7

CHAPTER 1

IN VIVO CONFOCAL MICROSCOPY OF THE HUMAN EYE

1.1 History of Optic Nerve examination

Since the first images of the fundus, obtained with the ophthalmoscope by Helm-
holtz [1] 145 years ago, examination methods have continued to improve. First, exami-
nation with direct ophthalmoscopy was used, followed by examination with binocular
indirect ophthalmoscopy, optic disc drawings, optic disc stereophotographs, planimetry,
the Takamoto & Schwartz’s stereophotogrammetry, neuroretinal rim measurements, Ai-
raksinen & Tuulonen’s evaluation of the retinal nerve fiber layer, optic disc pallor meas-
urements, angiofluoresceinography of the optic disc, image analyzer (Rodenstock Optic
Nerve Head Analyzer, Topcon Analyzer), the laser scanning ophthalmoscope, and Lot-
mar & Goldmann’s stereochronoscopy. All these methods are extensively explained in the
valuable book “The Optic Nerve in Glaucoma” by Rohit Varma & George Spaeth [2].
Of these methods, we have used, and continue to use retinofluoresceinography be-
cause it is very useful. We do not use stereoscopic photographs of the optic nerve in
adults any longer due to a significant interobserver variation in the interpretation of re-
sults, as reported in the literature [3, 35, 36]. But stereoscopic photographs are useful for
optic nerve examinations in children under 2 years of age because, due to their flat cor-
nea, with confocal tomography we have failed to obtain good images. In Boston we
learned about Schwartz & Takamoto's stereophotogrammetry [4], a very reliable method,
the results of which are consistent with those obtained with the HRT. However, it is a
time-consuming method. We later performed neuroretinal rim measurements according to
Airaksinen's [5] method, which we could apply thanks to Dannheim and Airaksinen's
personal communications. It turned out to be the most useful method. We also used Lot-
mar & Goldmann's optic disc stereochronoscopy [6]. All these methods require pupil
dilation.
For the above mentioned methods, particularly for the measurement of optic disc pa-
rameters: area, cup area, neuroretinal rim, the formula introduced by Littmann in 1982
allowed us, for the first time, to obtain the dimensions (length, surface) of any observable
object in the fundus: exudates, tumors, foreign bodies, optic discs, vessels, etc. We can
observe the image of these elements with considerable magnification produced by the
ocular system.
This morphometric magnification was corrected in order to find real values with the
Littmann formula [7]. Littmann was an engineer who worked for Zeiss and whom we had
the chance of meeting in Buenos Aires. Since 1982, thanks to his formula, we have been
8

able to obtain the real measurements in mm or mm2, of a body or structure on the retina.
For this formula, the corneal curvature, which is measured with an ophthalmometer, the
axial length, which is measured by echometry, and refraction are very important. Corneal
thickness, its posterior curvature, the lens face curvatures, the depth of the anterior cham-
ber and lens thickness are not required. This is because even if they varied, their influ-
ence on the measurement would be minimal. This formula does not apply for aphakia,
pseudophakia and refraction changes due to opacity of the lens.
It is necessary to approach the study of confocal microscopy from two different an-
gles: the study of equipment for retina exploration with laser ophthalmoscopes which use
the light emitted by the laser and the development of confocal microscopy and its appli-
cation to these ophthalmoscopes [8].

1.2 Laser ophthalmoscope for fundus examination

Before the first laser retina scanning ophthalmoscope prototypes were introduced in
1980, a TV camera was applied to a retinograph in order to obtain the retina’s image in
real time. Television ophthalmoscopy had the advantage of scanning, recording and dig-
itizing the image, yet the images were never clear. Then, the light from the laser beam,
which has two features: monochromaticity and coherence, was used. The latter feature is
divided into temporal and spatial coherence. The great directivity of the laser light is due
to spatial coherence. When laser light, with great directivity was used to illuminate the
retina, the image obtained improved considerably and the first prototypes were intro-
duced first in Boston and then in Heidelberg in 1980. Confocal microscopy was later
added to these machines and higher quality images were achieved. For this reason laser
scanning ophthalmoscopy may be carried out with or without confocal microscopy .
The first prototypes for the acquisition of two-dimensional retinal images using laser
operated ophthalmoscopes were presented 15 years ago, in 1980, first in Boston by R.H.
Webb [9, 10], then in Heidelberg by Klingbeil & Bille [11]. Similar machines were sub-
sequently developed in other countries [12, 13, 14]. However, it took 10 years for these
advances to be applied to clinical practice.
The laser ophthalmoscope is a special ophthalmoscope which linearly explores the
fundus point by point, with a low intensity laser beam. The laser beam is shifted periodi-
cally in two directions: horizontally first, point by point, then vertically in a given retina
area. The quick horizontal movement is achieved by means of a rotating polygon,
whereas a slower vertical movement is achieved by means of a mirror galvanometer [15]
(figure 1.1).
The laser light that is reflected from the retina reaches a mirror that sends it to a de-
tector transforming the luminous energy into electric energy. The image is finally recon-
structed on a television screen. It is important to bear in mind that the scanning is carried
out horizontally as well as vertically point by point. The size of one of these points is 10
µm.

1.3 Confocal microscopy

Confocal microscopy is a technique that has been around for the last 25 years. The
first commercially available prototypes were reserved for biological specimen observa-
 9

Fig. 1.1

tions [16, 17, 18, 20 and 21] and for industrial specimen observation [22]. This research
instrument has had extraordinary development in recent years due to its multiple applica-
tions and its original and surprising results.
Confocal microscopy allows us to study, in vivo, structures in the eye such as the
retina, the optic nerve, tumors, corneal tissue, chamber angle, and lens capsule with high
resolution and in greatly improved contrast conditions as compared to conventional mi-
croscopy. It thus allows for a new semiology of the ocular tissue at a histologic scale.
The images obtained with confocal microscopy have such high resolution and con-
trast that they call for optical microscopy limits redetermination. In the cornea it allows
us to reach a cellular resolution level, and in the retina and optic nerve the resolution is
between that of a magnifying glass and the microscope.
Confocal microscopy is the final stage of an evolving series of examination methods
used in ophthalmology that we shall briefly describe:
Slit lamp biomicroscopy yields images with improved quality and allows for ocular
structure contrast in vertical, horizontal and oblique sections. Retroillumination in a red
(retina) or yellow (iris) field allows us to better study the structures in the vitreous body
and the cornea, respectively. In addition, we can, to a small extent, observe the endothe-
lium with the specular reflection technique and with Eisner’s contact lens.
The specular corneal microscope solves the problem we had with the slit lamp by
focussing light on the most reflective surfaces of the cornea. The intensity of the light
reflected by these structures is greater than the luminous intensity reflected by under- and
overlying planes, like the tear layer, the aqueous humor, etc. Specular microscopy per-
mitted, mainly, endothelium study. When the stroma is pathological, diffraction produces
an increase of the light reflected by this tissue, which makes endothelium examination
difficult. This condition improves when the light beam is reduced in order to decrease the
light diffracted. The system’s resolution is thus improved but with a consequent substan-
tial loss in the observation field. To increase this field, scanning of the observation area
with a narrow slit was necessary to obtain a useful clinical image. To achieve this, either
the specimen could be moved, as done with the Scanning Slit Optical Microscope (de-
10

scribed by Maurice) [23, 24, 25], or the optical slit could be moved, as in the Scanning
Mirror Optical Microscope [26]. In both cases a small incidence angle was used to limit
diffraction. Image acquisition was very slow. A third possibility was to try to scan the
specimen through the optic beam. This method is the one used by almost all confocal
microscopes. This can be achieved in two ways: first, through a slit animated by periodic
vibrations (mirror galvanometer) [22], or through the Nipkow disc [27], a disc with a
series of perforations, arranged in a specific way that is animated by a quick rotation. The
main theoretical advantages of confocal microscopy over conventional biomicroscopy are
its better axial and lateral resolution as well as the possibility for dynamic analysis, thus
permitting three-dimensional temporal and spatial sequences. However, there are certain
limitations. Since the greatest advantages are the acquisition of the image of the studied
tissue in a single plane and the avoidance of noise from anterior and posterior planes
which deteriorates the image quality, a very strong light, not surpassing phototoxicity
limits, is required. The best resolution obtained by this system is at the expense of a sub-
stantial reduction in the working distance. Working distance is the one between the mi-
croscope’s front lens objective and the focal plane. Therefore, when examining the cor-
nea, the confocal microscope may be at a close distance, that is to say, less than 900 µm,
in which case cell observation becomes possible between 10 and 100 µm. The anterior
crystalline capsule may also be studied. In this case acetazolamide should be adminis-
tered to the patient, the day before, to decrease intraocular pressure, and thus allow cor-
nea depression with the microscope during examination, and to get closer to the anterior
surface of the lens. When fundus structures are studied the opposite occurs because we
can not get close to it. For this reason resolution at the tissue level is much less and does
not reach the cellular level.
For studying the cornea, the confocal microscope has an objective, as with the light
microscope, whereas in the confocal microscope for retina examination, the patient’s eye
is the objective. The optical features of the human eye are responsible for resolution
limitations. If the focus of the microscope is shifted forward and backward from the su-
perficial planes toward the deeper ones, different planes with which three-dimensional
images can be constructed, are scanned.

1.4 Description of the confocal microscope

Figure 1.2 shows the confocal principle. The light source is a power source that
emits luminous energy from a 670 nm laser diode. This laser beam is emitted by the
source in parallel beams that reach a positive lens that makes them converge at a certain
point. This point is located at the level of a pinhole diaphragm having 75 µm in diame-
ter. The image of the luminous points illuminates the retina in a tiny surface of approxi-
mately 10 µm. This special diaphragm is called confocal diaphragm.
The luminous beam thus emitted first goes through a beam splitter that is both trans-
parent and also serves as a mirror. It then goes through a scanning system that, as men-
tioned before, is basically composed of a quickly rotating polygon that deviates the light
beam horizontally and a slow galvanometer mirror that deviates it vertically. This is an
optical-mechanic and an optical-electric unit. The light beam later goes through a real
objective in the corneal confocal microscope and focuses on a point of corneal tissue at
 11

Fig. 1.2

the desired depth. The light reflected by that point is deviated by the mirror, and before
reaching the detector, it again goes through a pinhole confocal diaphragm like the one
described above. The first and second confocal diaphragms are conjugated with the focal
plane of the tissue examined. As shown in figure 1.2, the detector cannot be reached by
light reflected from any point located at a plane anterior or posterior to the one under
examination, because the small confocal diaphragm stops that light. This is referred to as
spatial filtering. These reflections from anterior and posterior points are responsible for
reflection or refraction interferences (noises and hales) undermining the image in ordi-
nary light microscopy, and not in confocal microscopy. An image with both high resolu-
tion and contrast can thus be obtained. Figure 1.2 also shows a corneal confocal micro-
scope at the top and a retinal microscope, in which the human eye serves as an objective,
at the bottom. If both microscopes are compared, it can be observed that in the retinal
one, the beams reach the anterior surface of the eye parallel and perpendicular to it, and
the eye itself is the objective making them converge on a retinal plane.
When comparing an ordinary microscope to a confocal microscope, the following
can be said: the luminous source is punctual; the first confocal diaphragm is the con-
denser; the human eye is the objective; the retina is the object and the detector is the
eye piece.

1.5 Resolution in confocal microscopy

Resolution in confocal microscopy of the eye was studied by Gaida, Ph.D., from
Zeiss in Oberkochen, Germany [28]. In order to calculate the resolution he used Gull-
strand’s model eye, with a 3 mm pupil and a laser beam with a 633 nm wavelength. Us-
ing the confocal microscope we have just made reference to for a three-dimensional ret-
ina study, Gaida described the lateral and depth resolutions as follows: he designed a
three-dimensional resolution element, which in the eye fundus, on the retina, is repre-
sented by a cylinder having a radius of 10 µm, which is the lateral resolution, and a
height of 300 µm, which is the depth resolution (z axis) (figure 1.3).
12

Fig. 1.3

The numerical aperture and the wavelength of the laser light are the most important
parameters for resolution determination [29, 30]. Gaida concludes that the internal retinal
structure can not be resolved.
The confocal microscope therefore increases the lateral and axial resolution, while
reducing the field of observation (Lukosz’s principle) [31]. This loss may be made up for
by mechanical or optical scanning of the object studied and by the computerized recon-
struction into 2 or 3 dimensions.
In brief, the confocal microscopy used in the laser ophthalmoscope for the study of
the human retina has the following advantages:
a) It is a non-invasive method.
b) The exposure is short: 1.6 seconds.
c) The tissue is studied in vivo without the artifacts produced by histologic sections
and stainings.
d) It is not necessary to dilate the pupil (except for a macula study). It should be
borne in mind that according to Campbell, the best fundus resolution can be achieved
with a 2.4 mm pupil diameter.
The image has:
a) Very good lateral and axial resolutions.
b) An absolute focal plane, excluding the light from the planes anterior or posterior
to the one studied. This spatial filter effect is what increases contrast in the structures
studied. The image obtained has a greater contrast due to a continuous point by point
selective illumination of the retina.
c) The detector’s high sensitivity allows for dim illumination.
d) The acquisition of temporal or spatial sequences leads to three dimensional
images.
e) The field depth is greater due to the laser light directivity.
The scale in table 1.1 shows the resolution of ordinary light microscopy, corneal
confocal microscopy and retinal confocal microscopy.
 13

MACROSCOPIC LEVEL
SUB-MACROSCOPIC LEVEL between cm and 1 mm = 1,000 µm
ECHOGRAPHIC LEVEL up to 0.19 mm = 190 µm
MICROSCOPIC LEVEL
Histologic between 0.5 and 0.1 mm = 100 µm
Confocal between 0.3 and 0.01 mm = 10 µm
Cytologic between 0.1 and 0.001 mm = 1 µm
SUBCELLULAR LEVEL between 1 µm and 15 Angstroms

Table 1.1: Dimensional levels of observation.

Finally, when studying confocal laser scanning ophthalmoscopy, it is important to

refer to the papers by Zinser, published in 1988, 1989 and 1990 [32, 33, 34].
14

Bibliography
1. Helmholtz HV: Beschreibung eines Augen-Spiegels zur Untersuchung der Netzhaut
im lebenden Auge. In: Engelking D (ed.): Dokumente zur Erfindung des
Augenspiegels durch Herrmann von Helmholtz im Jahre 1850. Bergmann Verlag,
München, 1950.
2. Varma R, Spaeth G: The optic nerve in glaucoma. JB Lippincott Company, Phila-
delphia, 1993.
3. Leydhecker W, Krieglstein GK, Colloni EV: Observer variation in applanation
tonometry and estimation of the cup disc ratio. In: Krieglstein GK, Leydhecker W
(eds) Glaucoma update: International Glaucoma Symposium, Nara, Japan, 1978.
Springer, Berlin Heidelberg, New York, pp. 101-117, 1979.
4. Takamoto T, Schwartz B: Photogrammetric measurements of the optic disc in
glaucoma. Int Arch Photogrammetry 1980; 23(B5):732.
5. Airaksinen PJ, Drance SM, Douglas GR, Schulzer M: Neuroretinal rim areas and
visual field indices in glaucoma. Am J Ophthalmol 1985;99:107.
6. Goldmann H, Lotmar W: Rapid detection of changes in the optic disc. Esterochro-
noscopy. Graefes Arch Clin Exp Ophthalmol 1977;202:87-90.
7. Littmann H: Zur Bestimmung der wahren Größe eines Objektes auf dem Hinter-
grund des lebenden Auges. Klin Monatsbl Augenheilkd 1982;180:286.
8. Plesch A, Klingbeil U, Rappl W, Schrödel C: Scanning Opthalmic Imaging. In:
Nassemann JE and Burk ROW (eds): Scanning laser opthalmoscopy and tomogra-
phy, pp 23-33, Quintessenz, München, 1990.
9. Webb RH, Hughes GW, Pomerantzeff O: Flying spot TV opthalmoscope. Appl
Optics 1980;19:2991-2997.
10. Webb RH, Hughes GW: Scanning laser opthalmoscope. IIEE Trans Biomed Eng;
BME 1981;28:488-492.
11. Klingbeil U, Rau H, Bille J: Ein hochauflösendes optisch-elektronisches Verfahren
zur Darstellung des Augenhintergundes. Ber Dtsch Opthalmol Ges 1980;77:337-
339.
12. Kobayashi K, Akiyama K, Yoshizawa I, Asakura T: Laser-beam scanning system
using acoustic-optic deflector: its application to fundus imaging. Meas Sci Technol
1990;1:151-157.
13. Norren D van, Kraats J van: Imaging retinal densitometry with a confocal scan-
ning opthalmoscope. In: Nasseman JE and Burk Row (eds): Scanning laser opthal-
moscopy and tomography, pp. 127-132. Quintessenz, München, 1990.
14. Sabban JC, Rodier JC, Roussel A, Simon J: Opthalmoscope a balayage optique. J
Optics 1984;15: 425-430.
 15

15. Ott D, Eckmiller R, Lades M: The Scanning Laser Opthalmoscope (SLO) as eye
movement measurement system. In: Nasemann JE and Burk ROW (eds): Scanning
laser opthalmoscopy and tomography, pp. 147-158. Quintessenz, München, 1990.
16. Brakenhoff GJ, Blom P, Barends P: Confocal scanning light miscroscopy with
high aperture immersion lenses. J Microsc 1977;117:219-232.
17. Egger MD: New reflected light microscope for viewing unstained brain and gan-
glion cells. Science 1967;157:305-307.
18. Frankhauser F, Kwasniewska S: Methods for structure analysis of the cornea.
Arch Soc Amer Oftalmol Optom 1987;21:33-37.
19. Koester CJ: Scanning mirror microscope with optical sectioning characteristics:
applications in opthalmology. Appl Optics 1980;19:1749-1757.
20. Petran M, Hadravsky M, Boyde A: The tandem scanning reflected light micro-
scope. Scanning 1985;7:97-108.
21. Petran M, Hadravsky M, Egger MD, Galambos R: Tandem scanning reflected
light microscope. J Optical Soc Am 1968; 58: 661-664.
22. Xiao GQ, Corle TR, Kino GS: Real-Time confocal scanning optical microscope.
Appl Phys Lett 1988:53:717-719.
23. Maurice DM: A scanning slit optical microscope. Invest Ophthalmol Vis Sci 1974;
13:1033.
24. Cavanagh HD, Petroll WM, Alizadeh H, HE Y-G, McCulley JP, Jester JV:
Clinical and diagnostic use of in vivo confocal microscopy in patients with corneal
disease. Ophthalmology 1993;100:1444-1454.
25. Cavanagh HD, Jestar JV, Essepian J, Shields W, Lemp MA: Confocal micros-
copy: of the living eye. The CLAO Journal 1990;16:65-73.
26. Koester CJ, Roberts CW, Donn A: Widefield specular microscopy: clinical and
research applications. Ophthalmology 1980;87:849.
27. Nipkow P: Brevet allemand No. 30105, 15/01/1984.
28. Gaida G: Perspectives and limits of three-dimensional fundus microscopy. In:
Nasemann JE and Burk ROW (eds): Scanning laser ophthalmoscopy and tomogra-
phy, pp. 253-257. Quintessenz, München, 1990.
29. Wilson T, Carlini AR: Theory and practice of scanning optical microscopy. Aca-
demic Press, London, 1984.
30. Hellmuth T, Seidel P, Seigel A: Spherical aberration in confocal microscopy. Proc
SPIE 1989;1028:28.
31. Lukosz W: Optical systems with resolving powers exceeding the classical limit. J
Opt Soc Am 1966;57:1190.
16

32. Zinser G, Wijnaendts-van-Resandt RW, Ihrig C: Confocal laser scanning mi-

croscopy for ophthalmology. Proc SPIE 1988;1028:127-132.
33. Zinser G, Wijnaendts-van-Resandt RW, Dreher AW, Weinreb RN, Harbarth
U, Schröder H, Burk ROW: Confocal laser tomographic scanning of the eye. Proc
SPIE 1989;1161:337-344.
34. Zinser G, Harbarth U, Schröder H: Formation and analysis of three-dimensional
data with the laser tomographic scanner. In: Nasemann JE and Burk ROW (eds):
Scanning laser ophthalmoscopy and tomography, pp. 243-252. Quintessenz,
München, 1990.
35. Lichter PR: Variability of expert observers in evaluating the optic disc. Trans Am
Ophthalmol Soc 1976;74:532-572.
36. Varma R, Steinmann WC, Scott IU: Expert agreement in evaluating the optic disc
for glaucoma. Ophthalmology 1992;99:215-221.
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