Confocal Scanning Laser Microscopy in Medicine

*Hasan Kiziltoprak, Dilara Ozkoyuncu, Kemal Tekin and Mustafa Koc*

## **Abstract**

Confocal Scanning Laser Microscopy (CSLM) offers high resolution morphological details and generates en-face images with excellent depth discrimination for visualizing different structures of the living human body non-invasively. There have been significant advances in technology since the CSLM was first defined. It has been used commonly, especially in ophthalmological area, in order to diagnose and give direction for the treatment of corneal pathologies. Ocular surface, corneal subbasal nerve plexus, filtering blebs of glaucoma surgery were also investigated widely by CSLM. With the improvements in CSLM technology over time, it is widely used in other fields than ophthalmology. The combined use of CSLM with the slit lamp biomicroscopy and optical coherence tomography will also lead to significant advances in the diagnosis and treatment of more diseases in the future.

**Keywords:** confocal scanning laser microscopy, laser imaging, medicine, ophthalmology, subbasal nerve plexus

### **1. Introduction**

Confocal Scanning Laser Microscopy (CSLM) is a non-invasive imaging method for visualizing different structures of the living human body [1]. CSLM provides morphological details with high resolution and generates en-face images with excellent depth discrimination [1, 2]. CSLM is compatible with three-dimensional (3D) live imaging provided by sequential acquisition of tomograms along the depth direction [3]. There is a broad range of experimental and clinical applications on corneal analysis with CSLM. The imaging procedure may assess stromal changes in keratoconus patients [4], experimental full-thickness corneal 3D imaging [5], the quantification of morphological features of epithelial cell layers, and the subbasal nerve plexus [6–10] that has become very popular in recent years.

Since its commercialization in the late 1980's, CSLM has become one of the most applied fluorescence microscopy techniques for 3D-dimensional structural studies of biological cells and tissues [3]. Recent technological breakthroughs have led to the development of CSLM, and it has reached the current level of high resolution that can be used in many areas today. In recent years, there has been a vast increase in researchers using CSLM in many fields of medicine, especially in ophthalmology.

In this chapter, we have attempted to summarize the principles of CSLM and the application in ophthalmological and non-ophthalmological areas of medicine. Finally, it was discussed how it could give an essential direction to medical development in the future.

## **2. Principles of confocal scanning laser microscopy**

Objects that share a conjugate focal plane are defined as with the term "confocal". In microscopic area, that means whereby the in-focus image plane can be seen from adjacent axial planes in case of coincidence between the focal plane of the objective lens and the detector. CSLM uses a diffraction-limited spot of light to illuminate the sample and an aperture in the collection light path at conjugate focus.

The first steps of CSLM were designed by Marvin Minsky in 1955 at his early education times and patented in 1957 [11]. However, there has been no significant improvement in CSLM technology over a long time as the required technologies which were either underdeveloped or non-existent at that time. Moreover, CSLM technology was new, and there was no pressing need for it by the scientific community. Therefore, the commercialization of CSLM occurred in the late 1980s. Petran *and colleagues* introduced the first tandem scanning confocal microscope in 1968 [12]. The Nipkow disk was used as the basis of a new sectioning microscope, and the field of view was achieved by simultaneously scanning multiple points on a stationary specimen using a rotating Nipkow disc. Even this allows for real-time imaging; it has the disadvantages of a very low light throughput and low image quality. In 1969, Svishchev produced the slit scanning confocal microscope based on an oscillating double-sided-mirror [13]. The Svishchev confocal microscope used two confocal adjustable slits. It was used to observe living neural tissue using an oscillating two-sided mirror for simultaneous scanning and de-scanning of the sample [13]. This design was subsequently further modified to enable real-time scanning. The slit scanning microscope was superior in tandem design in terms of shorter examining time and the requirement of low light intensity. The modern, and first commerciallysuccessful CSLM was developed by Brad Amos and John White at the University of Cambridge. With this new technology, precise 3D visualization of ocular microstructures was achieved [14]. Modern digital image processing technology enables quantitative data to be stored noninvasively, rapidly, and with a low level of illumination.

All confocal microscopes share the same basic principle in their designs that enable optical sectioning of a relatively thick light scattering object. A directed light is crossed through an aperture and focused with the help of an objective lens onto a small area of the specimen. At each tissue location, light is reflected or backscattered and travels the same way back. It is separated from the incident beam by a beam splitter. The reflected light from that specimen was then directed onto a second aperture by a second objective lens. By this method, out-of-focus light is strongly reduced, improving image resolution and contrast considerably. The ability of this system to distinguish between light out of the focal plane yields images of higher lateral and axial resolution compared with light microscopy. As the illumination and detection paths are at the same focal plane, the term confocal is used [2, 3, 15].

The precision of CSLM is mainly based on the concept of the confocality of the investigated object with the light source and the detector plane. Such a system was limited because of its small field of view. By the time, a larger field of view obtained either by moving the specimen whereas the microscope remains stationary, or by moving the confocal system over a stationary specimen. Modern CSLM devices use the second technique. The microscope's temporal resolution determined by the speed at which a single image of the field is acquired. Poor temporal resolution is important as increased motion artifacts inevitable because of pulse, respiration, and eye movement when examining living human subjects [16].

*Confocal Scanning Laser Microscopy in Medicine DOI: http://dx.doi.org/10.5772/intechopen.96771*

## **3. Ophthalmological applications**

Biological tissues usually slice with 2–5 μm thickness were cut, stained with various chemicals and examined by light transmission at high magnification as part of conventional microscopic evaluation. In ophthalmology, in vivo examination of semitransparent tissues is performed by slit lamp biomicroscopy thanks to the inventor Allvar Gullstrand [17]. With slit lamp biomicroscopy, optically cut planes are orientated sagittally and observed by a binocular microscope with the magnification up to 50-fold. However, single cell resolution is still impossible at this level of magnification. Nonetheless, a large number of corneal diseases could be diagnosed and followed up by slit lamp microscopy easily in many cases.

Since the corneal cells could not be evaluated by slit lamp biomicroscopy, CSLM has quite satisfactory use in this regard. CSLM provides the imaging of biological structures with up to a magnification of 800-fold that renders possible single cell evaluation (**Figure 1**). Secondly, the optical section is perpendicular to the slit lamp image as its direction is parallel to the corneal surface [2].

CSLM has been used in various ophthalmological conditions for corneal diagnostics. Corneal nerve degeneration and regeneration, assessment of corneal grafting and refractive surgery, contact lenses, diabetes mellitus, keratoconus, ocular surface disease, and normal anatomy are investigated by CSLM with a considerably high number of studies.

#### **Figure 1.**

*Normal endothelial cells. Endothelial pigment appeared as hyper-reflective spots in some frames of the central cornea, while peripheral endothelium appeared normal. (courtesy by Mustafa Kosker).*

### **3.1 Corneal fungal infections**

Fungal keratitis can be a significant problem in especially developing countries as its slow course and treatment resistance. The clinical findings are nonspecific, and there are difficulties in diagnosis due to its delayed growth even in specific cultures. Despite their infrequent nature, in industrialized countries proper management of fungal keratitis due to prolonged diagnostic procedures ends up with devastating results [18–21]. Moreover, after initiation of antimicrobial therapy, it is still difficult to assess therapeutic response of some ulcers based upon clinical appearances by slit microscopy alone.

CSLM has been reported to be useful in diagnosis and follow up of patients in fungal keratitis [18–21]. CSLM has provided instant diagnosis without long lasting preparations of sample cultures. Also, CSLM demonstrates activated keratocytes and directly proven fungi in the corneal ulcer in differential diagnosis of nonfungal keratitis [18–21]. Although it is a rapid and noninvasive method of diagnosis of routine as well as deep-seated corneal infiltrates, its use as a primary diagnostic modality may not be possible due to its cost and limited accessibility.

#### **3.2 Keratoconus**

Keratoconus is an ectatic corneal disorder characterized by progressive thinning of cornea, which leads to an apical corneal protrusion, irregular astigmatism, superficial scar formation, and progressive decreased vision [22]. The diagnosis of keratoconus is now easy with the development of corneal topography systems. However, CSLM is another approach for the diagnosis and follow up of keratoconus. Quantitative and qualitative structural alterations were seen in all corneal layers in eyes with keratoconus, and the alterations were more prominent as the severity of disease increased [22–25].

In the keratoconus, main pathologic changes evaluated by CSLM included elongated, exfoliating superficial epithelial cells; brightly reflective material deposition within the basal epithelial cells; prominent, thickened subbasal nerves; structural changes in subbasal nerve fibers; pronounced reflectivity and irregular arrangement of stromal keratocytes; structurally abnormal anterior stromal keratocyte nuclei; folds in the anterior, mid, and posterior stroma; folds in Descemet's membrane; pleomorphism and enlargement of endothelial cells; and endothelial guttata [22]. Moreover, keratocyte density is significantly lower in subjects with keratoconus and correlated with disease severity [26]. CSLM's noninvasive nature allows the opportunity to study early microstructural changes in the keratoconic cornea and to understand its pathophysiology (**Figure 2**).

#### **3.3 Subbasal nerve plexus**

There has been an increased interest in using CSLM, that non-invasive technique as an objective diagnostic tool for peripheral neuropathies due to the capability to acquire high-resolution in vivo images of the densely innervated human cornea [27–29]. Also, the evaluation of the subbasal nerve plexus of the cornea has led to a significant rise in CSLM use to help clinicians diagnose various diseases (**Figure 3**). Morphological alterations of the corneal subbasal nerve plexus may correlate with the progression of neuropathic diseases and even predict future-incident neuropathy.

Corneal nerves are affected in cases with limbal stem cell deficiency, infection, corneal surgery, keratoconus, diabetes mellitus, lysosomal storage diseases, and keratitis [2]. Moreover, the evaluation of systemic diseases could also be possible by

#### **Figure 2.**

*Keratoconus patient that underwent corneal cross-linking treatment. Hyperreflective cytoplasm, extracellular spaces, and anterior stromal edema give a honeycomb appearance and can be observed until the 3rd month. Although almost all of the keratocytes undergo apoptosis, sporadic keratocytes are observed (arrows). Demarcation line in confocal microscopy: The long, thin, hyperreflective, needle-like structures in the middle stroma and the transition area from the wide hyperreflective stromal bands to normal keratocytes appear as the demarcation line. These hyperreflective bands can be seen in the first six months. While these changes occur in anterior stromas, there is no significant change in the keratocyte density and endothelium count behind the demarcation line. (courtesy by Mustafa Kosker).*

observing corneal subbasal nerve plexus. In particular, the use of CSLM in diabetic patients, who are at risk of small fiber neuropathy leading to limb amputation, may be helpful in the early detection of small fiber neuropathy, and some preventions can be taken to slow down or eliminate the incident in both industrialized and developing countries [27].

The optical slicing of CSLM is parallel to the surface of the cornea. Therefore, it provides an ideal condition to display and to quantify structures of the subbasal nerve plexus which is located between Bowman membrane and the basal lamina of the corneal epithelial cells. It has been proposed that the imaging of the subbasal nerve plexus will be possible to find new treatment strategies and more effective prevention of serious disease.

### **3.4 Keratoplasty and refractive surgery**

Keratoplasty is still a common method in the treatment of corneal pathologies. It has been possible because of the increase in knowledge about corneal anatomy, improvement in instruments, and advancements in technology. Today, development of modern technologies, especially in microscopy, has reached a very good position in terms of success in keratoplasty. With the widespread use of CSLM, it was possible to image a graft's microstructure as well as calculation of endothelial cell density. CSLM detected some changes such as declining of subepithelial plexus nerves, keratocytes, and endothelial cells in the central clear graft following keratoplasty [30–32]. The graft is in a stress condition which affects the normal physiological function of keratocytes and leading to the graft failure [30–32]. Activated immune

#### **Figure 3.**

*Central mosaic dystrophy in a case with Megalocornea: Central cornea: The epithelium appeared normal morphologically. (white arrow): The subepithelial nerve fibers seemed to be thickened and appeared more prominent. In the stroma, starting just below Bowman's membrane, polygonal, moderately reflective areas of opacification separated by diagonal hyporeflective striations were observed. Peripheral cornea: The epithelium, bowman membrane, and anterior stroma appeared normal morphologically. (courtesy by Mustafa Kosker).*

cells could also be detected in some of the clear grafts, which clearly showed that the subclinical stress of immune reaction took part in the chronic injury of the clear graft failure without any rejection episode. Therefore, morphologic alterations of corneal grafts after keratoplasty detected by CSLM enables us to be aware of corneal graft rejection and to intervene early in a possible rejection.

Refractive surgical procedures are being used frequently in the light of the increasing incidence of myopia and technological developments in refractive surgical devices. It is possible to assess the wound healing response in the living human cornea that may help in unraveling the mechanisms of corneal haze and refractive regression observed following refractive surgery. Studies are carried out in the field of CSLM in order to increase the success rate of this surgery, to detect and manage possible complications at early period [33, 34].

## **3.5 Contact lens**

Contact lenses are used today for many different purposes. The effects of contact lenses on the eyes were evaluated with CSLM. Contact lens biocompatibility, its effects on cornea, limbal stem cells or conjunctiva, early diagnosis of devastating infections such as acanthamoeba keratitis are investigated by CSLM [35–38].

Acanthamoeba keratitis is a serious, sight-threatening corneal infection that can cause significant corneal damage and vision loss. Its incidence is on the rise because of the increasing usage of contact lenses. The diagnose of acanthamoeba keratitis is essential as its devastating nature, and CSLM can be used as an adjunct modality to the clinical data for diagnosing acanthamoeba keratitis [38].

## **3.6 Ocular surface diseases**

CSLM has been widely used to visualize the morphology of the cornea and conjunctiva and detect changes of the ocular surface in pathological conditions such as infectious, metabolic, and trauma. The micromorphology of the corneal epithelium and stroma can be changed by infections, metabolic diseases, and genetic disorders. The progression of diseases can be observed and monitorized via CSLM [39, 40]. Chemical burns, which may result in irreversible damage to the ocular surface, constitute a large part of ocular trauma. CSLM can provide images of the goblet cells on the corneal surface which is a hallmark of limbal stem cell deficiency. The application of CSLM on chemical burns also allows for evaluation of the limbal structures and ocular surface changes after reconstructive ocular surgery [39].

Dry eye disease is another area of research for CSLM. CSLM is an effective non-invasive tool for evaluation of phenotypic alterations of the conjunctival epithelium. The use of CSLM is also crucial in the diagnosis of meibomian gland disfunction [41, 42]. It demonstrated the importance of meibomian glands for the healthy ocular surface and was also used for the effective treatment modalities of dry eye disease.

## **3.7 Glaucoma surgery**

The formation of a filtering bleb, which attains by postoperative wound healing process, is a key factor in surgical procedures for glaucoma. Clinical and histological evaluation of these blebs has been investigated by CSLM to visualize functioning or nonfunctioning blebs at the cellular level [43, 44]. The CSLM images of filtering blebs have good consistency with the findings from previous studies. The implantation of CSLM in glaucoma surgery will be enlightened the histological processes responsible for filtration or failure.

## **4. Non-ophthalmological applications**

CSLM is mainly improved for ocular and ocular adnexal surface structures. However, it can be suited for analyzing any surface of the human body in case of convenient for the device to reach at. On the other hand, the application of CSLM in non-transparent tissue is limited due to light-tissue interaction including reflection and refraction, absorption, and scattering of photons. In human tissues, water molecules and macromolecules such as proteins and chromophores are the main

factors that affect penetration depths of the device. Therefore, CSLM images with cellular resolution can only be obtained at depths up to 300 μm [2].

CSLM has been used to evaluate the oral and pharyngeal mucosal membranes and showed promising results in dentistry applications [45–47]. Studies describing the cellular morphology and pathological alterations of the oral cavity, cervix, and esophagus also showed promising results [48, 49]. Cell morphology, tissue architecture of the epithelium, and a number of pathological skin conditions were investigated [50–52]. The amelanotic epithelial tissue of the gastrointestinal tract, lip and tongue, and the oropharynx demonstrated with CSLM [52]. CSLM identified intraepidermal blisters and acantholytic cells in pemphigus vulgaris [53]. Sinonasal inverted papilloma could also be detected noninvasively by CSLM [54]. The combination of CSLM with endoscopy is helpful in detection of schistosomiasis [55].

## **5. Future developments**

Presently, CSLM has been a potential source of many researches and has received a high level of scientific and clinical attention in ophthalmology. Since CSLM can show high resolution images of various cellular structures within the living cornea non-invasively, it is mainly used for diagnostic purposes. However, ongoing research on this area is under development in order to improve their diagnostic potential and the usability of this technology.

#### **5.1 Multiphoton microscopy**

Corneal cell differentiation can be evaluated under various conditions by CSLM. However, the detailed information will not be satisfactory. Multiphoton microscopy, which uses a non-linear interaction mechanism, can be more useful for evaluation of cellular morphology [2]. According to multiphoton absorption, the background signal is strongly suppressed and leads to an increased penetration depth for this technique [56]. Multiphoton microscopy can be a superior alternative to confocal microscopy due to its deeper tissue penetration, efficient light detection, and reduced photobleaching. Multiphoton microscopy is reported as a promising technique for non-invasive detection of diabetic neuropathy [56, 57]. Information derived from this technology may help to develop new drugs for the treatment of diabetic neuropathy.

#### **5.2 Slit lamp microscopy on a cellular level using CSLM**

Slit lamp microscopy is a revolution for ophthalmology. Despite whole anterior segment structures can be evaluated clinically, information on cellular level cannot be attained. Recently, an in vivo method for 3D volumetric reconstruction of the cornea on a cellular level with volume sizes up to around 250 × 300 × 400 μm3 has been reported. [58]. A piezo actuator is implanted to the microscope objective for image acquisition. Moreover, the automated, closed-loop control of the focal plane enables fast and precise focus positioning. Additionally, a novel contact cap with a concave surface has been presented that reduced eye movements by up to 87%. Therefore, the cuboid volume of the generated 3D reconstruction significantly increased. The possibility to generate oblique sections using isotropic volume stacks opened the window to slit lamp microscopy on a cellular level. The diagnosis can be made at cellular level during examination, and the treatment of diseases can be planned more effectively with the widespread useage of this technology,

## **5.3 Optical coherence tomography guided CSLM**

CSLM is valuable for studying corneal morphology at cellular level non-invasively. However, certain drawbacks such as small field of view limit its usability. The exact CSLM image location and orientation inside the cornea are difficult to locate. Therefore, a combination with optical coherence tomography (OCT) was adapted to the conventional CSLM in order to overcome this limitation.

The combination of both technologies renders it possible to track image position and orientation in real-time [2, 59]. Real-time evaluation of CSLM image plane position and its orientation within the cornea through the OCT section provides an enhanced location-based diagnosis. It is now possible to specify the angle between the corneal surface and the image. Further studies will be necessary for optimizing the system design and OCT scan patterns. In the future, the combination of these technologies will be used widely for diagnostic purposes and will give direction to the treatment.

## **6. Conclusion**

CSLM allows ocular structures and ocular surfaces to be assessed at cellular level. 2D tessellation or 3D reconstruction of the ophthalmic as well as nonophthalmic tissue evaluation is possible. This technology is still promising, and close and direct collaboration between clinical science and basic science as well as industry partners can help it to reach its potential. CSLM's combination with several other technologies will also affect our understanding of diseases, diagnosis, and treatment options in the near future.

## **Acknowledgements**

The authors thank to Assoc Prof. Dr. Mustafa Kosker from Ophthalmology Clinic of Health Sciences University Dışkapı Yıldırım Beyazıt Training and Researsch Hospital for sharing his valuable work in the field of CSLM with them.

## **Funding**

No funding was received for this research.

## **Conflict of interest**

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

## **Author details**

Hasan Kiziltoprak1 \*, Dilara Ozkoyuncu<sup>2</sup> , Kemal Tekin3 and Mustafa Koc4

1 FEBO, FICO, Bingol Maternity and Child Diseases Hospital, Ophthalmology Department, Bingol, Turkey

2 FEBO, Bilecik Training and Research Hospital, Ophthalmology Department, Bilecik, Turkey

3 Ophthalmology Department, Hatay Mustafa Kemal University, Hatay, Turkey

4 Kayseri Mayagoz Hospital, Kayseri, Turkey

\*Address all correspondence to: hsnkzltprk21@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Confocal Scanning Laser Microscopy in Medicine DOI: http://dx.doi.org/10.5772/intechopen.96771*

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## **Chapter 4**

## Cornea Confocal Microscopy: Utilities and Perspectives

*Eduardo Rojas Alvarez*

## **Abstract**

The cornea is the ocular refractive medium with the greatest refractive power of the eye. The study of it is of vital importance for the diagnosis and follow-up of ophthalmological diseases with the aim of achieving high standards of visual acuity in our patients. Confocal microscopy of the cornea allows in-depth study of it, quickly, safely, painlessly, obtaining high-resolution images of the corneal sublayers. This chapter summarizes the procedure for performing corneal confocal microscopy, the normal characteristics of the tissue with real images of our patients, as well as a brief explanation of the main applications of this technology in the study of corneal dystrophies (keratoconus), in refractive surgery, corneal transplantation, infectious keratitis, glaucoma filtration bulla, among other topics.

**Keywords:** microscopía confocal, cornea, epitelio, estroma, endotelio

## **1. Introduction**

The sense of vision is one of the most precious, the study of the visual phenomenon has always been in the sights of researchers at all times, with the common goal of finding the necessary tools to diagnose and treat ophthalmological disorders in a timely manner and therefore, achieve better results of visual acuity in patients.

Within the eyeball, one of the most important anatomical elements in terms of visual contribution is the cornea. This tissue is part of the outermost layer of the eyeball, it is made up of 5 layers, has a thickness in the central part of 0.5 mm and is thicker in the peripheral part. It is avascular and confers most of the ocular diopter power.In ophthalmology consultations, corneal diseases are a frequent reason for consultation, since they are the cause of decreased visual acuity. There are different diagnostic methods used by the ophthalmologist to study the cornea, such as: anterior biomicroscope, corneal topography, fluorescein staining, endothelial microscopy, keratometry, among others.

The advent of confocal microscopy has made it possible to study the corneal sublayers in greater depth, in vivo, without discomfort and with rapid obtaining of high resolution images. The principle of confocality has been very useful for the study of corneal dystrophies, patients with corneal refractive surgery, corneal infections, contact lens users, corneal transplantation, among other applications. All these results have positioned confocal microscopy as a method of great diagnostic importance in the study of the human cornea, aspects that we will summarize in the current chapter.

## **2. Development**

### **2.1 General principles of cornea confocal microscopy**

Until recently, the diagnosis of diseases of the cornea and the ocular surface has been based on the traditional anterior biomicroscopy. The exponential evolution of technology that has occurred in the last two decades has been led by the introduction of new instruments such as corneal topography, ultrasonic biomicroscopy and optical coherence tomography, among others, for the analysis of the ocular anterior segment [1–3].

These techniques offer details of the corneal curvature or macroscopic sections for the examination of structures. On the other hand, the microscopic morphology of the ocular surface was only performed by ex vivo histology, which presents limitations such as tissue degeneration, the presence of artifacts and the impossibility of evaluating the processes of disease.

Confocal microscopy is a non-invasive method for the study of microscopic images in living tissues, which has been used for the investigation of corneal microstructure since the beginning of the 1990s [4–6]. The study of images has evolved from experimental levels in laboratory research, to applications in healthy and sick patients [7, 8].

The confocal microscope for the study of cells of the nervous system in vivo, original from 1955, it was developed by Minsky in 1988. This allowed optical theory to be more formally developed and extended in the years of that decade (Wilson and Sheppard, 1984) and in the following decade (Hill; Masters and Thaer, 1994) [9, 10]. The basic principle of confocal microscopy is that an isolated point of tissue can be illuminated by a beam of light and simultaneously captured by a camera in the same plane. This produces a high resolution image [7, 10–13].

Currently, there are several types of confocal microscopes, for example: the Confoscan P4 (Tomey, United States), the Confoscan 4 (Nidek, Japan) and the confocal corneal laser microscope (Heidelberg Retina Tomograph II Rostock Corneal Module: HRTII) (Heidelberg, Germany), among others [3, 14, 15].

All confocal microscopes have the same basic principles of operation.8, 10 Light passes through an aperture and is focused on an objective lens in a small area. Light is reflected from this area and passes through a second objective lens. This light is focused on a second aperture, the out-of-focus light is eliminated. Illumination and detection are in the same focal plane, therefore the term confocal is used [10, 16–19].

The system has the ability to discriminate high-resolution lateral and axial images of light that is not in the focal plane, compared to light microscopes. Clearly as a system it is limited by a small field view. Image quality generally depends on two factors: contrast and resolution. It also depends on the numerical aperture of the objective lens, illumination levels, and the reflectivity of the studied structures and the wavelength of the illumination source [8, 14] (**Figures 1** and **2**).

#### **2.2 The normal cornea by confocal microscopy**

The superficial epithelium of the cornea is observed as hexagonal cells with bright edges with a defined nucleus and homogeneous cytoplasm [20–22]. The cells have a characteristic polygonal shape, almost hexagonal, which are characterized by a highly reflective cytoplasm since they are in a high continuous flaking process, with a shiny core and space perinuclear dark clearly visible. The superficial epithelium is five microns thick [11, 22–26] (**Figure 3**).

Intermediate stratum cells are characterized by bright edges and dark cytoplasm. The nucleus can be distinguished with great difficulty [27–29]. The average density

*Cornea Confocal Microscopy: Utilities and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.96272*

#### **Figure 1.**

*NIDEK ConfoScan 4 confocal microscope.*

#### **Figure 2.**

*Full focus quantitative confocal microscopy (CMTF), where the selected image is seen in the upper right: Apical epithelium one year after LASIK. The top left shows the parameters of depth, distance, intensity level, pressure of the Z ring, among others.*

is approximately 5000 cells/mm2 in the central cornea and 5,500 cells/mm2 in the periphery. The cells of the middle stratum have the lowest reflectivity in the entire corneal epithelium [12, 22, 30, 31].

The basal cells are located immediately above Bowman's membrane. They present bright cell borders in which the nucleus is not visible [7, 12, 23, 32–34]. Comparison between cells reveals inhomogeneous reflectivity of the cytoplasm. Like the cells of the intermediate stratum, basal cells show only minimal variation in their shape and size. In terms of cell density in normal subjects, the ratio

#### **Figure 3.**

*Apical corneal epithelium: Layer of polygonal cells with defined edges, with a bright nucleus that stands out from the homogeneous cytoplasm. Corresponds to patient 6 months after PRK.*

of surface, intermediate, and basal cells is 1: 5: 10. Basal cells have 10-15 μm in diameter and form a regular mosaic with dark cell bodies and brightness at the cell edges [14, 35] (**Figure 4**).

Langerhans cells appear as bright corpuscular particles with dendritic cell morphology and a diameter of 15 mm. Their distribution is in the form of a gradient from low numbers in the center to high cell densities in the periphery of the cornea [30, 36, 37].

The subbasal nerves are located between Bowman's membrane and the basal epithelium [38–40]. They appear as linear structures with homogeneous reflectivity, a dichotomous Y-shaped appearance, and fine H-shaped interconnecting fibers [7, 40, 41]. The membrane of Bowman is not visible by microscopy confocal. The subepithelial plexus is located between Bowman's membrane and the anterior stroma. This plexus has a diffuse, patchy distribution, apparently limited to the midperipheral cornea and probably absent in the central cornea [40, 42–47] (**Figure 5**).

#### **Figure 4.**

*Basal corneal epithelium: Layer of cells with darker homogeneous cytoplasm lacking a nucleus and defined borders. Corresponds to patient per year of PRK.*

#### **Figure 5.**

*Subbasal nerve plexus: Nerve fibers contrasting against the dark background, thin, bright, distributed in a parallel or oblique fashion with various interconnecting bifurcations. Corresponds to patient at LASIK year.*

The stroma is seen with images of keratocyte nuclei. The cell body, keratocyte processes, and stromal collagen are not usually visible on the normal cornea. Keratocytes in the anterior stroma are well-defined images with brightness, oval, objects with varied orientation that contrast with a dark background [46, 48–51]. In the middle stroma, keratocytes have a more regular oval shape. Keratocytes in the posterior stroma appear more elongated than those in the anterior layers [22, 45, 51, 52] (**Figures 6** and **7**).

Stromal nerves are located in the anterior and middle stroma, but they cannot be visualized in the posterior stroma: they appear as linear, thin, reflective structures, in various orientations, with a dichotomous pattern; internal details of the nerves are not observed [53–58].

#### **Figure 6.**

*Corneal stroma: Defined by the presence of bright oval bodies (keratocytes) that contrast against the dark background. Corresponds to patient 3 months after LASIK.*

#### **Figure 7.**

*Surgical interface: Presence of bright pinpoint bodies that stand out against the dark background. Corresponds to patient 1 month after LASIK.*

Descemet's membrane is not visible. Endothelial cells appear as a regular hexagonal cell line exhibiting bright cell bodies with darker edges. [8, 37, 52, 59–62] (**Figure 8**).

Taking as a reference the characteristics of the normal cornea, some variables have been described after corneal refractive surgery, through confocal microscopy, such as: the thickness of the epithelium [27] and the corneal flap [16], the keratocyte cell density in different strata [50], the characteristics of corneal nerves [41], endothelial cell density, pleomorphism, polymegatism [60], corneal haze thickness [29], among other variables (**Figures 9** and **10**).

#### **2.3 Applications of confocal microscopy of the cornea**

#### *2.3.1 Evaluation of cross linking treatment*

Confocal microscopy is a diagnostic means that allows the monitoring of histological modifications that occur due to the effect of corneal crosslinking. It allows to show the recovery of the tissue in the follow-up period of the treated cases.

Among the findings, a rarefaction of keratocytes accompanied by stromal edema in the first month can be observed in the anterior-middle stroma; This can take the appearance of a trabecular network, where small elongated keratocyte nuclei can be detected corresponding to masked necrotic keratocytes and apoptotic bodies, respectively. Initial restocking generally occurs within 3 months at this depth and they regenerate almost completely within 6 months [63].

#### *2.3.2 Corneal transplant*

In confocal microscopy, we observed, in most transplanted corneas, loss of continuity of corneal nerves, absence of nerve fibers, and reduction and activation of keratocytes. When confocal microscopy findings were related to the transparency status of the graft, the absence of nerve fibers was observed in all cases of non-transparent cornea and in the vast majority of transparent corneas [64].

#### **Figure 8.**

*Corneal endothelium: Hexagonal cells with defined edges, anucleated, with homogeneous cytoplasm. Corresponds to patient per year of PRK.*

#### **Figure 9.**

*Corneal haze, the keratocyte limits cannot be defined, with greater brightness than the rest of the stromal images. Corresponds to patient 3 months after PRK.*

### *2.3.3 Corneal dystrophies*

Sometimes it is difficult to make the differential diagnosis between different types of corneal dystrophies using only slit lamp biomicroscopy, hence the importance of confocal microscopy in the study of dystrophies.

For example, in a patient with a diagnosis of basement membrane dystrophy, reduplication of the basement membrane is observed, in Meesmann's dystrophies hyporeflectic areas in the basal layer of the epithelium, mostly circular, oval or drop-shaped, with a range of 48 and 145 μm in diameter [65].

In Thiel-Behnke dystrophy, deposits are observed in the basal layer of the corneal epithelium of homogeneous reflectivity with curved filaments accompanied

#### **Figure 10.**

*Pachymetry measurement. Pachymetry is the value illustrated as depth in the upper left of the CMTF curve image. The example is 6 months after LASIK.*

by dark shadows. Meanwhile, in corneas with Reis-Bücklers dystrophy, in the same layer, deposits with extremely high reflectivity are observed from small granular materials [65].

In granular dystrophy by confocal microscopy, the superficial epithelium is normal in appearance, while in the basal epithelium, highly reflective deposits are observed without defined borders. High reflectivity deposits are also observed in the superficial stroma. At Bowman's membrane level, sub-basal nerves with a raised fundus can be seen [65].

In keratoconus, the epithelium shows the following characteristics: elongated, spindle-shaped surface cells, larger and irregularly spaced nuclei of wing cells, and flattened basal cells. Images obtained using confocal microscopy reveal the disruption of Bowman's layer and the occasional presence of epithelial cells and stromal keratocytes [65].

#### *2.3.4 Corneal refractive surgery*

The confocal microscope has been widely used in the study of patients after refractive procedures. Modifications have been described at the level of different sublayers of the cornea, showing high reliability and reproducibility for these purposes [66–69].

Confocal microscopy allows the visualization of the stromal flap in LASIK, the analysis of its thickness and regularity, the density of corneal cells by stromal sublayers, the exact obtaining of the residual corneal bed, the study of the modifications of the subbasal nervous plexus and the nerves, stromal cells, as well as their recovery after this surgery. It allows the study of corneal haze in PRK, its evolution and response to treatment [66–69] (**Figures 11**–**19**).

*Cornea Confocal Microscopy: Utilities and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.96272*

#### **Figure 11.**

*Measurement of epithelial thickness. The most anterior apical corneal epithelium image is selected by CMTF curve (upper image) and the cursor is moved to the last basal corneal epithelium image. (lower image) epithelial thickness is the value illustrated as distance (*μ*m) = 41 in the upper left of the lower image. The example is 6 months after LASIK.*

#### *2.3.5 Infectious keratitis*

Confocal microscopy is emerging as an important technique in the early diagnosis of various corneal entities. It is capable of providing magnifications close to 200x-500x, providing images with high detail and contrast levels, even in opacified corneas. Its non-invasive nature makes it an especially useful technique in the diagnosis of infectious keratitis caused by Acanthamoeba. In addition, it allows repeated examinations, which helps the diagnosis, monitoring and taking therapeutic attitudes. Typical lesions correspond to amoebic cyst or trophozoite (hyperreflective lesions surrounded by a double wall and a peripheral halo), and radial keratoneuritis (linear hyperreflective thickening). Therefore, when faced with a patient who develops nonspecific manifestations of infectious keratitis, the possibility of performing this test should not be forgotten, in conjunction with histological studies, since it gives us the possibility of an early diagnosis and a better prognosis [70].

#### *2.3.6 Patients with retinal surgeries*

We can inspect corneal morphology at the cellular level in patients with emulsified silicone oil in the anterior chamber, which contributes to detecting

#### **Figure 12.**

*Flap thickness measurement. The first apical corneal epithelium image (upper image) is selected on the CMTF curve and the cursor is moved to the first surgical interface image (lower image). The thickness of the flap is the value illustrated as distance (*μ*m) = 147 in the upper left of the lower image. The example is 6 months after LASIK.*

early histological changes, both morphological and morphometric that guide us towards a behavior to avoid subsequent tissue damage.

Images obtained through confocal microscopy show different degrees of polymegatism and pleomorphism, hyperreflectivity determined by deposits of emulsified silicone oil on the corneal endothelium that are seen as bright or elongated stippling, activation of keratocytes in the stroma with loss of the matrix extracellular by contact with oil, multidots lesions and in some cases folds of the descemet [71].

#### *2.3.7 Diabetic patients*

Diabetes mellitus is the most common endocrine disorder in ophthalmic practice, and disorders of the anterior segment are less frequently described than those of the retina, although they are present in many patients. This disease affects the biomechanics of the corneal epithelium and endothelium, causes a significant effect on the morphology, metabolism and clinical and physiological aspects of the cornea. The metabolic disorders typical of diabetics constitute an important factor in the appearance of diabetic neuropathy and other subsequent pathologies.

*Cornea Confocal Microscopy: Utilities and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.96272*

#### **Figure 13.**

*Measurement of residual stromal bed (3 months after LASIK). Using the CMTF curve, the last surgical interface image (upper image) is selected and the cursor is moved to the last image of corneal endothelium (lower image). The residual stromal bed thickness is the value illustrated as distance (*μ*m) = 439.0 in the upper left of the lower image. The example is 3 months after LASIK.*

In confocal microscopy, a decrease in the density of the sub-basal nerve plexus can be observed with an increase in its thickness and tortuosity, a decrease in the density of the nerve fibers of the sub-basal nerve plexus, as well as the association of certain changes morphological of these as: fewer branches, shorter lengths, as well as increased tortuosity and presence of edema.

Greater total corneal thickness and layers are observed, the basement membrane is visible in diabetic patients. There is a decrease in the sub-basal nervous plexus with vertical disposition and an increase in the tortuosity of the nerve fibers [72].

#### *2.3.8 Filtering bull in glaucoma*

The confocal scanning laser microscope with the Rostock corneal module is a revolutionary element in the monitoring of glaucoma operated patients. The presence of encapsulated stromal cysts surrounded by a hyperreflective line that defines them and separates them from the rest of the structures (capsule) can be evaluated. Besides the visualization of thick conjunctival vessels that describe curves in their course (tortuous); On the other hand, the size of the filtering bullet is evaluable, through the measurement of the dimensions of the bullet in its largest diameter [73].

#### **Figure 14.**

*Calculation of the cell density of the apical epithelium. After selecting the image and the ROI area (0.0500 mm2 ), the cells are marked in the upper left image and the density value is obtained through the software, in this case 937.6 cells/mm2 . The example corresponds to 6 months after PRK).*

## **2.4 Method of use of the corneal confocal microscope. Calculations and images obtained**

The NIDEK ConfoScan four confocal microscope is shown for obtaining and studying in vivo images of corneal tissue. The Z ring must be attached for fixation of the eyeball with the 40x lens. It is programmed in automatic scanning mode, with central fixation, image acquisition speed of 25 images per second, 500x magnification, 0.6 μm/pixel lateral resolution, with 350 images per scan, 1.98 mm working distance.

Anesthetic eye drops and subsequently Viscotears (gel) are instilled as a coupling medium between the cornea and the Z ring. The lens is advanced until the ring is contacted with the coupling substance. The objective lens is aligned with the center of the cornea until the first images of corneal epithelium are observed. The digital images obtained are captured automatically and recorded on a computer for later analysis. Before and after each examination, the objective lens is cleaned with isopropyl alcohol.

Each image obtained is separated from the adjacent image by four microns, 25 μm depth of field, intensity level 0 to 255, Z-ring pressure 20%. All shots belong to the central four mm of the cornea.

The necessary examinations are performed in each patient until obtaining, by full-focus quantitative confocal microscopy (CMTF curve), scans and images of maximum stability in terms of pressure applied by the Z ring with variations of less than 10%, represented by the yellow curve. The selected images should not be modified in brightness and contrast, the analysis is carried out with the NAVIS software.

#### **Figure 15.**

*Calculation of basal epithelium cell density. After selecting the image and the ROI area (0.0500 mm2 ), the cells are marked in the upper left image and the density value is obtained through the software, in this case 4642 cells/mm<sup>2</sup> . Example corresponds to 1 year after PRK).*

Steps to follow in microscopy. A: Z ring for fixation of the eyeball. B: 40X lens. C: Coupling of A and B. D: The lens was advanced until contacting the ring with the coupling substance for examination.

#### **Figure 16.**

*Calculation of keratocyte density. In the upper right portion the selection of the area is observed, in the upper left portion the marking of each keratocyte. The first density quotient obtained is the one with red letters. This value is divided by the effective depth of field of the equipment: 25 to obtain the density in cells/mm3 .*

### **Figure 17.**

*Calculation of endothelial variables. In the upper right portion the selection of the area is observed, in the upper left portion the automatic marking of the cells. In the lower portion, the values obtained for endothelial cell density, pleomorphism and polymegatism are observed.*

*Cornea Confocal Microscopy: Utilities and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.96272*

#### **Figure 18.**

*Measurement of corneal haze, after PRK. In this case, 3 months after PRK, the upper image shows a CMTF curve, taking the first image of corneal haze, then the cursor is moved to the last image of corneal haze shown in the lower figure, where the parameter called distance = 61* μ*m.*

The lower right part shows the quality of the shot given by the constancy of the yellow line and the uniformity of the white line. The green line corresponds to the reflectivity in the form of peaks. An image of corneal epithelium belonging to the first image of the 4 existing ones is shown.

Full focus quantitative confocal microscopy (CMTF), where the selected image is seen in the upper right: Apical epithelium one year after LASIK.

The top left shows the parameters of depth, distance, intensity level, pressure of the Z ring, among others. The lower right part shows the quality of the shot given by the constancy of the yellow line and the uniformity of the white line. The green line corresponds to the reflectivity in the form of peaks. An image of corneal epithelium belonging to the first image of the 4 existing ones is shown.

#### **Figure 19.**

*Subbasal nerve plexus. (A) Short nerve, less than 200 μm in length. (B) Long nerve (greater than 200 μm without interconnections). (C) Nerves long with interconnections. (D) Measurement of the nerve.*

## **3. Conclusions**

The corneal study by confocal microscopy allows the differentiation of epithelium sublayers, subbasal nerve plexus, keratocytes, stromal nerves, and corneal endothelium cells.

*Cornea Confocal Microscopy: Utilities and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.96272*

Its usefulness has been demonstrated in the study of patients with corneal dystrophies, keratoconus, infectious keratitis, corneal refractive surgery, it has also shown usefulness in the study of patients with corneal transplantation, corneal crosslinking, diabetics, vitrectomized, as well as the study of the filtering bulla in glaucoma.

It is necessary to continue improving the methods of calculation and identification of structures by cornea confocal microscopy, in order to obtain better images and analyzes that contribute to more accurate diagnoses and broaden the spectrum utility of this novel technology.

## **Conflict of interest**

I confirm there are no conflicts of interest.

## **Author details**

Eduardo Rojas Alvarez1,2

1 Faculty of Medical Sciences, University of Cuenca, Ecuador

2 Exilaser Ophthalmology Center, Ecuador

\*Address all correspondence to: drerojasalvarez@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 3
