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1.1 Scope of the paper and additional resources
1. INTRODUCTION
1.1
Scope of the Paper and Additional Resources
Our goal is to present a critical evaluation
of the clinical utility of the confocal microscope for the observation
of the normal and the pathological cornea. The coverage discusses
confocal microscopy of the living human eye; however, we cite some relevant
ex vivo studies. We emphasize the studies performed by the authors.
Our focus precludes a complete review of the literature.
We develop the foundations of confocal microscopy
and show the development of optical techniques used for biomicroscopy of
the eye. The following topics are critically discussed: the practical
techniques of clinical confocal microscopy, the clinical findings of normal,
subclinical and pathological cases, and the consequences of confocal
microscopy. Confocal microscopy is compared with other optical techniques
that are used to study the eye. Three-dimensional computer visualization
techniques are also described.
The following reference materials
are sources of further knowledge. The morphology of the normal
and pathological cornea is the basis of its microscopic observation.
Corneal morphology can be studied with electron microscopy, light
microscopy, confocal microscopy, and clinically with biomicroscopy
using a slit lamp or a clinical confocal microscope (Beuerman and
Pedroza, 1966; Green and Muir, 1994; Maurice, 1984; Müller
et al., 1995, 1996, 1997). A user-friendly guide to light and
the optics of the eye is recommended (Miller, 1991). The fundamentals
of noninvasive instrumentation to investigate the eye are developed in
several books (Haugwitz, 1981; Masters, 1990a; Rosenblum and Benjamin,
1992; Straub et al., 1995). There are classic works which cover
biomicroscopy of the eye (Berliner, 1966; Vogt, 1930, 1981),
and slit lamp and photo slit lamp biomicroscopy (Martonyi et al., 1985).
We recommend a translation of the 1851 paper by von Helmoltz on the use
of the ophthalmoscope to examine the retina in the living eye (Hollenhorst,
1951).
Three useful books help explain the principles of microscopy (Bradbury, 1989; Gloede, 1986; Slayter and Slayter, 1992). Background information on the general field of confocal microscopy is presented in several sources (Corle and Kino, 1996; Masters, 1990b, 1992c, 1992d; 1995a, 1996b; Masters and Kino, 1990; Pawley, 1995; Wilson, 1990; Wilson and Masters, 1994; Webb, 1996). The history of confocal microscopy is revealed in a book which reprinted the key papers and patents from 1884 to the present (Masters 1996b). The following journals have published papers on the theory and applications of confocal microscopy: Applied Optics, Optics Letters, Journal of Microscopy, Scanning, Bioimages, Scanning Microscopy, Microscopy Research and Technique, Biophysical Journal, Bioimaging, Computerized Medical Imaging and Graphics, and the Journal of Biomedical Optics. Previous reviews on the use of confocal microscopy to study ocular tissue are an introduction to the subject of this paper (Böhnke and Masters, 1997; Böhnke and Thaer, 1994; Cavanagh et al., 1995; Masters and Böhnke, 1998; Masters and Kino, 1990, 1993; Masters et al., 1993, 1994; Masters, 1990a, 1990b, 1992c, 1993c, 1993d, 1995a, 1995b).
1.2.
Microscopy Development and Progress in Biology and Medicine
The technical development of the microscope contributed to many advances in biology and medicine. Progress in anatomy, morphology, bacteriology, pathology, immunology and public health was dependent on the improvements of microscope designs. The development of the electron microscope by Ruska, Knoll and other independent groups has had a major impact on cell biology and virology. The light microscope and the electron microscope are strongly linked to progress in biology and medicine (Gloede, 1986).
The story of the development of the light microscope started about 400 years ago and continues into the present time. Hooke described eukaryotic cells; plant cells and molds. Leeuwenhoek developed a single lens microscope and reported his microscopic observations to The Royal Microscopical Society of London. He described the structure of the lens, the cornea, the retina, and the optic nerve. He also observed protozoa, bacteria, striated muscle fibers, blood circulation in capillaries, and the motility of spermatozoa. Both Hooke and Malpighi observed the cellular structure of living organisms. Brown described the cell nucleus following his microscopic studies of plants. These observations were developed by Schleiden in Jena for plants, and by Schwann in Berlin for both animals and plants. This was the beginning of the cell theory.
The light microscope was crucial for other important discoveries in cell biology and pathology. For example, Metchnikoff made microscopic observations of cellular phagocytosis. Virchow, in Würzburg and later Berlin, used the microscope to develop the field of cellular pathology. Virchow’s book, Cellular Pathology was published in 1858. Koch, who worked in Berlin, is the father of medical bacteriology. He developed Koch’s postulates which form the basis of our understanding of the pathogenesis of disease. Koch used the microscope together with aniline dyes to stain microorganisms. These studies resulted in his description of the pathogenesis of anthrax, tuberculosis, and cholera. Another example is Pasteur who worked in Paris. He advanced microbiology with his microscopic investigations of stained preparations. Cajal, and Golgi both used the light microscope to investigate the histology and the embryology of the nervous system. Golgi also discovered the Golgi complex in cells.
Abbe worked out the role of light diffraction in image formation; these studies resulted in improved microscope designs. Zernicke invented the phase contrast microscope. This invention permits the microscope observation of unstained living cells to be observed with high contrast. Nomarski invented the principle of the differential contrast microscope. His microscope permits the observation of living cells without staining. More recently, Allen and Inoué developed the video-enhanced-contrast light microscope.
Ophthalmology has progressed with the developments
of microscopes that were designed for the observation of the living eye
(Duke-Elder, 1962). For example, the development of the slit lamp,
the ophthalmoscope, the specular microscope, the scanning laser
ophthalmoscope, and the clinical confocal microscope are instruments
which permit us to observe the living eye.
Gullstrand is credited with the invention
of the slit lamp; he actually invented the slit illumination system.
Babbage designed a direct view ophthalmoscope. In 1850 Helmholtz
introduced a practical device to permit the clinician to observe the living
retina. One hundred years later Ridley pioneered the development
of the television ophthalmoscope and an ophthalmoscope based on point scanning
a spot of light on the retina (Ridley, 1952).
Robert Webb developed the scanning laser
ophthalmoscope to image the retina. Goldmann invented the first confocal
microscope to image the cornea. Moreover, he made contributions
to photography, slit-lamp microscopy, gonioscopy, fluorometry,
the examination of the fundus, tonometry, the determination
of visual acuity, adaptometry and perimetry as well as studies of
cataract and glaucoma.
The specular microscope was developed to
image the corneal endothelium. It is based on the specular reflection
at the interface between the corneal endothelial cells and the aqueous.
Human endothelial cells were first observed by Vogt. During
the last three decades Maurice, Laing, Thaer, Bigar and Koester made important
contributions to its development. The clinical confocal microscope
was developed to optically section the living human cornea; its design,
construction, and clinical use are described in this paper.
1.3.
Increasing Importance of Early Diagnosis and Disease Prevention
During the last hundred years we have seen
the development of instruments which play important roles in the early
diagnosis and the prevention of disease. The development of the clinical
X-ray system by Röntgen has made possible the early diagnosis of tuberculosis
and cancer; today x-ray mammography is an important diagnostic
tool. Another example is the development of clinical magnetic resonance
imaging systems which permit the radiologist to image structure
and function within the living body. Examples for important
applications are the diagnoses of disseminated encephalitis and brain tumors.
The principles of magnetic resonance techniques as applied to ophthalmology
have been clearly explained (Seiler and Bende, 1990). Magnetic
resonance techniques have been used to monitor the hydration of the cornea
(Masters et al., 1982).
The development of the light microscope
was a major factor in the understanding and therefore the control of
infectious disease. In clinical ophthalmology the invention of the
Goldmann three-mirror lens has had wide utility for the diagnosis of retinal
breaks. The early diagnosis of pathology is a crucial factor in the
prevention of disease and the lowering of morbidity since treatment can
be initiated in the early stages of the development of pathology.
The clinical confocal microscope is predicted to have increasing importance
in the examination of the eye, the early diagnosis of eye disease,
as well as the monitoring and clinical evaluation of its treatment.
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