progression of keratoconus. Epithelial debridement during standard corneal collagen cross-linking leads to a complete loss of subbasal and anterior stromal nerves in an ex-vivo model, with preservation of nerves in corneas treated with the transepithelial approach [72]. Another study confirmed that subbasal and stromal nerves appeared intact after transepithelial

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cross-linking although the evaluation was qualitative [73]. Prospective evaluation by CCM has shown that subbasal nerves are absent from the radiation site at days 7 and 15, with preservation of the mid-stromal plexus and fine subbasal nerves were visible at 1 month and continued to regenerate until they reached pre-operative values at 24–36 months [74].

Corneal Nerve Alterations in Ophthalmic Disease Keratoconus Pathological alterations of the SNP have been implicated in the onset and progression of keratoconus [75]. Although corneal nerves are detectable in keratoconic eyes, their architecture differs substantially compared to the healthy cornea resembling the shape of the cone [76]. Subbasal nerves appear more tortuous with reduced density and correlates with reduced sensation, particularly in contact lens wearers [77, 78]. Histological characterization reveals loss of the radial pattern of subbasal nerves with increased tortuosity at the cone apex; a concentric arrangement at the cone base; and localized thickening at their point of origin beneath Bowman’s membrane [79]. Indeed, a study of 78 subjects with keratoconus demonstrated reduced epithelial, keratocyte and endothelial cell and corneal sub-basal plexus densities [78]. CCM could be used to identify patients at risk for progression of keratoconus and development of corneal neurotrophic ulcers.

Dry Eyes The International Dry Eye Workshop defines dry eye disease as a multifactorial disease of the tears and ocular surface that results in tear film instability, discomfort, visual disturbance, which may be harmful to the ocular surface [80]. Sensory signals from the ocular surface play a vital role in the maintenance of tear flow and deficits in any component of the lacrimal functional unit manifest as dry eye disease. It is unclear if dry eye disease is associated with reduced subbasal nerve density. Some studies show reduced corneal nerve fiber density along with increased beading, tortuosity and width, which correlate with reduced central sensitivity and the ocular surface disease index in non-Sjogren’s and Sjogren’s dry eyes [81, 82] (Fig. 2). By contrast, another investigation demonstrated increased nerve density, tortuosity and branching pattern in Sjogren’s dry eyes [83]. However, dry eye disease is heterogeneous and factors such as age, the presence and severity of Sjogren’s syndrome and diabetes may affect corneal nerve morphology. Whilst CCM visualize sensory nerves, the lacrimal functional unit may be affected by autonomic nerve damage contributing to tear film instability. Nevertheless, increased nerve tortuosity and branching are a common finding indicating nerve degeneration with regeneration and a recent study showed that the subbasal nerve status may predict the response to dry eye therapy [84].

Fig. 2  CCM images from the SNP of an age-matched healthy control (left) and a patient diagnosed with Sjogren’s syndrome (right eye—middle image; left eye—right image). Patient with Sjogren’s syndrome shows a moderate reduction in their SNP fiber and branch density which is more advanced in the right compared the left eye

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Corneal Neuropathic Pain Neuropathic pain is caused by a lesion or disease of the somatosensory system, including afferent nerves (Aβ, Aδ and C fibers) and central neurons with abnormal pain signaling [85]. Corneal neuropathic pain is encountered in neuroinflammatory disease, infectious disease and after refractive surgery or chemotherapy but a lack of abnormality on slit lamp examination makes the diagnosis challenging [86]. Corneal hyperalgesia is perceived as increased sensitivity to air and ambient light (photosensitivity) while allodynia manifests as burning sensation even to non-noxious stimuli [87]. In this context, corneal neuropathic pain represents a niche of the spectrum of neuropathic pain. Using CCM, abnormal subbasal nerve morphology with increased dendritic cell density and enlarged terminal nerve sprouts has been reported in a case of severe corneal pain with bilateral Bowman’s erosions and severe vitamin D deficiency [88]. Microneuromas are more prevalent in patients with corneal pain and may also help to differentiate from patients with dry eye disease with overlapping symptoms [89, 90]. The majority (~75%) are spindle neuromas, which appear as focal enlargements of stromal nerves followed by lateral neuromas (~44%), which represent enlargements of stromal nerves with sprouting of multiple tortuous subbasal nerves. Stump neuromas are less common (~15%) and appear as swollen terminations of stromal nerves [90]. Apart from decreased nerve fiber and branch density, tortuosity and reflectivity are also altered in patients with corneal neuropathic pain and may improve after treatment with autologous serum tears [91]. Subbasal nerve pathology of patients with corneal neuropathic pain after refractive surgery or due to herpetic infection appears to be comparable [92].

Infectious Disease Decreased subbasal nerve density has been demonstrated in the affected eyes of patients with herpes zoster ophthalmicus [93] and herpes simplex keratitis [94]. In unilateral keratitis, altered SNP morphology may also be present in the contralateral unaffected eye [94, 95] and decreased nerve density strongly correlates with reduced corneal sensitivity, superficial epithelial cell loss and enlargement of cell size [96]. Apart from herpetic infections, corneal nerve loss has been documented in Acanthamoeba keratitis, bacterial and fungal keratitis and cytomegalovirus endotheliitis [97–99]. In the acute phase of microbial infection patients demonstrate severe loss of main nerve fibers and branches with a significant increase in immune cell density even in the unaffected eye, greater than in acute herpetic keratitis [97, 98] (Fig. 3). In patients with cytomegalovirus endotheliitis, CCM demonstrates a decrease in subepithelial nerves and highly reflective keratocytes along with highly reflective dots, needle-shaped bodies and corneal opacities all of which resolve after treatment [99].

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Fig. 3  CCM images from the right eye of an age-matched healthy control (upper row) and a patient with bilateral acanthamoeba keratitis at diagnosis (middle row) and 12 months follow-up (bottom row). At baseline there is epithelial cell loss (left column), complete loss of subbasal nerve fibers with increased immune cells (middle column—white arrow) and appearance of thickened nerves in the anterior stroma (white arrowhead) with a moderate loss of anterior keratocyte density (right column). At follow-up epithelial cell density has been nearly restored with variations in cell size and shape still visible (white arrows). Regenerating nerve fibers are visible at the SNP (white arrowheads) and anterior stromal morphology appears normal

Other Conditions Associated with Corneal Nerve Alterations Patients with allergy commonly experience atopic disease and atopic keratoconjunctivitis is a bilateral chronic hypersensitivity disease associated with atopic dermatitis. Ocular surface inflammation leads to tear film instability and reduced corneal sensitivity. CCM reveals stromal and subbasal nerve abnormalities such as increased tortuosity, reduced branching and thickening with increased immune cells and loss of epithelial cells [100]. Reduced subbasal nerve density and beading with

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highly tortuous stromal nerves along with epithelial, endothelial and keratocyte cell changes is observed in vernal keratoconjunctivitis [101]. Recurrent corneal erosion syndrome is a condition associated with painful erosions of the corneal epithelium, with or without preceding injury, due to defective adhesion of the basal cells to Bowman’s membrane [102]. Reduced subbasal nerve density and reflectivity with increased tortuosity and immune cell density alongside epithelial cell pathology have been described [103] (Fig. 4). Corneal epithelial neoplasia is a relatively rare condition affecting older male patients, which includes dysplasia and carcinoma in situ. CCM shows a loss of subbasal nerves in areas affected by the condition, which is restored after treatment [104]. Stevens-Johnson syndrome, toxic epidermal necrolysis and limbal stem cell deficiency lead to severe subbasal nerve loss with an increase in immune cell density [105]. Chronic use of glaucoma medications may induce allergic or toxic alterations due to their active component or preservatives. There is a significant reduction in subbasal nerve density with increased tortuosity and endothelial cell changes with no difference between prostaglandin and beta-­ blocker analogues [106, 107]. A preservative-free prostaglandin analogue preserved corneal nerve density in treatment naïve and previously treated glaucoma patients [108].

Corneal Nerve Alterations in Systemic Disease A growing list of systemic conditions have been associated with pathological alterations in the SNP [109]. Moreover, in clinical trials of neuroprotective treatments in peripheral neuropathies, corneal subbasal nerve regeneration precedes the improvement in neuropathic symptoms and gold standard measures of neuropathy including nerve conduction and intraepidermal nerve fibre density in human and experimental models of disease [110].

Peripheral Neuropathies Diabetic Peripheral Neuropathy Diabetic peripheral neuropathy (DPN) is a length-dependent axonopathy which affects approximately 50% of patients with diabetes during their lifetime. DPN is the main risk factor for neuropathic pain and amputation and is associated with increased mortality [111]. Small sensory nerves (C-fibers) are involved early followed by involvement of large, myelinated nerves. The gold standard method to determine C-fiber pathology is skin biopsy for estimation of intra-epidermal nerve fiber density, which is invasive, costly and requires a dedicated facility for assessment. CCM represents a rapid, non-invasive method to assess for the presence and progression of DPN. Consistent with a distal, dying-­ back neuropathy, corneal nerve loss is symmetrical [112]; greater at the inferior

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Fig. 4  CCM image montage from the affected eye of a patient with a previously resolved unilateral corneal erosion. The Bowman’s membrane is disrupted with reduced subbasal nerve density (white arrows) and small epithelial cell patches (white arrowheads) of variable size scattered across the center of the erosion. A stromal nerve entering the Bowman’s membrane and giving rise to subbasal nerves is also noted (white star). The white ring around the erosion denotes the margins of the erosion. In-depth CCM scanning showed that the erosion extended into the stroma in a conical pattern with the cone apex terminating between the anterior and mid stroma

whorl compared to the central SNP and in patients with painful compared to painless DPN (Fig. 5). Subtle yet significant corneal nerve alterations are reported even in individuals with minimally elevated hemoglobin A1c (i.e. a measure of average blood sugar over the past 2–3 months), triglycerides and body mass index without diabetes [113] while patients with impaired glucose tolerance and newly diagnosed type 2 diabetes exhibit significant reductions in both corneal nerve and intra-epidermal nerve fiber density [114, 115]. Additionally, a lower baseline CNFL predicts subjects with impaired glucose tolerance who subsequently develop diabetes while patients with near normal CNFL revert to normal glucose tolerance [114]. The severity of corneal nerve loss is associated with the severity of symptoms and signs of neuropathy and correlates with loss of intra-epidermal nerve fiber density [116, 117] and corneal sensitivity [118] in patients with type 1 and type 2 diabetes. CNFL is lower in

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patients with cardiac autonomic neuropathy and is associated with reduced heart rate variability [119] and erectile dysfunction [120]. In a longitudinal cohort of patients with type 1 diabetes followed over 4 years a lower baseline CNFL predicted incident DPN.  Additionally, patients with a more rapid CNFL decline (>6% per year) exhibit faster clinical progression of diabetic neuropathy [121] and rapid CNFL loss without a change in quantitative sensory testing or nerve conduction has been associated with the development of foot ulceration and Charcot foot leading to amputation [122]. Simultaneous pancreas and kidney transplantation in patients with type 1 diabetes is associated with an increase in CNFD, CNBD and CNFL [123] followed by an improvement in symptoms and neurophysiology after 3 years [124]. An improvement in glycemic control with medication and dietary restriction improved SNP morphology, neurophysiology and albuminuria over 4 years [125] while patients with poor metabolic control continued to deteriorate [126]. Corneal nerve regeneration with no change in vibration perception or sudomotor function was recently shown in patients randomized to exenatide/pioglitazone or basal bolus insulin [127]. Bariatric surgery in morbidly obese patients (body mass index > 35) with or without type 2 diabetes results in an improvement in glycaemic control, body mass index and triglycerides and corneal nerve regeneration within 12 months [128, 129]. In two separate phase 2b trials of Cibinetide, an anti-inflammatory drug, patients with DPN and sarcoidosis-­ associated neuropathy showed a significant increase in CNFD and corneal nerve fiber area with an improvement in pain intensity and exercise capacity compared to placebo [130, 131]. An improvement in CNFL, but no change in quantitative sensory testing or neurophysiology has also been demonstrated with omega-3 supplementation which was paralleled by an increase in omega 3 plasma levels [132, 133].

Inflammatory Neuropathies Bechet’s disease is a relapsing inflammatory disorder characterized by oral aphthous and genital ulcers, uveitis, and lesions of the skin, gastrointestinal, vascular, and nervous systems and involvement of the vascular and nervous systems is associated with poor prognosis. Increased keratic precipitates and altered corneal biomechanical properties [134, 135] along with increased corneal immune cell density and a reduction in corneal nerves have been reported in keeping with underlying inflammation and neurodegeneration [136]. CCM shows a significant reduction in CNFD and CNFL with a characteristic change in the pattern of immune cell infiltration in patients with chronic inflammatory demyelinating polyneuropathy compared to diabetic neuropathy [137] and the size and distribution of immune cells is associated with the presence and severity of pain [138, 139]. An increase in corneal immune cell density and morphology without concomitant SNP pathology has been reported in patients with ankylosing spondylitis and rheumatoid arthritis with dry eye disease [140, 141].

Fig. 5  From left to right: CCM images of the inferior whorl from a healthy control; patient without diabetic peripheral neuropathy; and a patient with diabetic peripheral neuropathy indicating progressive loss of the subbasal nerves with increasing neuropathic severity. White star denotes the center of the whorl

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Fibromyalgia Fibromyalgia is an idiopathic, chronic pain syndrome associated with fatigue, morning stiffness, paresthesia, and anxiety in addition to widespread pain. Stromal nerve thinning and subbasal nerve loss is evident in women with fibromyalgia and is associated with pain descriptors [142]. Apart from reduced CNFD and CNFL, there is also a decrease in epithelial cell density with increased tear film instability, which correlate with the “widespread pain index” [143]. Abnormalities in SNP morphology are associated with signs of central sensitization in a sub-population of patients [144]. A large detailed phenotyping study showed significant CNFD loss in patients with fibromyalgia and higher pain intensity, more anxiety and paresthesias [145].

Amyloid Neuropathy Transthyretin familial amyloid polyneuropathy (TTR-FAP) is an autosomal dominant neuropathy characterized by limb paresthesias, pain, walking difficulty and weakness with cardiac and gastrointestinal symptoms [146]. CCM has been used to show a reduction in central CNFL in a cohort of patients with TTR-FAP which was associated with the severity of sensory neuropathy and cardiac autonomic neuropathy [147] and loss of corneal nerves has been reported in a case report of a patient with FAP [148] and a patient with light-chain amyloid neuropathy caused by multiple myeloma [149]. More recently, a reduction in the inferior whorl length was reported in patients with subclinical TTR-FAP with excellent diagnostic outcomes for both CNFL and IWL [150].

Other Peripheral Neuropathies Idiopathic small fiber neuropathy is a broad disease entity characterized by neuropathic pain and sensory neuropathy without large fiber involvement [151]. Patients exhibit decreased corneal sensitivity, CNFD, CNBD and CNFL in the absence of abnormalities in vibrotactile and thermal sensation and nerve conduction studies [152]. In a recent detailed phenotyping study of patients with small fiber neuropathy abnormal neurological examination (53/86, 62%), distal IENFD (60/86, 70%) and CCM (29/55 (53%) identified small fiber disease and whilst adding CCM further increased the identification of patients with small fiber impairment to 85%, quantitative sensory testing (22%) and quantitative axon sudomotor reflex test (9%) were of lower impact [153]. Pathological alterations in SNP morphology have also been reported in patients with hereditary forms of peripheral neuropathy such as Charcot-Marie-Tooth Disease Type 1A and nerve growth factor-β mutation [154, 155] and the latter is known to cause selective loss of Aδ and C-fibers. Fabry disease is an inherited metabolic disorder with an accumulation of glycolipids characterized by small fiber neuropathy [156]. CCM shows a reduction in CNFD, CNBD, corneal sensitivity and

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endothelial cell density, which correlates with the Mainz severity score index [157]. Corneal nerve loss has been demonstrated in patients with chronic migraine and photophobia [158]. Friedreich’s ataxia is an autosomal, recessive disease due to a homozygous guanine-adenine-adenine (GAA) trinucleotide repeat expansion on chromosome 9q13 that causes a transcriptional defect of the frataxin gene. A severe reduction in CNFD and CNFL has been demonstrated in patients with Friedreich’s ataxia, which correlates with the number of trinucleotide repeats and severity of neurological disability [159]. Patients with hypothyroidism have reduced CNFD which improves after treatment with thyroxine [160]. Chemotherapy-induced peripheral neuropathy is characterized by a sensory and painful neuropathy. Patients with upper gastrointestinal cancer showed a reduction in CNFD, CNBD and CNFL which correlated with the extent of lymph node involvement and treatment with platinum-based chemotherapy was associated with nerve regeneration. Human immunodeficiency virus-­ associated sensory neuropathy is a frequent and debilitating length-dependent axonopathy resulting from HIV or as a consequence of the earlier anti-retroviral therapies and is characterized by sensory loss with or without neuropathic pain. CCM reveals a reduction in CNFD, CNFL and CNBD with increased tortuosity [161]. Representative CCM images from patients with different peripheral neuropathies are presented in Fig. 6.

Central Neurodegenerative Diseases Axonal degeneration is a common feature of central neurological disorders and is implicated in disease onset, progression, and irreversible worsening of disability. Recent studies show that pathological alterations of the SNP may provide diagnostic utility and predict the disease course in central neurodegenerative diseases. Parkinson’s disease is characterized by bradykinesia, resting tremor, rigidity, and postural instability with a variety of non-motor sensory and autonomic symptoms. Corneal nerve loss occurs in treatment-naïve patients with newly diagnosed Parkinson’s disease without alterations in nerve conduction studies and intra-­ epidermal nerve fiber density [162]. Patients with Parkinson’s disease show a reduction in corneal sensitivity, CNFD, CNBD and CNFL in relation to dopaminergic therapy [163]. Corneal nerve loss is also related to disease severity based on the unified Parkinson’s disease rating scale, autonomic dysfunction and cognitive impairment [164, 165]; and a lower baseline CNFD is predictive of significant 1-year worsening in motor disability [166]. Axonal loss in multiple sclerosis is associated with irreversible worsening of disability and disease progression [167]. Patients with multiple sclerosis show significant corneal nerve loss and an increase in immune cell density which correlates with neurological disability [168–173]. Corneal nerve loss is progressive and correlates with change in disability severity [174]. A reduction in CNFD, CNBD and CNFL is also associated with cognitive impairment in patients with dementia [175]; and corneal nerve pathology and medial temporal lobe atrophy on MRI has a diagnostic

Fig. 6  CCM images from a healthy control and patients with various peripheral neuropathy showing a significant reduction in subbasal nerve density (white arrow) and/or an increase in immune cell density (white arrowhead). From left to right, top row: healthy control; patient with diabetic neuropathy; patient with chemotherapy induced peripheral neuropathy; patient with chronic inflammatory demyelinating polyneuropathy. Bottom row: patient with idiopathic small fiber neuropathy; patient with arthritis; patient with human immunodeficiency virus-associated sensory neuropathy; and a patient with Friedreich’s ataxia

Central Neurodegenerative Diseases 141

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Fig. 7  CCM images from patients with central neurodegenerative disorders showing a significant reduction in subbasal nerve density (white arrow) and/or an increase in immune cell density (white arrowhead). From left to right: patient with Parkinson’s disease; patient with relapsing-remitting multiple sclerosis; patient with dementia; and a patient with acute ischemic stroke

accuracy which is comparable for dementia but better for mild cognitive impairment [176]. Patients with acute ischemic stroke show a reduction in corneal nerves, which is associated with glycemic and lipid control [177]. Patients with amyotrophic lateral sclerosis also show a reduction in corneal nerves which is related to the bulbar disability score [178]. Representative CCM images from patients with central neurodegenerative diseases are presented in Fig. 7.

Summary In-vivo CCM has allowed us to undertake detailed quantitative studies of the corneal nerves to characterize changes in ocular surface disease and following surgical interventions e.g. LASIK which can lead to altered corneal innervation. Alterations

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in SNP morphology appear also to be a sensitive proxy for systemic neurological, autoimmune, metabolic and inherited diseases. Quantification of subbasal nerve morphology allows an objective means to diagnose, risk-stratify and predict progression as well as therapeutic response in patients with neurodegenerative disease.

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107. Martone G, Frezzotti P, Tosi GM, Traversi C, Mittica V, Malandrini A, et al. An in vivo confocal microscopy analysis of effects of topical antiglaucoma therapy with preservative on corneal innervation and morphology. Am J Ophthalmol. 2009;147(4):725–35.e1. 108. Rossi GCM, Blini M, Scudeller L, Ricciardelli G, Depolo L, Amisano A, et  al. Effect of preservative-free tafluprost on keratocytes, sub-basal nerves, and endothelium: a single-blind one-year confocal study on naïve or treated glaucoma and hypertensive patients versus a control group. J Ocul Pharmacol Ther. 2013;29(9):821–5. 109. Petropoulos IN, Ponirakis G, Khan A, Gad H, Almuhannadi H, Brines M, et al. Corneal confocal microscopy: ready for prime time. Clin Exp Optom. 2020;103(3):265–77. 110. Petropoulos IN, Ponirakis G, Ferdousi M, Azmi S, Kalteniece A, Khan A, et al. Corneal confocal microscopy: a biomarker for diabetic peripheral neuropathy. Clin Ther. 2021. 111. Selvarajah D, Kar D, Khunti K, Davies MJ, Scott AR, Walker J, et al. Diabetic peripheral neuropathy: advances in diagnosis and strategies for screening and early intervention. Lancet Diabet Endocrinol. 2019;7(12):938–48. 112. Petropoulos IN, Alam U, Fadavi H, Asghar O, Green P, Ponirakis G, et  al. Corneal nerve loss detected with corneal confocal microscopy is symmetrical and related to the severity of diabetic polyneuropathy. Diabetes Care. 2013;36(11):3646–51. 113. Sharma S, Tobin V, Vas PR, Malik RA, Rayman G. The influence of age, anthropometric and metabolic variables on LDIFLARE and corneal confocal microscopy in healthy individuals. PLoS One. 2018;13(3):e0193452. 114. Azmi S, Ferdousi M, Petropoulos IN, Ponirakis G, Alam U, Fadavi H, et al. Corneal confocal microscopy identifies small-fiber neuropathy in subjects with impaired glucose tolerance who develop type 2 diabetes. Diabetes Care. 2015;38(8):1502–8. 115. Asghar O, Petropoulos IN, Alam U, Jones W, Jeziorska M, Marshall A, et al. Corneal confocal microscopy detects neuropathy in subjects with impaired glucose tolerance. Diabetes Care. 2014;37(9):2643–6. 116. Malik RA, Kallinikos P, Abbott CA, van Schie CHM, Morgan P, Efron N, et  al. Corneal confocal microscopy: a non-invasive surrogate of nerve fibre damage and repair in diabetic patients. Diabetologia. 2003;46(5):683–8. 117. Quattrini C, Tavakoli M, Jeziorska M, Kallinikos P, Tesfaye S, Finnigan J, et al. Surrogate markers of small fiber damage in human diabetic neuropathy. Diabetes. 2007;56(8):2148–54. 118. Tavakoli M, Kallinikos PA, Efron N, Boulton AJ, Malik RA.  Corneal sensitivity is reduced and relates to the severity of neuropathy in patients with diabetes. Diabetes Care. 2007;30(7):1895–7. 119. Tavakoli M, Begum P, McLaughlin J, Malik RA. Corneal confocal microscopy for the diagnosis of diabetic autonomic neuropathy. Muscle Nerve. 2015;52(3):363–70. 120. Azmi S, Ferdousi M, Alam U, Petropoulos IN, Ponirakis G, Marshall A, et al. Small-fibre neuropathy in men with type 1 diabetes and erectile dysfunction: a cross-sectional study. Diabetologia. 2017;60(6):1094–101. 121. Lewis EJH, Lovblom LE, Ferdousi M, Halpern EM, Jeziorska M, Pacaud D, et al. Rapid corneal nerve fiber loss: a marker of diabetic neuropathy onset and progression. Diabetes Care. 2020;43(8):1829–35. 122. Dehghani C, Russell AW, Perkins BA, Malik RA, Pritchard N, Edwards K, et  al. A rapid decline in corneal small fibers and occurrence of foot ulceration and Charcot foot. J Diabetes Complicat. 2016;30(8):1437–9. 123. Tavakoli M, Mitu-Pretorian M, Petropoulos IN, Fadavi H, Asghar O, Alam U, et al. Corneal confocal microscopy detects early nerve regeneration in diabetic neuropathy after simultaneous pancreas and kidney transplantation. Diabetes. 2013;62(1):254–60. 124. Azmi S, Jeziorska M, Ferdousi M, Petropoulos IN, Ponirakis G, Marshall A, et  al. Early nerve fibre regeneration in individuals with type 1 diabetes after simultaneous pancreas and kidney transplantation. Diabetologia. 2019;62(8):1478–87. 125. Ishibashi F, Taniguchi M, Kosaka A, Uetake H, Tavakoli M.  Improvement in neuropathy outcomes with normalizing HbA1c in patients with type 2 diabetes. Diabetes Care. 2019;42(1):110–8.

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144. Oudejans L, He X, Niesters M, Dahan A, Brines M, van Velzen M.  Cornea nerve fiber quantification and construction of phenotypes in patients with fibromyalgia. Sci Rep. 2016;6(1):23573. 145. Evdokimov D, Frank J, Klitsch A, Unterecker S, Warrings B, Serra J, et  al. Reduction of skin innervation is associated with a severe fibromyalgia phenotype. Ann Neurol. 2019;86(4):504–16. 146. Planté-Bordeneuve V, Ferreira A, Lalu T, Zaros C, Lacroix C, Adams D, et al. Diagnostic pitfalls in sporadic transthyretin familial amyloid polyneuropathy (TTR-FAP). Neurology. 2007;69(7):693–8. 147. Rousseau A, Cauquil C, Dupas B, Labbé A, Baudouin C, Barreau E, et al. Potential role of in  vivo confocal microscopy for imaging corneal nerves in transthyretin familial amyloid polyneuropathy. JAMA Ophthalmol. 2016;134(9):983–9. 148. Bouaich K, Dufrane R, Youssfi A, Slim E, Ehongo A.  Corneal confocal microscopy and familial amyloidotic polyneuropathy. J Francais D’ophtalmologie. 2020;43(2):e81. 149. Sturm D, Schmidt-Wilcke T, Greiner T, Maier C, Schargus M, Tegenthoff M, et al. Confocal cornea microscopy detects involvement of corneal nerve fibers in a patient with light-­ chain amyloid neuropathy caused by multiple myeloma: a case report. Case Rep Neurol. 2016;8(2):134–9. 150. Zhang Y, Liu Z, Zhang Y, Wang H, Liu X, Zhang S, et al. Corneal sub-basal whorl-like nerve plexus: a landmark for early and follow-up evaluation in transthyretin familial amyloid polyneuropathy. Eur J Neurol. 2021;28(2):630–8. 151. Freeman R, Gewandter JS, Faber CG, Gibbons C, Haroutounian S, Lauria G, et  al. Idiopathic distal sensory polyneuropathy: ACTTION diagnostic criteria. Neurology. 2020;95(22):1005–14. 152. Tavakoli M, Marshall A, Pitceathly R, Fadavi H, Gow D, Roberts ME, et al. Corneal confocal microscopy: a novel means to detect nerve fibre damage in idiopathic small fibre neuropathy. Exp Neurol. 2010;223(1):245–50. 153. Egenolf N, Altenschildesche CMZ, Kreß L, Eggermann K, Namer B, Gross F, et  al. Diagnosing small fiber neuropathy in clinical practice: a deep phenotyping study. Ther Adv Neurol Disord. 2021;14:17562864211004318. 154. Tavakoli M, Marshall A, Banka S, Petropoulos IN, Fadavi H, Kingston H, et  al. Corneal confocal microscopy detects small-fiber neuropathy in Charcot–Marie–Tooth disease type 1A patients. Muscle Nerve. 2012;46(5):698–704. 155. Perini I, Tavakoli M, Marshall A, Minde J, Morrison I. Rare human nerve growth factor-β mutation reveals relationship between C-afferent density and acute pain evaluation. J Neurophysiol. 2016;116(2):425–30. 156. Politei JM, Durand C, Schenone AB. Small fiber neuropathy in fabry disease: a review of pathophysiology and treatment. J Inborn Errors Metab Screen. 2016;4:2326409816661351. 157. Bitirgen G, Turkmen K, Malik RA, Ozkagnici A, Zengin N.  Corneal confocal microscopy detects corneal nerve damage and increased dendritic cells in Fabry disease. Sci Rep. 2018;8(1):12244. 158. Shetty R, Deshmukh R, Shroff R, Dedhiya C, Jayadev C. Subbasal nerve plexus changes in chronic migraine. Cornea. 2018;37(1):72–5. 159. Pagovich OE, Vo ML, Zhao ZZ, Petropoulos IN, Yuan M, Lertsuwanroj B, et  al. Corneal confocal microscopy: neurologic disease biomarker in Friedreich ataxia. Ann Neurol. 2018;84(6):893–904. 160. Sharma S, Tobin V, Vas PRJ, Rayman G. The LDIFLARE and CCM methods demonstrate early nerve fiber abnormalities in untreated hypothyroidism: a prospective study. J Clin Endocrinol Metabol. 2018;103(8):3094–102. 161. Kemp HI, Petropoulos IN, Rice ASC, Vollert J, Maier C, Sturm D, et al. Use of corneal confocal microscopy to evaluate small nerve fibers in patients with human immunodeficiency virus. JAMA Ophthalmol. 2017;135(7):795–800. 162. Podgorny PJ, Suchowersky O, Romanchuk KG, Feasby TE. Evidence for small fiber neuropathy in early Parkinson’s disease. Parkinsonism Relat Disord. 2016;28:94–9.

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163. Anjos R, Vieira L, Sousa A, Maduro V, Alves N, Candelaria P.  Peripheral neuropathy in Parkinson disease: an in vivo confocal microscopy study. Acta Ophthalmol. 2014;92 164. Kass-Iliyya L, Javed S, Gosal D, Kobylecki C, Marshall A, Petropoulos IN, et al. Small fiber neuropathy in Parkinson’s disease: a clinical, pathological and corneal confocal microscopy study. Parkinsonism Relat Disord. 2015;21(12):1454–60. 165. Misra SL, Kersten HM, Roxburgh RH, Danesh-Meyer HV, McGhee CNJ.  Corneal nerve microstructure in Parkinson’s disease. J Clin Neurosci. 2017;39:53–8. 166. Lim SH, Ferdousi M, Kalteniece A, Mahfoud ZR, Petropoulos IN, Malik RA, et al. Corneal confocal microscopy identifies parkinson’s disease with more rapid motor progression. Mov Disord. 2021;36(8):1927–34. 167. Evangelou N, Konz D, Esiri M, Smith S, Palace J, Matthews P.  Size-selective neuronal changes in the anterior optic pathways suggest a differential susceptibility to injury in multiple sclerosis. Brain. 2001;124(9):1813–20. 168. Bitirgen G, Akpinar Z, Malik RA, Ozkagnici A. Use of corneal confocal microscopy to detect corneal nerve loss and increased dendritic cells in patients with multiple sclerosis. JAMA Ophthalmol. 2017;135(7):777–82. 169. Fernandes D, Luís M, Cardigos J, Xavier C, Alves M, Papoila AL, et al. Corneal subbasal nerve plexus evaluation by in vivo confocal microscopy in multiple sclerosis: a potential new biomarker. Curr Eye Res. 2021;1–8 170. Mikolajczak J, Zimmermann H, Kheirkhah A, Kadas EM, Oberwahrenbrock T, Muller R, et al. Patients with multiple sclerosis demonstrate reduced subbasal corneal nerve fibre density. Mult Scler J. 2017;23(14):1847–53. 171. Petropoulos IN, Kamran S, Li Y, Khan A, Ponirakis G, Akhtar N, et  al. Corneal confocal microscopy: an imaging endpoint for axonal degeneration in multiple sclerosis. Invest Ophthalmol Vis Sci. 2017;58(9):3677–81. 172. Testa V, De Santis N, Scotto R, Pastorino CE, Cellerino M, Olivari S, et  al. Neuroaxonal degeneration in patients with multiple sclerosis: an optical coherence tomography and in vivo corneal confocal microscopy study. Cornea. 2020;39(10):1221–6. 173. Khan A, Li Y, Ponirakis G, Akhtar N, Gad H, George P, et  al. Corneal immune cells are increased in patients with multiple sclerosis. Translational Vision. Sci Technol. 2021;10(4):19. 174. Bitirgen G, Akpinar Z, Uca AU, Ozkagnici A, Petropoulos IN, Malik RA. Progressive loss of corneal and retinal nerve fibers in patients with multiple sclerosis: a 2-year follow-up study. Transl Vis Sci Technol. 2020;9(13):37. 175. Ponirakis G, Al Hamad H, Sankaranarayanan A, Khan A, Chandran M, Ramadan M, et al. Association of corneal nerve fiber measures with cognitive function in dementia. Ann Clin Transl Neurol. 2019;6(4):689–97. 176. Al-Janahi E, Ponirakis G, Al Hamad H, Vattoth S, Elsotouhy A, Petropoulos IN, et al. Corneal nerve and brain imaging in mild cognitive impairment and dementia. J Alzheimers Dis. 2020;77:1533–43. 177. Khan A, Akhtar N, Kamran S, Ponirakis G, Petropoulos IN, Tunio NA, et al. Corneal confocal microscopy detects corneal nerve damage in patients admitted with acute ischemic stroke. Stroke. 2017;48(11):3012–8. 178. Ferrari G, Grisan E, Scarpa F, Fazio R, Comola M, Quattrini A, et  al. Corneal confocal microscopy reveals trigeminal small sensory fiber neuropathy in amyotrophic lateral sclerosis. Front Aging Neurosci. 2014;6:278.

Other Anterior Segment Applications of In Vivo Confocal Microscopy and Future Developments

Other Quantitative Evaluations of the Cornea Corneal Endothelial Cell Density Micromorphological evaluation of the corneal endothelium is important in screening and diagnosis of endothelial disease, and to determine prognostic outcome on cornea clarity, especially in eyes with a compromised endothelium, after anterior segment surgery. Historically, quantitative evaluation of endothelial cells and corneal endothelial cell density (CED) have been performed with specular microscopy. Although well established, and with the main advantages of instant image output and being noncontact, this technique has some limitations. The optical principle of specular microscopy is based on the concept of specular reflection. The instrument projects light onto the cornea and it captures the light that is reflected from the corneal endothelium and aqueous interface and a photograph of the endothelial layer is generated. However, this principle requires the cornea to be transparent and the endothelial surface to be plain and smooth in order to produce a clear image of the endothelium [1, 2]. Any corneal pathology such as corneal oedema or stromal opacity can increase light scattering in the stroma precluding the generation of a clear specular reflex, limiting the usefulness of specular microscopy to image the endothelium in these conditions. Even without the presence of oedema or opacity, endothelial abnormality such as corneal guttae changes seen in conditions like Fuchs corneal endothelial dystrophy, can make imaging the endothelium difficult with conventional specular microscopy. By contrast, IVCM obtains images by using the principle of confocality (see chapter “Principles of In Vivo Confocal Microscopy”) so it is less affected by abnormalities of the endothelial surface or in the presence of corneal oedema. It has been shown that both specular microscopy and IVCM are comparable in the evaluation of endothelial cells in normal corneas but IVCM is superior when there is abnormality on the endothelium [1, 2].

© Springer-Verlag London Ltd., part of Springer Nature 2022 G. Latifi, S. Hau, In Vivo Confocal Microscopy in Eye Disease, https://doi.org/10.1007/978-1-4471-7517-9_7

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The quantitative analysis of endothelial cells on IVCM can be performed manually or by automated segmentation systems. In manual analysis, the operator counts all the endothelial cells inside a selected area or region of interest (ROI) by using a digital image tool to mark each individual endothelial cell and the machine then calculates the CED. This method of cell counting can be quite laborious so automated cell counting has been introduced to speed up the process. One such commercially available software is the Nidek Technologies Advanced Vision Information software System (NAVIS) in the scanning-slit Confoscan 4. Cell analysis is performed via the automated option on the NAVIS software without the need for manual cell border correction. The software automatically identifies the cell boundary, defines the ROI based on the image size, and the CED is calculated. A manual cell count on the NAVIS system can also be carried out and it requires the operator to select the cells by placing a dot on the center of each cell by using the cursor and the machine then calculates CED based on the number of cells counted within the chosen frame area. The Heidelberg Retina Tomograph 3/Rostock Cornea Module (HRT 3/RCM) also has a semi-automated system for calculating the CED. Similarly, after manually marking the centre of the endothelial cells in a ROI, the cell density (cells/ mm2) is automatically calculated [3, 4] (Fig. 1).

Corneal Thickness Measurement Confocal optics can be utilised to obtain corneal thickness measurement. The method used is called confocal microscopy through focusing (CMTF) for collecting and quantifying three-dimensional information of the cornea. The basis of this technique relies on the fact that different reflective intensities are generated by the various corneal layers. The objective lens or its focal plane in the Z axis moves rapidly from epithelium to endothelium and the movement is registered by computer. The intensity of light backscattered by the central section of each image is recorded to produce an intensity profile curve and a special software program is used to measure the distance between any two points on the curve [5, 6]. High peaks correspond to the corneal endothelium and lower peaks are related to anterior keratocytes in the stroma and the corneal epithelium. Since different layers of the cornea reflect different amounts of light, the intensity profile is able to determine the corneal sublayer thickness including Bowman’s layer, epithelial thickness and potentially flap thickness in patients after laser in situ keratomileusis (LASIK). There is a difference in the precision of the thickness measurement between the various confocal optical techniques. In Tandem scanning confocal microscope (TSCM), the focal plane is changed by moving lenses inside the objective casing so that the tip of the objective remains stationary during scanning. With the HRT 3/RCM, the focal plane is changed by rotating the RCM but because the Tomocap is in contact with the cornea, rotating the RCM may change the amount of pressure on the Tomocap and this may produce compressive artifacts like folds and ridges which can affect the reproducibility and accuracy of the measurements. TSCM has been reported to be more accurate and more repeatable with thickness measurements when compared to

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Fig. 1  Example of endothelial cell analysis using the HRT 3/RCM. The image shows the centre of each cell has been highlighted by a blue dot and an analysis of the region of interest (ROI) is shown below the image. The number of cells calculated in this example is 3882 cells/mm2

Focus position [µm]:

0.0129

Number of cells counted:

ROI area [mm2]:

3882 ± 109

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HRT/RCM [5, 6]. Slit-scanning confocal microscopy has been reported to be poorly repeatable for corneal thickness measurement but the use of a low vacuum suction cup is said to improve the accuracy [5–8].

Corneal Transparency Measurement In addition to thickness measurement, the amount of backscattered light (intensity units) provided by CMTF imaging can be used to objectively measure corneal transparency as well as to estimate corneal haze [6]. As the focus of the objective lens is moved along the Z-axis, the amount of back-scattered light, corresponding to each sublayer of the cornea, generates a series of intensity profile curves that allow cornea transparency to be defined. The amount of backscattered lights are measured in arbitrary intensity units and this gives an estimate of the level of cornea haze. In addition to IVCM, other optical methods are available or have been described to quantify cornea transparency including Scheimpflug photography, slit lamp photometry, opacity lensometers and charge coupled device camera systems. The repeatability of corneal haze measurements by IVCM has been reported to be moderate with the average coefficients of variation and intrasession coefficients of repeatability to be 35% and 8.2 intensity units, respectively [6, 9, 10]. It must be emphasised that all these methods quantify corneal transparency by determining the amount of back-reflected or back-scattered light towards the observation system rather than measuring the degree of forward scattered light, which is more representative of the retinal image quality and the patient’s symptoms.

Corneal Keratocyte Density Measurement There is a slow attrition of keratocyte density with age (see chapter “Normal Anatomy”). In addition, a reduction in keratocyte density also occurs in certain cornea diseases such as keratoconus and after laser refractive surgery [11, 12]. However, most of these studies estimated keratocyte density from single two-­ dimensional images and the drawback with this approach is the inherent variability of keratocyte density with different depths. CMTF allows the true three-­dimensional position of each cell to be determined and it provides a more accurate estimation in keratocyte density compared to averaging multiple sectional two-dimensional images. To perform a true three-dimensional count, the entire CMTF data set, compensated for eye movements, will need to be used and the density can be obtained either manually or by automated means [6].

I n Vivo Confocal Microscopy in Iridocorneal-Endothelial Syndrome The iridocorneal-endothelial (ICE) syndrome is a group of conditions caused by proliferation of corneal endothelial cells that migrate toward the drainage angle and onto the surface of the iris causing potential corneal decompensation and secondary

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ocular hypertension or glaucoma [13]. Clinically, a ‘beaten-bronze’ appearance is seen on the endothelium associated with iris atrophy, peripheral anterior synechiae, corectopia and ectropion uveae. The condition can be confused with iris melanoma, oculodermal melanocytosis and neurofibromatosis [14]. Traditionally, the endothelial changes are observed with specular microscopy with the classic dark-light reversal pattern in that the cell centre is dark and the cell boundary is light. However, the major drawback with specular microscopy is it is difficult to obtain clear endothelial images when there is sign of cornea thickening or oedema or if there is gross change on the endothelium that affects the quality of the specular reflex that is required to generate the image. By contrast, IVCM is not affected by corneal thickening or mild corneal oedema so it is the method of choice for the diagnosis of ICE syndrome. In addition, the image quality with IVCM is far superior compared to specular microscopy. On IVCM, characteristic epithelioid-like cells with prominent central hyper-reflective nuclei and a loss of hexagonality are seen (Fig. 2). In addition, the cells are polymorphic and often the migrating leading edge of the ICE cells can be seen at the boundary between normal endothelium and the abnormal ICE cells (Fig. 2). In established disease, the endothelium can be partially or completely a

b

c

d

Fig. 2  In vivo confocal microscopy (IVCM) of a patient with iridocorneal endothelial syndrome (ICE). (a) Low magnification—slit lamp examination of the right eye showing corectopia and an area of peripheral anterior synechiae at 11 o’clock. (b) High magnification—slit lamp examination showing ‘beaten—bronze’ appearance on optical section (arrow). (c) Specular microscopy showing light- dark reversal with the bright cell boundary (arrow) and the dark centre (star). (d) IVCM of the affected cornea showing the boundary between the less affected endothelium inferiorly and the superior migrating ICE cells. The cells are typified by the bright central nuclei

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replaced by the ICE cells with the severity of the affected endothelium to be related to the risk of developing glaucoma [15–17]. In addition, two types of ICE cells have been described with the smaller ICE cell type to be associated with elevated intraocular pressure [15].

 ther Anterior Segment Applications of In Vivo O Confocal Microscopy I n Vivo Confocal Microscopy of Other Anterior Segment Structures In contact IVCM with the HRT/RCM setup, because of its limited working distance (1.5 mm), it is incapable of imaging intraocular tissues [18]. However, various studies have attempted to evaluate the peripheral iris in eyes with peripheral synechiae by contact IVCM [19, 20]. Li et al. reported that iris structure in synechia can be divided into five specific types: (1) tree trunk-like structure, (2) tree branch/bush-­ like structure, (3) fruit-like structure, (4) epithelioid-like structure, and (5) deep structure that is difficult to analyze due to limited penetration of laser light [19]. A novel non-contact IVCM using the HRT/RCM combination, has previously been developed to examine intraocular anterior segment tissues. It can visualize anterior segment structures from the ocular surface all the way to the posterior lens capsule. In this set-up, a 50 X non-contact objective lens (Nikon, CF plan 0.45), with a working distance of 13.8 mm, gives a 500 × 500 μm field of view, and an estimated 1–2 μm in transverse resolution. During image acquisition, the subject needs to look straight ahead and the focal plane should be parallel to the central cornea. Then the focal plane can be moved towards the iris and lens capsule with a z-axis drive control. Image quality can be improved by the use of artificial tears and lubricant gels [18, 21]. Sbeity et  al. used non-contact IVCM to visualize structural alterations of the cornea, lens, and iris in patients with exfoliation syndrome. They could detect the fibrillar exfoliation material as hyperreflective deposits on the iris, anterior capsule of the lens and aqueous humor [18, 21]. Compared to contact—IVCM, the image quality of the ocular surface is inferior with non-contact IVCM but the main advantages are the ability to image the tear film and intraocular structures in the anterior segment.

In Vivo Confocal Microscopy of Skin Lesions Over the last decade, in vivo reflectance confocal microscopy has developed into a widespread and useful adjunctive diagnostic tool in the field of dermatology. The commonly available reflectance IVCM device include the VivaScope 1500® and VivaScope 3000® (MAVIG GmbH, Munich, Germany), which uses an 830 nm near-­ infrared laser light. Both instruments provide non-invasive, real time, high

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resolution images of the epidermis and the superficial collagen layers of the skin. Similar to the HRT/RCM device, the system detects the back-reflected or back scattered light of the different cellular structures of the skin. The VivaScope 3000® model is handheld and the main benefit of this is it allows the clinician to freely navigate over the skin, which is particularly relevant when scanning around the eyelid and periocular region. This model also has a larger field of view 750 × 750 μm compared to 500 × 500 μm with the Vivascope 1500. The imaging depth in normal skin is 200–300 μm and morphological details can be defined up to a lateral resolution of 0.5–1.0 μm [22, 23]. Due to the optical design, the depth of imaging with current reflectance IVCM is limited to about 250 μm, which reaches into the papillary dermis and upper reticular dermis. Therefore, the examination of the dermal–epidermal junction, which is at depths of 50–150 μm is possible. The dermal–epidermal junction is an important region in diagnosing dermatologic problems as the histopathological changes of inflammatory skin disease mainly occur in the epidermis and dermal papilla and skin cancers originate and spread from the basal cell layer in this location [22, 23]. As such, the penetration depth satisfies the coverage of commonly encountered skin lesions. Cinotti et  al. evaluated the utility of the Vivascope 3000® in the diagnosis of cutaneous eyelid tumors including basal cell carcinoma (BCC), squamous cell carcinoma, and melanoma. When comparing reflectance IVCM with clinical examination for diagnosing malignant tumours, they found a sensitivity and specificity of (98% versus 92%) and (74% versus 462%), respectively [24]. Specific IVCM features for the various types of benign and neoplastic lesions have been characterised. For example in BCC, dark silhouette, lobular nests of tightly packed cells, peripheral palisading of elongated cells, peritumoral clefts, convoluted and dilated blood vessels, and polarized elongated keratinocytes have been described [24]. In malignant melanoma, large dendritic or roundish hyper-reflective cells at the epithelial— stromal junction and/or in the stroma associated with the large pagetoid type cells are seen [24]. In non-malignant lesions such as a dermal naevus, reflectance IVCM shows hyper-reflective, medium-sized (10–20 μm), nests of round cells in the stroma with the absence of pagetoid cells and atypical cells at the epithelium/stromal junction [24] In an earlier study, Cinotti et al. evaluated the confocal microscopy features of 47 eyelid margin lesions with a suspicion of malignancy, 35 lesions were excised and when compared to histopathological diagnosis, they found a sensitivity and specificity of 100% and 69.2%, respectively [25]. Limitations with current reflectance IVCM include the difficulty in differentiating the various types of normal tissues such as melanin from melanocytes, and also the difficulty in differentiating normal cells from dysplastic or neoplastic cells. The main reason is because all the cellular structures are present in grayscale (black/ white) that lacks specificity for cell types and tissue structures [23]. Furthermore, it is important to emphasise that observer experience is essential in improving diagnostic accuracy of skin lesions when using these devices [22, 23]. Future directions include the development of various modes of contrast such as 1—photon and

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2—photon fluorescence microscopy, which is analogous to the use of multiple diagnostic stains in histology, would aid in the identification of the different cell types [23].

In Vivo Confocal Microscopy of Demodex spp. IVCM has been used to characterise the eyelid changes in eyes with blepharitis and meibomian gland dysfunction (see chapter “Conjunctiva and Limbus”). Demodex spp. is a common parasite in humans and can be found in the eyelids, cilia, meibomian glands and the face. There are a few different species but the two that affect humans are Demodex folliculorum and Demodex brevis [26]. Various reports have described the use of IVCM in imaging Demodex spp. Kojima et  al. investigated the applicability of IVCM in the diagnosis of ocular demodicosis. They reported that IVCM showed the mites in the terminal bulbs of the eyelashes, which were not observed clinically, and they disappeared after treatment. Marked inflammatory infiltrates around the meibomian glands and conjunctiva in Eyelids with demodicosis infestation were also noted, which subsequently disappeared after tea oil treatment (Fig. 3) [27]. In a separate study by Randon et al. they found 100% mite infestations in patients with anterior blepharitis and only 12% in healthy subjects [28]. In addition, they found that Demodex infestations were associated with meibomian gland dysfunction and were well characterised using IVCM [28]. Despite the promising imaging potential of the parasite in situ, false positive results can occur due to the fact that on IVCM, the mite can look similar to an eyelash or dandruff-like material [28].

 ecent Advances and Future Developments of In Vivo R Confocal Microscopy Confocal Fluorescence Microscopy Vital staining of specific structures can provide important information in microscopy. Heidelberg Retina Angiograph (HRA) and HRA II which were established for fluorescence angiography of the fundus, can be modified with the addition of the Rostock Cornea Module (RCM) that allows the laser beam to focus on the plane of the anterior segment of the eye to visualize intrinsic or stimulated (sodium fluorescein labeled) fluorescence of cells. It is possible in this method to change the laser beam stimulation with blue laser light (wavelength 488 nm) and add a blocking filter (500 nm) to visualize the intracellular fluorescence [29]. Using this design, it is possible to characterize the staining properties of cells and the distribution of cell spaces after corneal staining with fluorescein [29]. Mocan et al. have evaluated fluorescein enhanced IVCM of the epithelium using the Confoscan 3.0 (Vigonza, Italy). The corneal epithelium was assessed pre and

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Fig. 3 (a, b) Representative in vivo confocal microscopy images of the eyelash bulbs in a 72-yearold female patient with demodicosis and a healthy female control subject. Note the heavy Demodex infestation of the eyelash in the patient (a) and the absence of mites in the control subject (b). (c, d) Representative in vivo confocal microscopy images of the meibomian gland acinar units before (c) and after treatment (d) in the same patient. Note the dilatation of meibomian gland acinar units surrounding the infested eyelashes with periglandular inflammatory infiltrates mainly consisting of dendritic cells. Reprinted from ‘In vivo evaluation of ocular demodicosis using laser scanning confocal microscopy.’ Kojima, T., Ishida, R., Sato, E.  A., et  al. Investigative ophthalmology & visual science, (2011). 52 (1), 565–569. copyright permission under the terms of Creative Commons CC BY [27]

post instillation of 2% fluorescein solution and they found that there is approximately a 500 cells/mm2 difference in superficial epithelial cell density between pre and post instillation of fluorescein. In addition, they found hyper-reflective epithelial cells were much more prevalent in eyes with keratoconus and overt epitheliopathy [30].

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Optical Coherence Tomography-Guided In Vivo Confocal Laser Scanning Microscopy The drawbacks with current IVCM include a relatively small field of view, only en face images are possible, and a lack of precise data on the exact orientation and position of a point on the cornea. By contrast, optical coherence tomography (OCT), which is based on the principle of low coherence interferometry, provides more accurate estimation of corneal thickness and depth data, cross-sectional images that provides better correlation with histology, and improved precision of locating a point on the cornea in relation to other anterior segment structures. To overcome these limitations of confocal laser scanning microscopy, a multimodal imaging platform has been described in the form of OCT-guided confocal scanning laser microscopy (CSLM). In this platform, a microscope lens is attached to a SPECTRALIS OCT module (Heidelberg Engineering, Germany), with a modular lens adapter and a piezo actuator allocated for controlling image focus. The light from both imaging modalities (CSLM and OCT) are combined within the camera, and they have the same beam path through the SPECTRALIS objective and the added microscope lens [31]. With this set-up, the bright reflection in the OCT cross-­ sectional image determines the orientation of the confocal en-face image and this image plane can reveal the exact location inside the cornea (Fig. 4). The piezo actuator can shift the confocal plane up to 600 μm and the image position and orientation can be tracked in real time enabling the clinician to locate the actual position of interest on the cornea in real time. The combined OCT-CSLM utilises OCT optics to guide CSLM in accurately locating a position on the cornea but the resolution of current commercially available OCTs is insufficient for visualising cellular or subcellular structures [31]. To improve OCT resolution, curved-field OCT [32] and micro-optical OCT [33] have recently been developed. The curvature of the cornea limits the field of view of existing high resolution imaging devices including both CSLM and OCT due to the fact that only flat field optical sections are obtained. Curved-field OCT, which can capture optical sections of arbitrary curvature, enables the field of view to increase to 1.13 mm × 1.13 mm and with a high en face imaging speed of 0.6 billion pixels/s; this ensures the images are free of eye movement artifacts [32]. To capture optical sections that match the corneal curvature, the flat mirror in the additional optical reference arm is replaced with a curved optical lens, the camera then captures all the pixels within the viewing area at the same time. Micro-optical OCT is a spectral-domain OCT that uses a superluminescent diode array, it has a spectral bandwidth of 350 nm centred at 930 nm, achieving high axial resolution of 1.3 μm in air. A preliminary study has shown it was able to produce ‘histology’ resolution in both the cross-sectional view of the cornea and en face view of the endothelial surface, with the latter providing similar cellular structural detail that is similar to CSLM and scanning electron microscopy [33].

Fig. 4  Example of Optical coherence tomography (OCT) guided confocal scanning laser microscopy (CSLM): confocal image (left) and cross-sectional OCT image (right). The bright reflection within the OCT image reveals the position of the confocal en-face image with respect to the cornea’s anterior and posterior interface. Reprinted from ‘High Resolution Imaging in Microscopy and Ophthalmology’, Chap. 12, p281, by Oliver Stachs, Rudolf F. Guthoff, 2019. Copyright permission under the terms of Creative Commons CC BY [31]

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In Vivo Confocal Laser Scanning Guided-Slit Lamp Microscopy In vivo confocal laser scanning guided-slit lamp microscopy is a method for reconstruction of three-dimensional corneal images at a cellular level and volumetric reconstruction up to around 250  ×  300  ×  400  μm3. The microscope has a piezo actuator for precise focusing in image acquisition and automated controlling of the focal plane [31, 34]. This method of reconstruction potentially enables the possibility of IVCM guided—slit lamp microscopy on a cellular level. In vivo corneal three-­ dimensional imaging on a cellular level and cornea cross-section reconstruction is limited by involuntary eye movements, which induce image distortion and image shift during acquisition. A novel contact cap element with a concave surface has been designed to reduce involuntary eye movements and also it increases the cuboid volume of three-dimensional image reconstruction (Fig. 5) [31, 34].

Multiphoton Microscopy Combining reflectance IVCM with multiphoton microscopy (MPM) can provide further structural information at a cellular level. MPM was invented by Denk et al. in 1990 [35] and it is uniquely suited in imaging living biological tissues at the molecular level to produce subcellular resolution. This technology can measure details with minimal invasion over long periods of time so it is capable of providing accurate specifications of biological processes. Similar to CSLM, it provides optical sections but with enhanced depth penetration [36]. MPM and CSLM are complementary in that they detect different cellular structures and they provide additional structural information about the cornea architecture. In MPM, near infrared excitation signals are exploited that allows deep tissue penetration and reduced photo-­ damage. Highly focused near infrared (NIR) femtosecond lasers are utilized to induce non-linear signals in the visible range. The NIR excitation reduces light scattering, and it enhances the ability to capture images from deep layers of samples. Moreover, the fluorescence emission signals are detected from different molecules and can be used to monitor cell metabolic activities. Nonlinear multiphoton excitation can induce second harmonic generation (SHG) signals that are produced by non-centrosymmetric molecular structures which is dependent on the macromolecular arrangements particularly the fibrillar collagen type I. Therefore, with the combination of SHG microscopy and reflectance confocal microscopy both the cellular and extracellular information can be visualized [37]. The epithelial cells and nuclei, the Bowman’s layer and the keratocytes are detectable with CSLM. While the autofluorescence signal shows the cytoplasm of epithelial cells; the second harmonic generation signal is used to detect collagen found mostly in the stroma of the cornea [36–38]. If only morphological information is needed, one can select the second harmonic generating wavelength mode to omit cellular autofluorescence excitation (Fig. 6) [36–38].

b

c

Fig. 5  Conventional slit lamp (a), histology (b) and cross-section after three-dimensional reconstruction (c) exemplifying the potential of the laser based slit lamp. Reprinted from ‘High Resolution Imaging in Microscopy and Ophthalmology’, Chap. 12, p 280, by Oliver Stachs, Rudolf F. Guthoff, 2019. copyright permission under the terms of Creative Commons CC BY [31]

a

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Fig. 6  Intravital four-dimensional image of the subconjunctival tissue of the LysM-eGFP knockin mouse using multiphoton microscopy. The intravital LysM-eGFP positive cells, blood vessels, conjunctival and scleral collagen fibers, are labelled in green, red, and blue colour, respectively. Reprinted from ‘Visualization of intravital immune cell dynamics after conjunctival surgery using multiphoton microscopy.’ Kojima, S., Inoue, T., Kikuta, J., Furuya, M. et al. Investigative ophthalmology & visual science, (2016). 57 (3), 1207–1212. copyright permission under the terms of Creative Commons CC BY [44]

Subbasal Nerve Plexus (SNP) Mosaicking One of the most important applications of IVCM has been to quantify morphological changes of the SNP in ocular or systemic diseases such as in patients with diabetes (see chapter “Corneal Nerves Useful for Ophthalmology But Indispensable for Neurology”) [39, 40]. The major challenges in this regard are the inhomogeneous distribution of nerve fibers across the cornea and the restricted field of view with current IVCM (0.16 mm2), which it is insufficient for proper morphometric SNP characterization. To increase the examined area of the SNP, a concept was developed to compose mosaic images from several overlapping IVCM images to expand the examined corneal area range [31, 41–43]. In this approach, a modified RCM and a new software are used to control the focal plane rapidly. In addition, in the newer design, a piezo actuator is used to move a lens inside the modified RCM, so that the focal plane is controlled without moving the TomoCap. In this technique, the focal plane oscillates around the SNP layer, and the shifts in focal planes are fast and precise enough for real time image acquisition. A cornea tissue classification (CTC) algorithm is used to distinguish different tissues and defines the optimal center position for the piezo oscillation around the SNP. By excluding images of other cell layers, CTC can significantly increase the quality of SNP mosaics [31, 44]. Preliminary results are promising for reliable SNP quantification that can be used as a biomarker for peripheral neuropathies (Fig. 7).

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Fig. 7  Large scale subbasal nerve plexus mosaicking of a healthy human subject. Reprinted from ‘High Resolution Imaging in Microscopy and Ophthalmology’, Chap. 12, p 279, by Oliver Stachs, Rudolf F. Guthoff, 2019. copyright permission under the terms of Creative Commons CC BY [31]

Conclusion Confocal microscopy has come a long way since it was first invented by Marvin Minsky in 1957. Applied research using IVCM has advanced our understanding in both normal anatomical, physiological and pathological processes of the cornea and adnexal structures. The translational research from “bench -to -bedside” of using IVCM over the last 10–15 years have seen our ability to diagnose conditions such as keratitis in real time, using subbasal nerve plexus as a surrogate biomarker for neurodegenerative diseases, and to diagnose adnexal conditions such as skin tumours. There are limitations with the technique such as the inability of reliably differentiating host cellular structures from pathogenic organisms and the restricted field of view of the images. Promising new innovations and technologies are at their ‘bench’ stage in development at present and accordingly over the next few years, the potential use of multimodal imaging platform for example, combining CSLM with OCT or MPM would further enhance our understanding of anterior segment structures at a cellular level offering the potential of real time in vivo functional histology. Acknowledgement  We wish to thank Maryam Kasiri for her assistance in providing images used in this chapter.

Disclosures  None to declare.

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Index

A Acanthamoeba keratitis (AK), 42, 43, 46, 133, 134 Anterior segment applications, 158–160 Anti-retroviral therapies, 140 Atopic keratoconjunctivitis, 134 B Bacteria keratitis (MK), 40–42 Bechet’s disease, 137 Bleb morphology, 109, 111, 112 Bowman’s layer, 16 Bowman’s membrane, 136 Bulbar conjunctiva, 91, 92 C Central neurodegenerative diseases, 140, 142 Chemotherapy-induced peripheral neuropathy, 140 Confocal fluorescence microscopy, 160–161 Confocal optics, 13 Confocal scanning laser microscopy (CSLM), 162 Congenital stromal corneal dystrophy (CSCD), 77, 81, 82 Conjunctiva, 93–96 Conjunctival melanoma, 109 Conjunctival nevus, 105, 106 Contact lens wear, 127, 129 Cornea nerves, 19, 21, 22 Corneal anatomy, 14 Corneal endothelial cell density, 153–155 Corneal epithelial neoplasia, 135 Corneal hyperalgesia, 133 Corneal innervation atopic keratoconjunctivitis, 134 Bowman’s membrane, 136

central human subbasal innervation, 128 central neurodegenerative diseases, 140, 142 contact lens wear, 127, 129 corneal collagen cross-linking, 130, 131 corneal epithelial neoplasia, 135 corneal nerve branch density (CNBD), 127 diabetic peripheral neuropathy (DPN), 135–138 dry eye disease, 131, 132 embryology, 125 external stimuli, 126 fibromyalgia, 139 glaucoma medications, 135 high-resolution, laser-scanning CCM, 127 inflammatory neuropathies, 137 keratoconus, 131 keratoplasty, 130 light-based CCM models, 127 mechano-nociceptors, 126 median corneal nerve fiber density (CNFD), 127 myelination pattern, 126 neuropathic pain, 133 ocular surface inflammation, 134 organization, 126 origin, 125 peripheral neuropathies, 139–141 polymodal nociceptors, 126 recurrent corneal erosion syndrome, 135 refractive surgery, 129, 130 stromal and subbasal nerve abnormalities, 134 in systemic disease, 135 thermal nociceptors, 127 TTR-FAP, 139 ultrastructural ex-vivo studies, 127 Corneal keratocyte density measurement, 156 Corneal thickness measurement, 154–156

© Springer-Verlag London Ltd., part of Springer Nature 2022 G. Latifi, S. Hau, In Vivo Confocal Microscopy in Eye Disease, https://doi.org/10.1007/978-1-4471-7517-9

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Index

172 Corneal transparency measurement, 156 Cytomegalovirus endotheliitis, 56 D Demodex spp., 160, 161 Dendritic cells, 19 Descemet’s membrane, 22 Diabetic peripheral neuropathy (DPN), 135–138 Dry eye disease, 93–95, 131, 132 E Endothelium, 22, 23 Endothelium-keratic precipitates, 38 Epithelial basement membrane dystrophy (EBMD), 61, 63 Epithelial-stromal TGFBI dystrophies, 64–74 Erythrocytes, 34, 35 F Fibromyalgia, 139 Friedreich’s ataxia, 140 Fuchs’ endothelial corneal dystrophy (FECD), 80–85, 87, 88 Fungal keratitis (FK), 47, 49, 50 G Gelatinous drop-like corneal dystrophy (GDLD), 64, 66, 67 Glaucoma, 109–112 Granular corneal dystrophy, type 1 (Classic) (GCD1), 74–76 H Heidelberg Retina Angiograph (HRA), 160 Heidelberg retina tomograph (HRT), 6 Helium Neon diode laser, 6 Herpes simplex keratitis, 54 Herpes zoster ophthalmicus, 54, 56, 133 Human cornea and adnexa Bowman’s layer, 16 corneal anatomy, 14 cornea nerves, 19, 21, 22 dendritic cells, 19 Descemet’s membrane, 22 endothelium, 22, 23 epithelium, 14, 16 limbus, 24

precorneal tear film, 13, 14 Rete cell diameter and density, 25 stroma, 19 I Inflammatory neuropathies, 137 International Dry Eye Workshop, 131 In vivo confocal laser scanning guided-slit lamp microscopy, 164, 165 In vivo confocal microscopy (IVCM) basic components, 2, 3 LSCM, 3, 6, 7, 9, 10 non-contact high resolution laser scanning microscopy, 10, 11 principle of confocality, 1 real-time sectional biopsies, 1 reflection, diffusion and diffraction, 1 section mode, 3 slit-scanning confocal microscope, 5 swept-field confocal microscopy (SFC), 5, 6 temporal mode, 3 three-dimensional visualization of biological tissues, 1 TSCM, 3–5 volume mode, 3 Iridocorneal-endothelial (ICE) syndrome, 156–158 K Keratitis Acanthamoeba keratitis, 42, 43, 46 acute inflammation, 29 bacterial keratitis, 40–42 corneal nerve and dendritic cellular response, 50 cytomegalovirus endotheliitis, 56 dendritic cells, 30 diagnostic accuracy, 53 endothelium-keratic precipitates, 38 erythrocytes, 34, 35 fungal keratitis, 47, 49, 50 granular leucocytes, 29 herpes simplex keratitis, 54 herpes zoster ophthalmicus, 54, 56 hyper-reflective deposits, 54 microbial keratitis, 38 microsporidia keratitis, 50 neutrophils (polymorphonuclear neutrophils), 30 stroma, 35, 38

Index viral keratitis, 54 Keratoconus, 131 Keratoplasty, 130 L Langerhans cell density, 30, 35 Laser scanning confocal microscope (LSCM), 3, 6, 7, 9, 10 Lattice corneal dystrophy, type 1 (Classic) (LCD1), 69–74 Lid margin, 112–114 Limbal stem cell deficiency, 100–102 Limbus, 24 M Macular Corneal Dystrophy (MCD), 74, 77, 78 Mainz severity score index, 140 Meesmann corneal dystrophy (MECD), 61–63, 65 Meibomian gland dysfunction (MGD), 115–116 Meibomian glands (MG), 112–114 Microbial keratitis (MK), 38 Microneuromas, 133 Micro-optical OCT, 162 Microsporidia keratitis, 50 Multiphoton microscopy (MPM), 164, 166 N Neutrophils (polymorphonuclear neutrophils), 30 Nocardia spp., 42 Non-contact high resolution laser scanning microscopy, 10, 11 Normal corneoscleral limbus, 95, 97–99 O Ocular demodicosis, 160 Ocular surface inflammation, 134 Ocular surface squamous neoplasia (OSSN), 102–104 Optical coherence tomography-guided in vivo confocal laser scanning microscopy, 162, 163

173 P Palpebral conjunctiva, 91, 94, 112–114 Peripheral neuropathies, 139–141 Pigmented lesions of conjunctiva, 105–108, 110 Platinum-based chemotherapy, 140 Posterior polymorphous corneal dystrophy (PPCD), 77–80, 83, 84 Precorneal tear film, 13, 14 Proteus mirabilis, 41 R Racial melanosis, 106 Recurrent corneal erosion syndrome, 135 Reis-Bücklers Corneal Dystrophy (RBCD), 64–66, 68, 69 Rostock Cornea Module (RCM), 6, 160 S Schnyder corneal dystrophy (SCD), 74–77, 79, 80 Slit-scanning confocal microscope, 5 SPECTRALIS OCT module, 162 Stevens-Johnson syndrome, 135 Stroma, 19, 35, 38 Stump neuromas, 133 Subbasal nerve plexus density (SBND), 21 Subbasal nerve plexus (SNP) mosaicking, 166–167 Swept-field confocal microscopy (SFC), 5, 6 T Tandem scanning confocal microscope (TSCM), 3–5 Thiel-Behnke corneal dystrophy (TBCD), 66–71 TomoCap®, 7, 9 Trachoma, 119, 120 Transthyretin familial amyloid polyneuropathy (TTR-FAP), 139 V Vernal keratoconjunctivitis (VKC), 116–119 Viral keratitis, 54 VivaScope 3000® model, 159