ANTERIOR OCULAR MICROSCOPY 18

PART 1 BIOMICROSCOPY
S. Zantos and 1. Cox

18.1 INTRODUCTION subject of biomicroscopy of the eye, the

first books being published in the early
The transparent nature of the ocular tissues
1920s by Koby, Vogt and Koeppe. Other
enables non-invasive examination of the liv­
noteworthy treatises were later published
ing eye that is unique in clinical work. The
by Butler (1924), Berliner (1943) and Dog­
slit lamp microscope combines a clever illu­
gart (1949). This was a period of discovery­
mination system with a convenient stere­
the various slit lamp techniques for illumi­
omicroscopic viewing system. Its great
nating and observing the ocular tissues
variety of attachments make it an extremely
were discussed meticulously, and the nor­
versatile instrument for examining the
mal and abnormal appearances of the eye
human eye.
were beautifully described and illustrated,
The first binocular corneal microscope
with detail similar to that seen using mod­
was constructed by H. Aubert in 1891.
em instrumentation.
Improvements were made by S. Czapski,
A significant advance in slit lamp evolu­
and in particular by Alvar Gullstrand who,
tion came in 1965 with the introduction of
in 1911, presented his design of an illumi­
instrumentation capable of photographing
nation system that made slit lamp micros­
copy practical by providing a sufficiently
bright and focused beam of light to the ~ye.
.",¥~
A subsequent modification, the so-called
;';'.
Kohler illumination system, served to
enhance the homogeneity of the slit beam
projected on the eye, enabling optical sec­
tioning of the eye tissues, and it became
the basis of today's instruments. Over the
next 40 years numerous improvements
were made by Henker, Koeppe, Vogt,
Koby, Poser, Goldmann, Littman and oth­
ers, culminating in instrumentation that
resembled and operated similarly to
today's slit lamps (Fig. 18.1). Concurrently,
numerous publications appeared on the Figure 18.1 A typical modern slit lamp.

Contact Lens Practice. Edited by Montague Ruben and Michel Guillon.

Published in 1994 by Chapman & Hall, London. ISBN 041235120 X

360 Anterior ocular microscopy

optical sections of the eye. This coupled 18.2 INSTRUMENTATION
with the development of numerous attach­
Many well-designed and well constructed slit
ments (pachometer, goniolens, Hruby fun­
lamps are available today. The main features of
dus lens, applanation tonometer, filters,
slit lamps can be seen in Figures 18.2, 18.3 and
polarizers, fluorophotometer, endothelial
18.4. The three essential components of all
lens, video, etc.), have made the modern
instruments are the slit illumination system,
slit lamp microscope an extraordinarily
the stereomicroscope viewing system and the
useful instrument in research and in clini­
mechanical system for adjusting position and
cal practice. Its application to contact lens
height of the instrument and of the patient.
work is extensive.
Detailed reviews of these are available in sev­
With regard to biomicroscopic examina­
eral textbooks (e.g. Brandreth, 1978),and famil­
tion of the eye, it is worth adding that in
iarization is best done using the actual
recent years two new instruments have been
instrument. However, in selecting a brand of
investigated in the search for higher magni­
slit lamp, we recommend that particular atten­
fication and resolution of tissue structures:
tion be given to certain features.
the specular microscope and the confocal
(tandem scanning) microscope. Both instru­
ments can be used for the living eye, though
the confocal microscope is presently in the
research and development phase, and not yet
used in clinical practice. The specular micro­
scope is widely available for clinical work,
having been extensively used for evaluation
of the corneal endothelium before and after
cataract surgery and intra-ocular lens
implantation; in contact lens research, the
instrument has been used for investigating
long-term changes in the endothelium, as
well the effects of lens wear on the corneal
epithelium. Where the specular microscope
reveals cellular patterns only at tissue layers
differing in refractive index (e.g. epithelium­
to-air; endothelium-to-aqueous), the confo­
cal microscope reveals tissue structure at any
layer. Moreover, it does so with considerably
more detail, for example, stromal keratocytes
and cell nuclei can be seen in uiuo with the
confocal microscope. Numerous fine review
articles exist on the optical principles and
applications of the specular microscope Figure 18.2 The slit lamp illumination system. Slit
(Koester & Roberts, 1990; Mayer, 1984) and beam width and height are adjusted by the
the confocal microscope (Cavanagh et al., knurled rings at the front, while the knobs on the
1990; Masters & Kino, 1990). Still, the slit rear of the housing allow coloured filters to be
introduced into the light beam. The diffusing
lamp microscope remains the instrument of
filter can be seen at the top of the illumination
choice for clinical practice, and its main housing ready to be flipped into place in front of
features, applications, and procedures will the prism. Slit beam offset is adjusted by turning
now be discussed. the prism housing laterally.

Figure 18.3 The stereomicroscope viewing sys­
tem. The eyepieces are adjustable to compensate
for the viewer's refractive error and pupillary
distance. Magnification is adjusted by turning the
handle located on either side of the lens housing.
18.2.1 THE SLIT LAMP ILLUMINATION
SYSTEM
1. High illumination is required to enhance
detection of subtle conditions in the eye
tissues when using optic section or indi­
rect illumination techniques (e.g, for
assessing corneal thickness or for evaluat­
ing changes in corneal transparency). A
slit illumination up to 600 000 lux is desir­
able, and a halogen or xenon light source
is preferred to a tungsten lamp for reasons
of longevity. colour, temperature and low
heat generation.
2. The ability to displace (offset) the slit
beam sideways is invaluable for perform­
ing indirect illumination techniques (e.g.
sclerotic scatter, retro-illumination), and it
is necessary that a control device for doing
this be present, conveniently and finely
adjustable, and steady. A click-stop posi­
tion is useful for re-setting the beam to its
normal position.
3. A diffusing filter and a cobalt blue filter
should always be incorporated into the
instrument, and other filters are desir­
able, e.g. green (red-free) and polarizer.
Instrumentation 361
Figure 18.4 The mechanical system. Lateral move­
ments are controlled by the joystick, while vertical
adjustments are made with the electric switch
located in front of the joystick. This adjustment
may be manual on many slit lamps. Illumination
intensity can be altered using the rheostat located
on the left of the joystick.
18.2.2 THE STEREOMICROSCOPE VIEWING
SYSTEM
The optical design of today's slit lamps is
quite good, so that the practitioner need not
be too concerned with the resolution, bright­
ness, and stereoscopic results. Detailed dis­
cussion oithese items is given in other books
(e.g. Muller; Schmidt, 1975). From a more
practical standpoint, the magnification, focus
adjustment and parfocality, and the working
distance are more important considerations:
1. The magnification should be easily
changeable, and it should preferably
cover the range approximately 5X to 30X
before one has to change eyepieces to
achieve higher magnification.
2. It is advisable to check periodically that
the object of regard remains in focus as
362 Anterior ocular microscopy
the magnification is changed through
the range available. A well-constructed
focusing rod accompanying the slit lamp
can be invaluable in checking the parfo­
cality of the slit illumination system and
the microscope viewing system. It is also
worthwhile to check for absence of
vertical prismatic effect between the
two eyepiece views as the objective
magnification is changed.
3. The working distance of the microscope
(from object plane to the front lens of the
microscope) should be approximately Figure 18.6 Diffuse illumination. Low magnifica­
110 mm, long enough to allow for tion overview of the whole eye.
manipulation of the eye or of accessory
attachments, but not too long so as to
require an uncomfortable ann position
during such manipulations.

18.2.3 lHE MECHANICAL SYSTEM

Accurate and convenient positioning of the

instrument in relation to the patient's eyes is
vital to efficient biomicroscopy:

Figure 18.7 Diffuse illumination. Medium magni­

fication view of the edge of a soft lens.

Diffusing
Filier
1. It is preferable that the height adjust­
ment of the slit lamp and the joystick
for lateral adjustment and focus be
combined, or be near enough to each
other so that one hand can control all
Illumination these functions, leaving the other hand
System
free to manipulate the patient's eyelids
Observation or an accessory device, or to manoeu­
System vre the illumination system or the
microscope.
2. It is important that the friction in the
Figure 18.5 Diffuse illumination. The diffusing
sliding stage be not so loose that slip­
filter in front of the illumination system spreads a page occurs as the instrument is moved,
wide beam of unfocused, scattered light uni­ and not so tight that the joystick might
formly across the field of view. be jerky or excessively tilted from its
 Instrumentation 363

Normal to
Cornea

Illumination Actual
System View

Observation
System

Figure 18.8 Optic section. The illumination system sections the cornea with a thin, beam of light,
focused co-incidentally with the axis of the observation system.

vertical position as the instrument is In addition to the slit lamp microscope,

moved during the examination.
3. The well-constructed instrument will
have the slit illumination system and
microscope system nearly perfectly
coaxial, so that as the former is rotated
(swung) from one side to the other, the
slit beam will not shift significantly
sideways in the field of view, and
parfocality can be maintained. The
focusing rod can be used to check this,
but it must be perfectly constructed
and positioned in the coaxial slot to be
of any value.
illumination system and mechanical stage,
the construction of the patient's headrest
and of the instrument table are obviously
important to the ease of performing biomi­
croscopy. As with other ophthalmic instru­
ments, the comfort and adjustability of
these functions should be tried for suitabil­
ity to the practitioner's personal preference
before purchase.
Reviews of the specifications of the vari­
ous commercially available instruments have
been published elsewhere, and the reader is
referred to these articles for comparison of
364 Anterior ocular microscopy
instrument features (Stockwell, 1983 Marto­
nyi, 1989;).
18.3 SLIT'LAMP ILLUMINATION AND
OBSERVATION TECHNIQUES
The ability to detect and diagnose the vari­
ous anterior segment conditions which may
occur depends to a large extent on the ability
of the observer to correctly adjust and posi­
tion the illumination system of the slit lamp.
In many cases, subtle changes to anterior
segment structures can only be detected and
thoroughly assessed by using a combination
of illumination techniques. Slit lamp illumi­
nation can be categorized into four main
groups:
1. Diffuse illumination.
2. Direct illumination.
3. Indirect illumination.
4. Filtered illumination.
Figure 18.9 Optic section. Medium magnification
view of a central corneal scar. Note that the optic
section allows the depth of the scar to be easily
determined.
Figure 18.10 Optic section. High magnification
view of infiltrates invading the peripheral cornea.
Note that the optic section reveals the infiltrates to
be concentrated towards the anterior stroma.
Within each of these categories are several
techniques which are defined and described
in the following section according to the
adjustment and positioning of both the illu­
mination and observation systems. This
differentiation is important for the inex­
perienced observer to ensure that the opti­
mal observation technique is used for
viewing each particular condition or struc­
ture. However, it should be understood that,
in reality, several different methods of illu­
mination may be simultaneously present in
the field of view at anyone moment during
the slit lamp procedure, providing the expe­
rienced observer with the necessary differen­
tial information without resorting to several
different physical adjustments of the slit
lamp. This speeds up the slit lamp examina­
tion and reduces patient fatigue and discom­
fort.
18.3.1 DIFFUSE ILLUMINATION
Diffuse illumination is so named because a
ground glass diffusing filter is placed in the
focused light beam of the slit lamp. This
defocuses and scatters the light to give a
broad even illumination over the entire field
of view of the observation system (Fig. 18.5).
 Slit lamp illumination and obsertiation techniques 365
The angle of the illumination arm is not 18.3.2 DIRECT ILLUMINAnON
critical when the diffuser is in place and can
Direct illumination describes any illumina­
be anywhere from 10 to 70 degrees in relation
tion technique where the slit beam from the
to the observation arm. Brightness is con­
illumination arm and the optics of the obser­
trolled by both the slit width and the illumi­
vation system are focused co-incidentally on
nation rheostat of the slit lamp.
the object or area under view. The following
Diffuse illumination is typically used for
set-ups are considered to be direct illumina­
low magnification views of the anterior seg­
tion techniques:
ment, particularly for assessing conjunctival
redness, contact lens fitting performance, or 1. Optic section.
for initial gross scanning views of anterior 2. Parallelepiped.
ocular structures (Figs 18.6 and 18.7) 3. Broad beam.

Normal to
Cornea

Illumination Actual

System View

Observation
System

Figure 18.11 Parallelepiped. Essentially a broadened optic section. Note that the size of an object
within a parallelepiped can be estimated by comparing the width of the parallelepiped to its depth
(which is approximately 500 lAID).
366 Anterior ocular microscopy
4. Conical beam.
5. Specular reflection.
6. Oblique.
Optic Section
Optic section desaibes an illumination tech­
nique which utilizes an extremely thin beam
of light (e.g. 0.02-0.1 mm) to 'cross-section'
the corneal tissue in much the same way as
would be seen with a transverse histological
section. This provides the ability to assess
the depth of an object within the corneal
layers or the depth of the tear film between a
rigid lens and the anterior cornea.
To set up an optic section, the illumination
arm is positioned on the side of the micro­
scope that corresponds to that section of the
cornea to be viewed (i.e. temporal side for
temporal cornea) and is set at an angle
between 30 and 60 degrees to the observation
arm. Increasing the angle creates a wider
section but may not allow the intersection of
several objects separated by depth (Fig. 18.8).
The illumination rheostat is turned fully
on and the slit beam is narrowed as much as
possible while still providing sufficient illu­
mination for viewing. The slit width can be
Figure 18.12 Parallelepiped. High magnification
view of the central cornea, the parallelepiped
being almost twice as wide as it is deep. Note the
microcysts seen to the right of the parallelepiped
in indirect retro-illumination.
Figure 18.13 Parallelepiped. High magnification
view of endothelial folds caused by stromal
oedema. Note that the anterior portion of the
cornea is out of focus when the plane of focus is at
the endothelium. This demonstrates the short
depth of field that accompanies higher magnifica­
tion views.
refined while viewing the cornea through
the microscope.
Optic section is used for viewing anything
where a sense of depth will enhance the
appreciation of the object's size and location,
such as a foreign body embedded in the
cornea, corneal haziness and oedema, endo­
thelial bedewing or pigment cells, or the
clarity of the corneal epithelium (which
appears dark when it is clear and grey when
it is oedematous) (Figs. 18.9 and 18.10).
Parallelepiped illumination
A parallelepiped is perhaps the most com­
monly used form of direct illumination used
in slit lamp examinations. It is essentially the
same in setup as an optic section, except that
the slit beam is widened until it is approxi­
mately the same width as the apparent cor­
neal depth (e.g. 0.1-0.7 mm) (Fig. 18.11). This
view allows an appreciation of an object in
true three dimensions, since its width,
height and depth can be assessed simulta­
neously.
Parallelepiped is used to assess the corneal
 Slit lamp illumination and observation techniques
endothelium, corneal scarring, corneal stain­
ing, neovascularization, corneal infiltrates,
and corneal striae and folds (Figs. 18.12 and
18.13).
Broad beam illumination
Broad beam illumination is the next logical
'step from the optic section and parallelepi­
ped, and refers to a similar setup, except that
the slit beam is now widened to somewhat
broader than the apparent corneal depth (e.g.
1-5 mm) (Fig. 18.14). Light intensity should
be reduced using the illumination rheostat.
The angle between . . the illumination and
Illumination
System
Figure 18.14 Broad beam. An extremely wide parallelepiped, used mostly for looking at large corneal
defects.
367
observation arms is not critical and should
be adjusted to provide the optimum view of
the object in question.
Broad beam illumination is particularly
useful for viewing corneal nerve fibres,
debris trapped beneath soft and rigid lenses,
conjunctival abnormalities and pterygia, and
larger scars and opacities within the cornea
(Figs. 18.15 and 18.16).
Conical beam illumination
Conical beam is useful primarily for observ­
ing inflammatory cells and protein in the
anterior chamber as a result of a uveal
Normal to
Cornea
Observation
System
 368 Anterior ocular microscopy
inflammatory response. The conical beam is
formed by setting up an optic section and
then reducing the height of the slit beam
until it is about 1-2 mm in height. Light
intensity should be maximized using the
illumination rheostat. Since the illumination
intensity of the conical beam is low, observer
sensitivity will be increased if the room
illumination is turned off. The anterior
chamber should then be scanned at low to
medium magnification from side to side.
Cells (inflammatory cells) will appear as
bright, white reflections as they pass through
the conical beam, while flare (protein) will
appear as yellowish particles. These reflec­
tions will be more easily detected if viewed
against a dark background, it is therefore
best to reduce light scattering by avoiding
having the conical beam strike the iris, and
by enhancing pupil dilatation by directing
the conical beam entering the pupil away
from the foveal area. Some observers suggest
oscillating the slit beam using the offset
control to enhance the visibility of cells and
flare (Fig. 18.17).
Specular reflection
Specular reflection is a specific case of the
parallelepiped setup where the angle of the
incident slit beam to the corneal surface
r
t clear view of the endothelial mosaic or
Figure 18.15 Broad beam. High magnification
view of debris trapped beneath a soft lens.
Figure 18.16 Broad beam. High magnification
view of corneal nerves.
equals the angle of the observation axis
through one of the oculars (Fig. 18.18). At
this specific angle the illumination beam is
reflected from the smooth surfaces of the
anterior segment (tear film, contact lens
posterior surface, endothelium and ante­
rior lens capsule) and provides a bright,
mirror-like reflection. To achieve specular
reflection, set-up the slit lamp to form a
parallelepiped. Adjust the magnification to
the lowest available setting. Move the
observation arm up to 20 degrees away
from the illumination arm, and then begin
to move the illumination arm in the oppos­
ing direction while observing the corneal
surface. At the point where specular reflec­
tion is achieved, a bright reflex will fill one
of the oculars. Note that specular reflection
cannot be achieved binocularly. Having
located the specular reflex, increase the
magnification of the observation system
stepwise to its highest setting to provide a
other object of interest.
Specular reflection is used for observing
the quality of the tear layer, since it allows
the lipid layer of the tears to be viewed.
Obviously, lens front surface wetting can
also be observed in this manner. It is also
important to note that specular reflection is
the only technique which allows observa­
 Slit lamp illumination and observation techniques 369

Cells & Flare

Illumination
System

Observation
System

Figure 18.17 Conical beam. A narrow optic section with the height reduced to form a small oval beam.
Note that the dilated pupil is used as a dark field background to enhance observation of any cells or
flare in the anterior chamber.

tion of the corneal endothelial mosaic in Oblique illumination

vivo, along with guttae, folds and blebs.
However, it is often difficult for the inexpe­
rienced observer to clearly view the endo­
thelial reflex because the significantly
brighter reflex from the tears causes visual
distraction. Increasing the overall angle
between the two arms of the slit lamp will
increase the separation between the two
specular reflexes and minimize this inter­
ference. For this same reason, viewing the
endothelial specular reflex is easier for the
peripheral areas of the cornea than for the
central region, since the cornea's greatest
thickness is in the periphery (Figs. 18.19
and 18.20).
Although infrequently used in contact lens
practice, oblique illumination provides a
unique view of contour changes which
would not be visible by other methods of
illumination. As the name suggests, oblique
illumination is achieved by setting up a
parallelepiped and then moving the illumi­
nation arm away from the observation sys­
tem until the angle between them is close to
90 degrees. The illumination arm is adjusted
so that the light beam is almost tangential to
the object of regard. In this way, very small
deviations in the topography of the iris, for
example, will cast large areas of shadow,
370 Anterior ocular microscopy

Normal to
Cornea

.Illumination
System
Observation
System
Figure 18.18 Specular reflection. Note that specular reflection can only be achieved when the angle of
the incident illumination to the normal of the cornea equals the angle of the observation system, and
that specular reflection is viewed through one eyepiece only.

clearly defining the high and low points of a
surface which appears to be relatively flat
under other more frequently used methods
of illumination.
Oblique illumination is used to examine the
uniformity of the iris surface, as well as any
changes in the corneal structure such as Fleis­
cher's ring, which may otherwise be hard to
assess using other methods of direct illumina­
tion. In contact lens practice, oblique illumina­
tion may also be used to investigate the
elevation height of front surface deposits, lens
edge lift, and the location of bifocal lens optic
zones (Figs. 18.21 and 18.22).
18.3.3 INDIRECT ILLUMINATION
Indirect illumination refers to any technique
where the focus of the illumination beam
does not coincide with the focal point of the
observation system (Fig. 18.23). To achieve
this the slit beam must be offset (i.e. dis­
placed sideways). This can be achieved
manually by turning the prism or mirror
controlling the slit beam away from the axis
of the illumination arm, or, as is often done
by experienced observers, by achieving
direct illumination and focusing the sli t
beam to one side of the object to be viewed
and utilizing a different part of the micro­
scope field of view to observe the object
which is now indirectly illuminated (Fig.
18.24).
As with direct illumination, indirect illu­
mination can be divided into several specific
illumination techniques. The following set­
 Slit lamp illumination and observation techniques
Figure 18.19 Specular reflection. High magnifica­
tion view of endothelial guttae.
ups are considered indirect illumination
techniques:
1. Proximal.
2. Sclerotic scatter.
3. Retro-illumination (direct and indirect).
Figure 18.20 Specular reflection. Low magnifica­
tion view of front surface wrinkling on a soft lens
fitted too steeply. Note that the wrinkles are only
visible in the area of specular reflection from the
tear film.
371
Figure 18.21 Oblique illumination. Medium mag­
nification view of a pinguecula. Note that the
shadow cast by the pingecula when illuminated
by oblique illumination provides an indication of
the height of the growth.
Figure 18.22 Oblique illumination. High magnifi­
cation view of the iris architecture. The radial
folds seen in this photograph were not visible
with frontal direct illumination. Note also that the
small areas of pigmentation do not cast a shadow,
indicating that they are not raised from the sur­
face of the iris.
Proximal
Proximal illumination is very similar to par­
allelepiped direct illumination, except that
the parallelepiped is offset slightly to one
side of the object being viewed. This allows
the object and its immediate surrounding
area to be illuminated by light scattered
through the cornea, uncovering subtle
372 Anterior ocular microscopy
Illumination
System
Figure 18.23 Indirect illumination. The illumination and observation system axes do not coincide in
indirect illumination. Note that in this case indirect illumination has been achieved by offsetting the
slit beam.
changes in the corneal transparency which
may not have been visible using direct illu­
mination. One example of this is the obser­
vation of the leading edge of a pterygium as
it moves onto the cornea. Direct illumination
will only display the pterygium itself, while
proximal indirect illumination allows the
changes in the corneal tissue in advance of
the pterygium to be viewed and assessed.
Proximal illumination can be used to view all
objects and conditions within the cornea
which are viewed with direct illumination.
Normal to
Cornea
Observation
System
Sclerotic scatter
Sclerotic scatter is a specific case of indirect
illumination used to investigate any subtle
changes in corneal clarity occurring over a
relatively large area, such as the central cor­
neal clouding associated with steeply fitted,
low Dk rigid lenses. Sclerotic scatter is
achieved by setting the slit lamp up for a
wide angle (45 to 60 degrees) parallelepiped
view of the central cornea, and then manu­
ally offsetting the slit beam until it is focused
 Slit lamp illumination and observation techniques
Illumination
System
Figure 18.24 Indirect illumination. Note that in this case indirect illumination has been achieved by
utilizing the microscope field of view.
on the nasal or temporal limbus (Fig. 18.25).
Correct positioning of the slit beam is sig­
nalled by a bright glow from the limbal
region directly opposite that being illumi­
nated. This glow results from the fact that the
light from the slit beam is being totally
internally reflected between the epithelial
and endothelial layers of the cornea and then
scattered as it hits the scleral tissue of the
opposing limbus. Obviously any area of the
cornea which is altered in such a way as to
increase light scatter will interrupt the total
internal reflection of the slit beam and can be
seen either through the observation system
at low magnification or by the naked eye as a
373
Normal to
Cornea
Observation
System
grey hazy area within a clear background.
Sclerotic scatter can also be used to observe
corneal scars, foreign bodies and deposits
within the corneal layers. It should be noted
that widespread corneal oedema, such as that
seen with soft lenses, will not be detected
using sclerotic scatter because there is no
portion of non-oedematous cornea with
which to compare it (Figs. 18.26 and 18.27).
Retro-illumination (direct and indirect)
Retro-illumination refers to any indirect illu­
mination technique where light is reflected
from the iris, anterior lens surface or retina
374 Anterior ocular microscopy
and used to illuminate an object or area in
the cornea from behind. It should be noted
that in retro-illumination there is no light
being reflected directly from the object of
regard.
Direct retro-illumination
This is that specific situation where the
object of regard is illuminated entirely from
behind (by light reflected from a deeper
tissue), and is viewed against an illuminated
background, causing the object to be seen in
shadow. Therefore, objects seen as white or
opaque against a darker background in
Illumination
System
Figure 18.25 Sclerotic scatter. Corneal illumination is achieved by directing the illumination beam to
the limbal region of the cornea and creating total internal reflection of the light beam within the cornea.
direct illumination are seen as black or dark
objects on a lighter background in direct
retro-illumination. Direct retro-illumination
is achieved by setting the slit lamp as if
forming a direct illumination parallelepiped,
then offsetting the, slit beam until the
reflected light of the slit beam from the
surface of the iris is directly behind the
object under observation when viewed
through the eyepieces (Figs. 18.28, 18.29 and
18.30).
Direct retro-illumination of objects within
the central region of the cornea and lens/
vitreous can also be achieved by aligning the
slit lamp centrally as if forming a direct
Normal to
Cornea
Observation
System
 Slit lamp illumination and observation techniques
illumination optic section on the geometric
axis of the eye. The slit lamp is focused in the
plane of the object under observation and
then the slit beam is offset to pass just inside
the edge of the pupil. The slit beam will be
reflected from the retina/choroid and the
object under observation will be seen against
the red glow of the fundus. This procedure is
more easily performed with large or dilated
pupils.
Indirect retro-illumination
Like direct retro-illumination, indirect retro­
illumination has the object of regard being
illuminated entirely from behind (by light
reflected from a deeper tissue) but now it is
being viewed against a dark background.
Indirect retro-illumination can be achieved
using a similar setup to that used for direct
retro-illumination, except that the illumina­
tion system is offset such that the reflected
image from the iris is not directly behind the
object under observation (Fig. 18.31). It is
interesting to note that if several objects are
viewed in direct retro-illumination, such as
epithelial microcysts, those to the left and
right of the centrally viewed microcysts are
seen in indirect retro-illumination,
Retro-illumination is used to investigate
Figure 18.26 Scleroticscatter. Medium magnifica­
tion view of cellular debris trapped behind a soft
lens.
375
~? -­
i
Figure 18.27 Scleroticscatter. Medium magnifica­
tion view of a pterygium on the cornea. Note that
the full extent of the encroachment of the ptery­
gium is visible under this illumination.
the full extent of changes in the corneal
layers, such as epithelial microcysts, vacu­
oles, scars, degenerations and dystrophies,
and trauma. Direct retro-illumination from
the retina is also used for estimating the
extent of lenticular opacities and changes,
and also the location of optic zones in bifocal
contact lenses. It should be noted that, even
though stereoscopic perception is present,
the depth of anomalies or lesions cannot be
assessed reliably when only retro-illumination
is used; therefore, the optic section technique
is used to complement the retro-illumination
technique (see Figs. 18.32 and 18.33).
18.3.4 FILTERED ILLUMINATION
Slit lamps are typically fitted with two
coloured filters which can be introduced into
the light path of the illumination system - a
cobalt blue filter to aid in fluorescein obser­
vation and a green ('red-free') filter to
enhance the contrast of blood vessels.
Fluorescein stain is routinely added to the
tear film to aid in the observation of corneal
and conjunctival staining, rigid contact lens
fittings and qualitative assessment of the tear
film itself (soft lenses require the use of
special high-molecular-weight fluorescein).
376 Anterior ocular microscopy
.Illumination
System
Figure 18.28 Direct retro-illumination, The illumination beam is directed so that the axes of the light
reflected from the iris and the observation system coincide.
Following fluorescein instillation, illumi­
nation of the tear film with cobalt blue light
of about 490 nm maximum causes a greenish
light with a maximum of about 520 nm to be
emitted by the excited fluorescein molecules.
The brightness of the emitted light is an
indicator of the amount of fluorescein accu­
mulated in a local area, and hence variations
in the brightness of the area under observa­
tion can be used as an indication of the
relative thickness of the tear film or, in the
case of epithelial staining, the severity of the
tissue damage, at that location. Addition of a
yellow filter in front of the microscope objec­
tive (e.g. Kodak Wratten No. 15), which
Normal to
Cornea
Microscope
Field of View
Observation
System
allows transmission of the green light emit­
ted by the fluorescein but blocks most of the
blue light reflected from the ocular surface,
greatly enhances the contrast between adja­
cent areas covered by fluorescein (Figs. 18.34
and 18.35). This allows a more accurate
assessment of subtle variations in tear film
thickness beneath a rigid lens, and areas of
corneal staining which would otherwise go
undetected. When fluorescein is instilled, the
cobalt blue filter is typically used with broad
beam illumination and maximum slit lamp
intensity since the combination of blue and
yellow filters significantly decreases image
brightness through the oculars. It should be
 Procedure for the slit lamp examination of the anterior segment
Figure 18.29 Direct retro-illumination. High mag­
nification view of limbal vessel spikes growing
towards the centre of the cornea. Note that the
looping of the vessels is visible under the direct
retro-illumination from the iris.
Figure 18.30 Direct retro-illumination. High mag­
nification view of epithelial microcyst seen ill
direct retro-illumination from the retina.
noted that the majority of cobalt blue filters
provided by the slit lamp manufacturer are
not ideal in their spectral and overall light
transmission characteristics. Typical glass
cobalt blue filters transmit unwanted longer
wavelengths which are not blocked by the
yellow filter, and often transmit less than
377
50% of the overall available light energy.
This can be improved by using long pass
interference filters in place of the regular
cobalt blue filter (Fig. 18.36). Long pass inter­
ference filters typically have an extremely
sharp 'cut-off' around the 490 nm region
with essentially no transmission above that
value. Average light transmission is typically
above 75% for the short wavelength region.
These characteristics enhance the contrast
and brightness of the area under observation
above and beyond that seen with the typical
cobalt blue filter.
Cobalt blue filters used without fluores­
cein and the yellow filter can also be used to
enhance the view of certain intracorneal
anomalies such as Fleischer's ring (some­
times seen in keratoconic patients) and the
Hudson-Stahli line.
'Red-free' filters are actually greenish-blue
in colour and are so-named because they
block all wavelengths of light at the red end
of the visible spectrum. Red-free filters
enhance the contrast of any structure which
is red in colour, such as arteries and veins, or
stains such as rose bengal, by displaying
them as dark against the lighter green back­
ground of the sclera, lids and iris.
18.4 PROCEDURE FOR THE SLIT LAMP
EXAMINAnON OF THE ANTERIOR
SEGMENT
Examination of the anterior segment of the
potential contact lens patient with the slit
lamp should be done in a systematic and
ordered fashion to ensure that all structures
are investigated, not just those indicated by
the patient's history and symptoms. In the
case of the pre-fitting baseline examination,
this is especially important, since this infor­
mation will be the basis for all future deci­
sions regarding patient management and
care.
The procedure outlined here is designed to
flow smoothly from one group of structures
to the next, allowing adequate observation of
378 Anterior ocular microscopy
Illumination
System
Figure 18.31 Indirect retro-illumination, The observation system is directed so that the axes of the light
reflected from the iris and the observation system do not coincide.
all the tissues in the minimum possible time.
If however, a particular point of interest is
noted, it behoves the observer to deviate
from this routine and use any other illumina­
tion and observation techniques appropriate
to that condition or structure before return­
ing to the outlined procedure.
18.4.1 SEITING UP TIiE SLIT LAMP
It is essential that the illumination and
observation systems of the slit lamp be
focused coincidentally, or clear, direct obser­
vation of an object will not be possible and
the light beam will appear offset whenever
an object is viewed. To achieve this, place the
focusing rod supplied with the slit lamp in
Normal to
Cornea
Observation
System
the appropriate mounting hole, illuminate
the rod with a narrow slit beam and focus
each eyepiece of the microscope on the slit
beam individually. Always adjust the eye­
pieces from positive to negative to avoid
accommodative spasm. Ensure that the slit
beam is in the centre of the field of view,
otherwise the illumination system is out of
alignment and must be serviced.
18.4.2 SEITING UP THE PATIENT
Adjust the height of the slit lamp and chair to
provide a comfortable height for the patient
so that their neck is not extended or their
head tilted. Take note of the patient's outer
canthus relative to the canthus mark on the
 Procedure for the slit lamp examination of the anterior segment 379

vertical bar of the slit lamp headrest, and if

necessary, adjust the height of the chinrest so
that they are aligned. Instruct the patient to
look straight ahead and use either the fixa­
tion target provided with the slit lamp or a
distant target to provide a fixation point for
the eye not under examination. Instruct the
patient not to focus on the fixation target
since this may induce fatique and conver­
gence. Set the illumination arm temporally at
about 45 degrees and open the slit wide.
Have the patient close their eyes and align
the light beam over either their right or left Figure 18.33 Indirect retro-illumination, High
globe (start with either the right or left eye, magnification view of debris trapped behind a
but try to maintain consistency in which eye soft lens. Note that the debris in the light beam is
is examined first). Insert the diffuser into the in direct illumination, while that seen against the
light beam. Select the lowest magnification. dark background of the pupil is in indirect retro­
illumination.

Figure 18.34 Filtered illumination. Low magnifi­

cation view of a rigid lens fitting with fluorescein
instilled in the tears. Note that only the cobalt
blue illumination filter was used in this photo­
graph.

Look through the observation system and

Figure 18.32 Indirect retro-illumination. Medium
magnification view of deposits on the anterior
surface of a soft lens. Note that the deposits can be
seen in three different types of illumination. The
deposits in the light beam are under direct illumi­
nation, those seen against the dark background of
the pupil are in indirect retro-illumination, and
those illumination from behind by the light
reflected from the iris (which are just barely
visible) are in direct retro-illumination.
ensure that their eye is centred in the field of
view. Now instruct the patient to open their
eyes.
18.4.3 EXAMINATION ROUTINE
Initially, using low magnification (e.g. 5-8x),
examine the gross appearance of the eye and
lids with diffuse illumination, paying par­
380 Anterior ocular microscopy
Figure 18.35 Filtered illumination. Low magnifi­
cation view of a rigid lens fitting with fluorescein
instilled in the tears. Note that a yellow Wratten
filter has been placed over the objective lens of the
observation system in addition to the cobalt blue
illumination filter in this photograph. The
enhanced contrast of this filter combination is
obvious when compared to Figure 18.34.
Figure 18.36 Filtered illumination. Low magnifi­
cation view of a rigid lens fitting with fluorescein
instilled in the tears. Note that in addition to the
yellow Wratten filter over the objective lens of the
observation system, the cobalt blue illumination
filter was replaced by a long wavelength cut-off
interference filter in this photograph. This filter
combination has slightly enhanced contrast when
compared to Figure 18.35/ but most importantly
the flash reflexes have been eliminated, improv­
ing the view of the central fitting pattern.
ticular attention to any apparent abnormali­
ties such as redness of the lid margins,
matted or missing lashes, any localized dis­
coloration or redness of the bulbar conjunc­
tiva/ clarity of the cornea and unusual
colourings of the iris. If a lens is worn, note
the centration and movement of the lens
both in primary gaze and upgaze.
Move the slit lamp and focus on the tem­
poral canthus. Decrease the light beam to a
wide parallelepiped and remove the diffuser.
Increase the magnification to a medium level
(1~15X) and begin scanning along the lower
lid towards the nasal canthus. Note the
cleanliness of the lashes (makeup, etc.), the
patency of the meibomian glands, the
amount of debris in the tear film and the size
of the lower tear meniscus. At the mid-point,
pull down on the lower lid and focus on the
palpebral conjunctiva. Note its redness and
the presence or absence of papillae or fol­
licles. Have the patient look up and scan the
bulbar conjunctiva for any undue redness or
discoloration, move the slit up and scan the
inferior limbus, estimating vessel ingrowth
and severity of injection. Also note any
undue hazing or arcus senilis near the lim­
bus. Release the lid and swing the illumina­
tion arm to the nasal side of the microscope,
again to about 45 degrees. If the patient is
wearing soft toric lenses, this is an ideal time
to assess the lens orientation. Continue the
scan to the puncta and caruncle, noting anv
abnormalities or debris.
Continue back along the upper lid margin,
paying particular attention to the redness of
the lid. Again, at the mid-point, have the
patient look down, and assess the superior
bulbar conjunctiva while holding the lid up.
Complete the scan to the temporal canthus
remembering to swing the illumination arm
back to the temporal side after passing the
mid-point.
Direct the patient to look nasally and scan
the temporal quadrant of the bulbar conjunc­
tiva. Assess the injection and sheen of this
tissue since this is a possible indicator of
reduced tear volume. Have the patient look
straight ahead once again and move the slit
 Procedure for the slit lamp examination of the anterior segment 381
nasally to the limbus. Again assess the
degree of vessel ingrowth and injection. If
the patient is wearing soft lenses, estimate
the amount of lens/cornea overlap and
observe whether there is any conjunctival
compression by the lens edge. Continue to
scan the cornea, noting any peripheral haze
or arcus and the general clarity of the cornea.
If lenses are worn, note any deposits on the
lens front surface. At the corneal mid-point,
swing the illumination arm nasally once
more and continue the scan of the nasal
cornea, limbus and conjunctiva.
Repeat this examination on the other eye.
At this point if the patient is wearing soft
lenses, remove them. Instill a drop of fluores­
cein into each eye. Introduce the cobalt blue
filter into the illumination system and open
the beam to its full width. Place the yellow
Wratten filter in front of the observation
system. Increase the illumination using the
slit lamp rheostat. Using low to medium
magnification, scan the ocular surface and
estimate the completeness of the tear cover­
age of both cornea and conjunctiva following
a blink. Look for any epithelial basement
membrane irregularities, particularly near
vessel ingrowth around the periphery of the
cornea. If rigid lenses are worn, assess the
fitting pattern of each lens. Scan the cornea
for any apparent areas of staining. If any are
noted, increase the magnification and assess
their density and size. Move back to low
magnification, and view the central cornea.
Have the patient blink several times. Assess
the completeness of their blinking pattern.
Have the patient then hold their blink and
estimate the tear break up time. Repeat these
observations on the other eye. If rigid lenses
are worn, remove them.
Decrease the width of the slit to that of a
parallelepiped, remove the cobalt blue and
yellow filters and move the light beam to the
temporal side. Increase the magnification to
medium or high (15-20X). Starting at the
temporal limbus, scan the cornea slowly,
looking for irregularities in corneal thick­
ness, corneal striae or folds, nerve fibres or
any other abnormalities or observable mark­
ers. When the point of specular reflection is
reached, observe the smoothness of the
endothelium, any guttae or, bumps and
degree of polymegethism. Also assess the
presence and quality of the lipid layer in the
specular reflection from the tears. Continue
the scan, remembering to move the illumina­
tion arm nasally after the corneal mid-point
is reached. Move up and then down to com­
plete a full scan of the entire cornea in a
similar manner. If corneal staining was
detected earlier, narrow the slit beam and
assess the depth of the staining.
Finally, widen the slit to form a broad
beam, move the illumination arm nasally,
and reduce the magnification to low or
medium. Evert the upper eyelid by
approaching from the temporal side. Hold
the everted lid and observe the upper palpe­
bral conjunctiva. Assess the level of injec­
tion, smoothness, presence of papillae,
follicles or concretions. Lower the eyelid and
repeat these observations on the other eye.
Record the findings, preferably on a record
card designed for this purpose. This saves
time and acts as a prompt when needed. It is
useful to use grading systems such as the
example listed in Table 18.1. Such systems
provide quantitative information regarding
findings which would otherwise need to be
laboriously described. Record all informa­
tion, even that which seems normal, such as
the visibility of corneal nerve fibres or vis­
ibility of pigmented areas on the sclera.
Sketch any abnormalities and try to estimate
size and location as well as shape. Careful
record keeping will not only provide a base­
line against which to compare future find­
ings, but in this age of increased litigation
proceedings against health care profession­
als, they may prove extremely useful as sup­
porting evidence when confronted by such a
circumstance. Finally, it should be noted that
the efficiency with which the entire biomi­
croscopy procedure is carried out can be
Table 18.1 Expected slit lamp signs in contact lens wearers
Location Condition Type of lens wear
. -­ ----­ _.-~._,-
- -­
No Lens Wear Daily Wear Extended Daily Wear Daily Wear Extended
RGP Wear PMMA Soft Lens Wear
RGP Soft Lens
Palpebral Papillae Few papillae Few papillae Mild papillae Few papillae Mild papillae Mild papillae
conjunctiva and redness No redness No redness Mild redness Mild redness Mild redness Mild redness
Corneal Staining None 3 and 9 o'clock 3 and 9 o'clock 3 and 9 o'clock Chemical 5PK Dehydration
epithelium Foreign body Foreign body . Foreign body Dehydration Metabolic 5PK
Indentation Dimple veil-
from lens ad­ ing
herence
Microcysts None None Few" Few" Few" Numerous
Corneal Striae Absent Absent Possible" Probable" Possible" Possible"
stroma
Limbal vessel Absent < 0.5 mm < 1.0 mrn" < 1.5 mm" < 0.75 mm" < 1.0 mm"
ingrowth
Central cor- Absent Absent Possible" Present" Absent Absent
neal clouding
Corneal Increased Absent Absent Probable Present" Absent Probable"
Endothelium polymeg­
ethism
Folds Absent Absent Possible" Probable" Absent Possible"
" Indicates that the presence or absence of the condition is determined by lens Dk/L, patient metabolic needs, and/or the time of day of
the examination.
 Procedure for the slit lamp examination of the anterior segment 383

Table 18.2 Suggested grading scale for recording or slit lamp signs
Sign/ Grade (0-4) Description
condition
Bulbar o No hyperaemia present. A few major vessels present but small vessels
conjunctival and capillaries are not filled and bulbar conjunctival region is essentially
injection clear and white.
1 Trace. Very slight hyperaemia of the bulbar conjunctival vessels. Several
minor vessels now apparent in bulbar region.
2 Mild. Hyperaemia of the bulbar conjunctiva and limbal region. Con­
junctival capillaries are apparent with filling of some limbal vessels.
3 Moderate. Hyperaemia of the bulbar conjunctiva obvious when viewed
with the naked eye; large numbers of capillaries and larger vessels of the
bulbar conjunctiva are apparent, although they may be localized to one
quadrant of the conjunctiva. Limbal vessels are filled and engorged,
although again, this may be localized to one area of the limbus.
4 Severe. Hyperaemia of the bulbar conjunctiva obvious when viewed
with the naked eye, all quadrants of the bulbar conjunctiva are signifi­
cantly injected with vessels of all sizes apparent. Limbal vessels are filled
and engorged around a majority of the limbus.
Palpebral o None. No papillae of follicles visible. Specular reflex from palpebral
conjunctival conjunctiva reveals the tissue surface to be smooth. Palpebral conjunc­
changes tiva appears clear and white, several major vessels may be apparent but
no capillaries.
1 Slight. No papillae or follicles visible and the specular reflex from the
palpebral conjunctiva reveals a slight unevenness across the tissue
surface. Palpebral conjunctiva has a slight pink hue, but no distinct
capillary engorgement is visible.
2 Mild. Small papillae or follicles visible and the specular reflex begins to
be appear slightly mottled due to the increased surface unevenness.
Palpebral conjunctiva has a light red hue; slight capillary engorgement
can be seen.
3 Moderate. Marked papillae and/or follicles across the majority of the
palpebral conjunctival surface. Specular reflection from the tissue is
broken up due to marked roughness of the tissue surface. Palpebral
conjunctiva has definite red hue, capillaries are engorged across the
majority of the lid.
4 Severe. Entire surface of palpebral conjunctiva is covered with large
papillae and/or follicles, specular reflection is essentially absent and is
only seen as bright pinpoints from the crests of the papillae. Palpebral
conjunctiva has a bright red hue; capillaries and larger vessels are less
distinct because of their engorgement and close proximity to each other.
Corneal 0 None. No fluorescein staining visible.
staining 1 Trace. Few localized discrete stipples, non-coalescing, in any corneal
area. Includes superficial foreign body staining.
2 Mild. Lightly coalescent punctate staining in any corneal area. Superfi­
cial, with no staining diffusion into the underlying stromal tissue.
3 Moderate. Densely coalescent punctate staining in any area of the
cornea. Slight diffusion of fluorescein into the underlying stroma after
several minutes.
4 Severe. Abrasion or erosion causing noticeable loss of the epithelial
substance over any area of the cornea. Marked and rapid diffusion of
fluorescein stain into the underlying stromal tissue.
384 Anterior ocular microscopy

Sign! Grade (0-4) Description

condition
Epithelial -0 None. No microcysts seen using retro-illumination,
microcysts 1 Trace. Fewer than approximately 50 microcysts over the central or
paracentral cornea. No overlying staining or surface anomaly.
2 Mild. More than approximately 50 microcysts over the central or
paracentral cornea. No overlying staining or surface anomaly.
3 Moderate. More than approximately 50 microcysts, tending to be coales­
cent. Surface anomaly manifesting itself as faint staining and/or dry
spots when fluorescein is used.
4 Severe. Very numerous, dense, coalescent microcysts, visible using
direct and retroillumination, with overlying staining and erosions.
Corneal o None. Normallimbal vessel dilation and advancement.
neovascular­ 1 Trace. Slight dilation and less than 1.5 mm of advancement into the
ization cornea in one quadrant only.
2 Mild. Slight dilation and less than 1.5 mm of advancement into the
cornea in more than one quadrant.
3 Moderate. 1.5 mm to less than 3.0 mm of advancement in any quadrant.
4 Severe. More than 3.0 mm of advancement in any quadrant.
Lens deposits o No surface deposits.
1 Presence of 5 or less, small «0.1 mm) individual deposits.
2 Presence of more than 5 small, individual deposits and/or one indi­
vidual deposit O.lmm to O.5mm in diameter.
3 Multiple deposits O.lmm to O.5mm in diameter or one individual
deposit larger than O.5mm in diameter.
4 Multiple deposits of O.5mm in diameter or larger.

enhanced if the practitioner knows in
advance the type of contact lens wear that the
patient is using, since there are expected slit
lamp findings for the different types of lens
wear (Table 18.2).
18.5 SLIT LAMP PHOTOGRAPHY
Since one of the main uses of the slit lamp is
to enable assessment of the structures of the
anterior segment as an aid to the detection
and diagnosis of abnormal conditions, it is
beneficial if a photographic record of any
abnormalities can be made as an aid to
identifying any changes over time.
Ideally, a photographic slit lamp with an
inbuilt flash and camera mounts should be
used for taking anterior segment photo­
graphs (Fig. 18.37). However, a less expen­
sive alternative is to attach a camera and
ordinary flash unit to a non-photographic slit
lamp (Fig. 18.38) (Khaw & Elkington, 1988).
Before describing these two methods of achiev­
ing photographic slit lamp records in detail, it
is worth introducing a few terms for the
photographic neophyte.
Film speed or ASA rating is an indicator of
how much light a particular film type needs
to provide a correctly exposed image. 'Fast'
films are those which need lower amounts of
light for correct exposure, while 'slow' films
need more light. ASA 1000 or ASA 400 films
are considered fast, while ASA 25 or ASA 64
are considered slow. Other typical film
speeds are ASA 100 and ASA 200. Fast films
usually produce grainier images with lower
resolution than do slow films. Since the
majority of changes to structures in the ante­
rior segment are subtle, such as endothelial
polymegethism or stromal striae, it is best to
use the slowest film which still provides the
correct exposure.

Figure 18.37 A dedicated photo slit lamp. Note
the camera attached to the objective housing, and
the flash charging unit attached to the table.
Figure 18.38 A non-photo slit lamp adapted for
photographic use. Note that the camera is
attached to the eyepiece, and that the flash is
positioned to provide additional illumination.
Film colour temperature describes the
spectrum of light to which the film was
designed to respond. All light sources emit a
range of wavelengths described by their
colour temperature. All these light sources
may be described as 'white' in general terms,
but, just as their colour temperatures differ,
so does the composition of their emitted
spectra. Therefore, there are more blue wave­
lengths in the apparently 'white' light emit­
ted by a fluorescent light source than in the
'white' light provided by an equivalent halo­
gen light source. Colour films are matched to
Slit lamp photography 385
the colour temperature of specific light
sources. Hence, films may be described as
'daylight' films or 'tungsten' films. Since
preferred films are often not matched to the
colour temperature of the light source
being used, specific filters can be used to
compensate for the difference in colour
temperature between film and light source
and provide a more realistic rendition of
the true colour of the subject being photo­
graphed. Daylight film should be used with
flash photography.
Exposure describes the amount of light
which is allowed to fall on the film. Expo­
sure can be controlled by the shutter speed,
i.e. the fractions of a second that the camera
shutter is open, such as 1I60th or 1I125th.
Ideally, exposure times should be kept as
short as possible since even the slightest
eye movement will cause a blurred photo­
graph. Many of the anterior segment condi­
tions which clinicians would like to
photograph at high magnification cannot
be correctly exposed using the available
illumination of the slit lamp. Therefore,
dedicated photographic slit lamps have
in-built flash systems which provide suffi­
cient light to enable correct exposure with
very short shutter speeds.
Aperture size refers to the size of the iris in
the camera lens and, along with the shutter
speed, controls the exposure of the film.
Aperture sizes are usually described in
'f-stops', where the larger the number, the
smaller the aperture size. It is important to
note that large apertures also shorten the
depth of field. However, in most commer­
cially available photo slit lamps, the camera
is used without a lens, so that lens aperture is
not a factor. In slit lamps where the camera
and lens are positioned behind the eyepiece
(e.g. the Holden-Zantos technique, described
later), changing the aperture setting on the
camera lens changes the size of the field of
view but not the brightness. In this situation,
film exposure is controlled by camera shutter
speed or flash intensity.
386 Anterior ocular microscopy
18.5.1 DEDICATED SLIT LAMP
PHOTOGRAPHY
Slit lamps designed for photography have
two additional facilities - an in-built flash
system which allows a powerful beam of
high intensity light to be generated and
flashed through the slit beam as illumination
for the photograph, and a camera mount
somewhere on the slit lamp microscope.
The flash system usually uses a semi­
reflecting mirror in the illumination system
of the slit lamp to allow both the regular
incandescent/halogen light and the flash
tube generated light to follow identical path­
ways through the slit aperture. Therefore, all
structures illuminated by the regular light
source will be illuminated in the same pat­
tern by the flash tube. However, the inten­
sity of the flash will be very different, and
this is usually controlled
. by
. a switch on the
flash generator housing. Typically there are
four settings for flash intensity, and the best
one to use will vary according to the reflec­
tance of the structure being viewed and the
slit lamp set-up chosen.
Thus, a photograph of the sclera and bul­
bar conjunctiva will require a low intensity
flash setting, whereas a high magnification
shot of an optic section of the cornea will
require a high flash intensity. The optimum
flash settings for a particular photographic
slit lamp are best determined by trial and
error, and they will depend on the film ASA
rating and the structure being photographed.
However, a good starting point is to use 200
ASA film and the lowest intensity flash set­
ting for diffuse and broad beam, low magni­
fication shots; and the highest flash intensity
setting for low magnification parallelepiped
and optic section photographs, and for all
medium to high magnification shots. It
should be noted that good fluorescein photo­
graphs of corneal staining or rigid lens fitting
patterns can only be taken using a slit lamp
equipped with a high output flash tube
when the cobalt blue and yellow barrier
filters are used. Film exposure guidelines for
photographic slit lamps have been published
elsewhere (Spivak, 1977; Zantos et al., 1980).
The camera mount for most photographic
slit lamps is placed between the objective
lens and the magnification tumbler of the
microscope; a right-angled prism or semi­
reflecting mirror is put into the optical path­
way of the microscope to divert the images to
the camera during photography but not dur­
ing normal viewing. A bayonet-type mount
is usually provided to which the camera
body (without lens) is mounted. The mount
is designed in such a way that the image
seen by the observer in the eyepieces is
focused simultaneously on the film plane of
the camera, ensuring correct focus if the
microscope eyepieces have been correctly
adjusted for the observer. Drawbacks to this
type of mounting system is that the magnifi­
cation of the camera image is fixed, and
relatively low (approximately 3X), since the
light rays from the objective lens are diverted
before entering the magnification tumbler of
the microscope head. This limitation can be
overcome by mounting the camera and lens
over the eyepiece of the microscope, as
described by Zantos and Holden. This tech­
nique takes full advantage of the increased
magnification available through the magnifi­
cation tumbler of the microscope and alter­
nate high powered eyepieces. A special
adapter must be fabricated which clamps
over the eyepiece and screws into the filter
thread of the camera lens to utilize the
Holden-Zantos technique, but this inconve­
nience is far outweighed by the versatility
and enhanced convenience that variable
magnification and through-the-camera
focusing bring to any photographic slit lamp.
18.5.2 PHOTOGRAPHY USING A
NON-PHOTOGRAPHIC SLIT LAMP
While a dedicated photo slit lamp is the
preferred method of taking photographs of
the anterior ocular segment, quite useful

photographs can be obtained by using an
auto-exposure camera mounted over the eye­
piece of a regular non-photographic slit
lamp. The 35 mm camera used should have
an aperture priority, auto-exposure mode.
The major limitation of this method of pho­
tography is the lack of flash, and hence a
relatively high speed film should be used
(e.g. 400 ASA) in conjunction with the high­
est light settings of the slit lamp. Ideally, the
slit lamp should have a halogen or other high
output light source, and the film colour tem­
perature should be matched to the light
sources. Since the exposure is automatic and
controlled by shutter speed, a good estimate
of correct exposure can be judged by listen­
ing to the shutter open and close. For shutter
speeds of slower than 1I8th second, move­
ment of the eye will blur the photograph.
Faster shutter speeds can be produced by
increasing the light intensity of the slit lamp
or by increasing the width of the slit beam.
This technique is limited to white light
photographs, and cannot provide high mag­
nification images of optic sections, but it can
provide reasonable quality photographs of
many features of the anterior segment which
are of interest to the contact lens clinician.
18.5.3 VIDEO PHOTOGRAPHY WITH TIfE
SLIT LAMP
The recent availability of relatively inexpen­
sive video cameras and recorders has made
the rather appealing concept of recording slit
lamp observations on video tape a clinical
reality. The major advantage which such a
system provides over 35 mm photography is
the ability for immediate playback. This is
particularly useful in describing the appear­
ance of many conditions such as lens depos­
its or lens fitting to patients or colleagues.
Video cameras are usually attached to slit
lamps using the same beam-splitters incor­
porated for 35 mm photographic attach­
ments. Ideally the camera should be compact
and light-weight to minimize any interfer-
References 387
ence with the balance and normal function­
ing of the slit lamp. Cameras with remote
heads are ideal for this use. Contemporary
video cameras have sufficient gain to allow
good images to be captured with the incident
light from the slit beam, negating the need
for additional light sources or low illumina­
tion 'surveillance-type' cameras. The other
parameter which should be considered is the
resolution capabilities of the camera. Black
and white cameras generally have higher
resolution capabilities than colour cameras
but, for the majority of clinical applications,
the advantages of a colour image far out­
weigh the loss of resolution. It should be
noted, however, that while the majority of
colour cameras have sufficiently high resolu­
tion to demonstrate lens fitting performance
and front surface deposits, oniy the most
expensive have the capability of capturing
high resolution images such as the endo­
thelial mosaic. The interested reader IS
directed to recent publications regarding
clinical videography both with and without
slit lamps (Robboy & Hammack, 1987).
REFERENCES
Berliner, M.L. (1943) Biomicroscopy of the Eye. Paul
B. Hoeber, Inc.
Brandreth, R.H. (1978) Clinical Slit Lamp Biomicros­
copy, Blaco Printers, Inc.
Butler, T.H. (1924) Focal illumination of the eye.
Br. J. Ophthal., 8(12), 561-90.
Cavanagh, H.D. et al. (1990) Confocal microscopy
of the living eye. CLAO L 16, 6~73.
Doggart, J.H. (1949) Ocular Signs in Slit Lamp
Biomicroscopy. C V. Mosby Co., St Louis.
Goldberg J.B. (1984) Biomicroscopyfor Contact Lens
Practice: Clinical Procedures. The Professional
Press, Inc.
Holden, B.A., Zantos, S.G. and Jacobs, K.J. (1978)
The Holden-Zantos technique for endothelial and
high magnification slit lamp photography. Bausch
& Lomb.
Khaw, P.T. and Elkington, A.R. (1988) Slit-lamp
photography made easy by a spot metering
system. Br. J. Ophthalmol., 67, 63-6.
Koester, CJ. and Roberts, CW. {199m Wide-field
388 Anterior ocular microscopy
specular microscopy. In Non-invasive Diagnostic
Techniques in Opthalmology (ed. B.R. Masters),
Springer-Verlag, New York, pp. 91-121.
Long, W.F. (1988) How to choose and use a
photoslitlamp. Review of Optometry, Septem­
ber, 69-73.
Martonyi, CL. (1989) Photographic slit-lamp
biomicroscopes. Ophthalmology - Instrument
and Book Issue, pp. 6-19.
Masters, B.R. and Kino, G.S. (1990) Confocal
microscopy of the eye. Noninvasive Diagnostic
Techniques in Opthalmology (ed. B.R. Masters),
Springer-Verlag, New York, pp. 152-71.
Mayer, D.J. (1984) Clinical Wide-Field Specular
Microscopy. Balliere Tindall.
Muller, O. Ocular Examination with the Slit Lamp.
Carl Zeiss Publication I<3O-1l5-e.
Nom, M.S. (1983) External Eye: Methods of Exami­
nation. Scriptor Publisher ApS.
Robboy, M.W. and Hammack, G.G. (1987) Video­
tape recording system for use with the slitlamp
biomicroscope. j. Am. Optom. Assoc., 58(4),
290-2.
Schmidt, T.A.F. (1975) On slit-lamp microscopy,
Doc. Ophihalmol., 39, 117-53.
Spivak, T.W. (1977) Photography through the
Nikon slit lamp biomicroscope. Rev. Opiom.,
November, 56-60.
Stockwell, H.J. (1983) Considering the slit lamp and
biomicroscope, The Optician, June 10, 16-20.
Terry, J.E. and Kercheval, D.B. (1979) Interpretive
biomicroscopy. J. Am. Optom. Assoc., 50, 79J-..­
803.
Zantos, S. G. (1984) Slit Lamp Examination of Con­
tact Lens Wearers. Bausch & Lomb.
Zantos, S.G. and Pye, D.C. (1979) Clinical photog­
raphy in optometric practice, Aust. j. Opiom.,
62,279-85.
Zantos, S.G., Holden, B.A. and Pye, D.C. (1980)
A guide to ocular photography with the
Nikon photo slitlamp. Aust. J. Optom., 63,26­
32.
PART 2 REAL-TIME CONFOCAL MICROSCOPY OF THE IN VIVO HUMAN CORNEA

B.R. Masters, A.A. Thaer and a.-c. Geyer

18.6 ABSTRACT and devoid of any digital image processing

A new, non-applanating, real-time slit scan­
ning confocal microscope is described for the
in vivo examination of the living human eye.
This confocal microscope produces real-time
video images of the in vivo human cornea. In
contrast to other confocal microscopes
designed for in vivo ocular imaging, which
are based on either the Nipkow disk (pin­
holes) or the modified wide-field specular
microscope (a photographic system which is
not real-time), this new instrument produces
images which are captured as single video
frames which have superior contrast. This
new real-time slit scanning confocal micro­
scope produces en face, high contrast, high
resolution, images of superficial epithelial
cells, wing cells, epithelial basal cells, corneal
innervation, nuclei of stromal keratocytes,
and the cell bodies of the stromal keratocytes
in the posterior stromal region.
18.7 INTRODUCTION
This chapter presents the principles of in vivo
coniocal microscopy and applications to the
observation of the in vivo human cornea. A
new, real-time, scanning slit confocal micro­
scope is described. Examples are presented
of confocal microscopic examination of the
normal human cornea which illustrate the
resolution and the contrast of single video
frames. The photographs are unprocessed
Contact Lens Practice. Edited by Montague Ruben and Michel Guillon.
enhancements.
The primary instrument for the examina­
tion of the cornea is the slit lamp. This
important instrument was developed in its
present form by Gullstrand who developed
the system to condense the illumination
light into a slit aperture and then project
the image of the slit into the eye. The
modern slit lamp uses a narrow slit which
removes the light scattered from adjacent
tissue and provides a degree of optical
sectioning. However, the slit lamp pro­
vides an oblique view across the cornea
and provides limited contrast of cellular
components.
A more recent development for imaging
the cells of the corneal endothelium is the
specular microscope. If the illumination and
the observation axes are adjusted correctly,
then the specular reflection from the bound­
ary cf the corneal endothelium and aqueous
humor will show the cell outlines of the
endothelial cells. These cells were first
observed and described by Vogt who used
the modified Gullstrand slit lamp to examine
the human corneal endothelium. There are
several problems with the specular micro­
scope; the surface reflection from the tear­
corneal interface degrades the contrast of the
endothelial cells. An edematous stroma will
also reduce contrast of the endothelial cells.
The field of view is small with the standard
specular microscopes. The development of a

Published in 1994 by Chapman & Hall, London. ISBN 041235120 X

390 Anterior ocular microscopy

scanning wide-field specular microscope by
Koester provided high-contrast, wide-field
views of about one square millimeter of
corneal endothelium. This microscope
scanned a narrow slit over a larger area of the
endothelium and recorded the composite
image on film.
Both the slit lamp and the specular
microscope are limited in their ability to
observe cellular and subcellular details
throughout the full thickness of the cornea.
In this chapter we introduce a new tech-
nique, real-time scanning slit confocal
microscopy, with the capability to over­
come the limitations of the slit lamp and
the specular microscope. In vivo real-time
scanning sli t confocal microscopy provides
a new approach to the observation of the
human eye.
18.8 CONFOCAL MICROSCOPY
This section provides a brief introduction to
the principles of confocal microscopy and

Figure 18.39 In vivo confocal microscopic en face image of human cornea. The U-matic tape was
stopped on a single video frame, using the pause mode, and a 35 mm film camera was used to
photograph the television monitor to produce the image. No digital or analog image enhancement or
processing was used. There was no frame averaging applied to the video images. The photograph
shows the raw data directly photographed from the screen of the television monitor. This figure shows
a single video frame of the superficial cells of the corneal epithelium. This cell layer, which is just below
the tear film, has the following characteristics: the cell nucleus appears as a highly reflecting bright oval
which is surrounded by a darker band; the focal plane is set to optically section the most superficial
cells which appear brightest; cells slightly below this focal plane appear darker. Some of these
superficial epithelial cells are in the process of desquamation. Objective 25X, NA 0.6.
 Confocal microscopy 391

Figure 18.40 In vivo confocal microscopic en face image of human cornea. This optical section is in the
plane of the wing cells which are located between the superficial epithelial cells and the basal epithelial
cells of the human cornea. The image of these cells is characterized by the bright cell nuclei which are
devoid of the darker band which is observed in the superficial epithelial cells. Objective SOX, NA 1.0.

describes the basic types of confocal micro­
scopes (Wilson, 1990; Pawley, 1990). First we
answer the question: What is a confocal
microscope?
In 1957 Minsky filed a patent application
for a confocal microscope which elucidated
the principles of operation upon which
today's commercial confocal systems work.
A confocal microscope has two sets of pin­
holes, or apertures; which are arranged in
conjugate, or confocal, planes. One pinhole
or slit is placed in the illumination path
and another set is placed in the detection
path. These sets of conjugate apertures only
illuminate a narrow field of the specimen
and detect the back-scattered and reflected
light from the same narrow field of view.
The confocal microscope has a scanning
mechanism such that the narrow field of
illumination, which is identical to the
detection field, is scanned over the speci­
men to generate a composite image of it.
This is the principle of confocal micros­
copy.
In a standard microscope - which is
operated in the epi-illumination mode ­
the lateral and axial resolutions are func­
tions of the wavelength of the light used for
the illumination and of the numerical aper­
ture of the microscope objective. In a confo­
cal microscope there is an increase in both
the axial and lateral resolutions of the
microscope objective. While the increase in
the axial resolution of a confocal micro­
392 Anterior ocular microscopy

Figure 18.41 In vivo confocal microscopic en face image of human cornea. The optical section is in the
plane of the basal epithelial cells. The image shows the bright cell borders and the darker cell interiors.
The cell borders appear thick since there is a high degree of invagination between the adjacent cells.
Water immersion microscope objective sox, NA 1.0.

scope is important, it is the increase in the degraded in sharpness and contrast as in a

lateral resolution which is of prime impor­
tance for imaging thick specimens. The
increased lateral resolution gives the confo­
cal microscope the unique ability to opti­
cally section the specimen. It is important
to note that the axial resolution is about
three times the lateral resolution.
There are several advantages of the
optically-sectioning capability of a confocal
microscope as compared to the standard
microscope. The first advantage is that
images are obtained with enhanced con­
trast; this is because the confocal micro­
scope excludes light scattered from optical
sections above and below the section in the
focal plane, therefore the focal plane is not
standard light microscope. The second
advantage is that thick sections of living
tissues and cells can be optically sectioned
for observation and subsequent three­
dimensional reconstruction.
There are several types of confocal micro­
scopes. Some designs are based on a rotating
disk which contains many sets of pinholes
arranged in a spiral pattern. This design is
called a Nipkow disk-based microscope;
such designs are further divided into
tandem-scanning confocal microscopes and
one-sided Nipkow disk-based confocal
microscopes.
The tandem-scanning confocal microscope
uses two sides of the disk which contains
 Confocal microscopes for in vivo clinical examination of the cornea 393

Figure 18.42 In vivo confocal microscopic en face image of human cornea. This is an optical section of
the basal epithelial cells similar to that shown in Figure 18.41. The wider field is obtained with the use
of a microscope objective with a lower magnification. Water immersion microscope objective 25X, NA
0.6.

two sets of conjugate pinholes. One set of They are also real-time confocal systems.
pinholes is in the illumination path, and the
other is in the detection path. As the Nipkow 18.9 CONFOCAL MICROSCOPES FOR IN
disk rotates, the full field of view is swept out VIVO CLINICAL EXAMINAnON OF THE
by both the illumination spots of light and CORNEA
the detec.tion spots. Since both sets of pin­
holes are conjugate to each other the system The advantages of confocal microscope
is a confocal microscope. It is called a imaging systems as compared to standard
tandem-scanning confocal microscope since microscopy include enhanced x, y and z
both sets of pinholes rotate in tandem. resolution and improved contrast of the
Another type of Nipkow disk-based images. These factors permit the optical
microscope uses the same set of pinholes sectioning of thick living tissue. The result­
on one side of the rotating Nipkow disk for ing images are sharp and free from signifi­
both the illumination path and the detec­ cant blur due to contributions of scattered
tion path. This is the basis of the so-called light from adjacent sections. In contrast to
one-sided, real-time, confocal microscope. slit lamp observations, and other standard
Other designs are based on conjugate slits. imaging techniques, confocal microscopy is
394 Anterior ocular microscopy
Figure 18.43 In vivo confocal microscopic en face image of human cornea. This optical section shows the
corneal nerves in the anterior stroma just below Bowman's membrane. The large bifurcating nerves are
easily observed. Objective 25X, NA 0.6.
a new paradigm for ocular visualization.
Confocal microscopy permits real-time
observation across the full thickness of the
cornea (Lernp et al., 1986). The use of confo­
cal microscopic imaging systems permits
two-dimensional, high-resolu tion imaging
of the cornea and ocular lens which is sharp
and in the plane of the cornea perpendicu­
lar to the optic axis (Masters, 1990a; Mas­
ters and Kino, 1990) and in the plane of the
cornea perpendicular to the optic axis. The
advances made possible with ocular confo­
cal microscopy are not limited to corneal
imaging. A major development in oph­
thalmic imaging has been the adoption of
confocal microscope imaging systems to
image ocular structures (Beuerman et al.,
1992; Wegener et al., 1992; Masters, 1988).
Confocal microscopy has been used to
observe living specimens and provides high
resolution, high contrast images with good
optical sectioning capability (Koester and
Khanna, 1989; Koester and Roberts, 1990;
Florakis et al., 1992; Koester et al., 1992;
Koester, 1993). Confocal microscopy has also
been used to examine the ex vivo eye.
Tandem-scanning confocal imaging of ill
vivo ocular tissue has been reported. A one­
sided Nipkow disk confocal imaging system
has been used to observe the excised rabbit
cornea and ocular lens.
The in situ observation and visualization
of the ocular lens with confocal microscopy
is a major technological advance in ocular
lens imaging. The feasibility of confocal
microscopy is to produce high contrast,
 A new real-time scanning slit system for clinical examination of the cornea 395

Figure 18.44 In vivo confocal microscopic en face image of human cornea. This optical section shows the
corneal nerves in the anterior stroma just below Bowman's membrane. Objective 25x, NA 0.6.

high resolution images across 1.7 mm of
ocular tissue. The in situ observation of the
lens capsule, lens epithelium.. lens suture
and detailed structure of the lenticular
fibers provides a new approach to lens and
cataract research.
The development of the wide-field specu­
lar microscope by Koester was limited by the
low numerical aperture of the applanating
cone objective (Florakis et al., 1992; Koester et
al., 1992; Koester, 1993). Recent develop­
ments of a high numerical aperture for the
wide-field specular microscope have resulted
in a confocal microscope for the eye. How­
ever, this confocal microscope has two disad­
vantages - it is a photographic system (and
therefore not real-time) and the microscope
objective flattens (applanates) the cornea.
The corneal flattening, due to the applanat­
ing cone of the microscope objective, limits
its use in the clinic and tends to modify the
normal structure of the cornea.
18.10 A NEW REAL-TIME SCANNING SLIT
SYSTEM FOR CLINICAL EXAMINATION OF
THE CORNEA
The in vivo observation of the living cornea
by the technique of confocal microscopy pro­
vides en face images of high contrast and
resolution. Slit-based confocal systems col­
lect more light from the eye as compared to
Nipkow disk pinhole confocal microscopes.
Therefore the light incident on the cornea
can be reduced.
We describe a new flying slit, real-time
confocal microscope which has unique imag­
ing characteristics for in vivo human confocal
396 Anterior ocular microscopy
Figure 18.45 In vivo confocal microscopic en face image of human cornea. This optical section shows a
large corneal nerve in the anterior stroma. The bright oval objects are the nuclei of the stromal
keratocytes. Objective 25X, NA 0.6.
microscopy (Thaer et al., 1990; Masters and
Thaer, 1993a,b,c). This system uses non­
applanating microscope objectives and the
illumination is provided by a halogen
lamp.
This new real-time confocal microscope
has several unique and advantageous fea­
tures which differentiate it from other in vivo
confocal imaging systems.
1. The standard light source is a halogen
lamp which has many advantages over
the mercury or xenon arc lamps used in
other confocal designs. The mercury or
xenon arc lamps have the problem of
arc jitter which results in changing
illumination intensity. The halogen
lamp offers steady levels of illumina­
tion. Filters are inserted in the lamp
housing to remove both short ultravio­
let and infrared light. The light levels
at the cornea are less than those from
the wide-field specular microscope.
The light intensity is sufficiently low
so that after 30 minutes of continuous
examination of a subject's cornea the
subject has no after image from the
microscope.
Other in vivo confocal microscopes
for corneal examination use a mercury
or xenon arc lamp as the source of
illumination. These sources are much
brighter than the new real-time system
described in this chapter.
2. The microscope uses standard non­
applanating microscope objectives with
 A new real-time scanning slit system for clinical examination of the cornea 397

Figure 18.46 In vivo confocal microscopic en face image of human cornea. This optical section shows the
oval shaped nuclei of the stromal keratocytes. Objective 25X, NA 0.6.

RMS threads which are readily inter­
changeable. This permits the use of sev­
eral different microscope objectives and
provides the capability to vary the field
of view and the magnification. The stud­
ies we describe in this chapter used a
Leitz sox, NA 1.0 water immersion
objective. Other studies which required
a larger field of view used a Leitz 2SX,
NA 0.6 water immersion objective.
Other in vivo confocai systems are
limited to a built-in objective which is
not removable. Some microscopes
require custom designed microscope
objectives which are of high cost.
3. The confocal microscope is based on two
sets of adjustable conjugate slits which
are located in conjugate planes in front
of the halogen source and in front of the
detector. Scanning of the image of the
slit over the plane of the cornea is
accomplished with the use of an oscillat­
ing two-sided mirror which is used for
beth scanning and descanning. The
scanning bilateral system based on a
double-sided mirror was published over
24 years ago (Svishchev, 1969). However,
there are alternative methods of scan­
ning and des canning which may have
advantages over the bilateral scanning
system which is described here.
4. The detection system consists of an
intensified video camera with video out­
put to a Sony U-Matic tape recorder. The
PAL video format provides 62S lines.
The U-Matic video tape recorder pro­
vides a high bandwidth. In parallel with
the video recording there is a video
398 Anterior ocular microscopy

._~''::::::-
._-------~
-~-_._-

------­
_------­
_..

Figure 18.47 In vivo confocal microscopic en face image of human cornea. This optical section shows the
polygonal corneal endothelial cells. This image is 500 microns below the surface of the cornea (Figure
18.39). The focal plane is at the interface between the cornea and the aqueous humor. Objective 2Sx,
NAO.6.

monitor in order that -the operator can
observe the confocal images of the sub­
ject's eye in real time.
5. Hard copy of individual video frames is
obtained immediately with a video
printer. Individual video frames can also
be digitized. These digital images can be
registered and preprocessed by digital
image processing techniques for further
analysis and quantification. However, in
this chapter all the figures are photo­
graphs of single video frames of the raw
data - no analog or digital enhancements
were used.
6. The real-time confocal microscope is
mounted on a special ophthalmic head
rest (Leitz). The microscope stand pro­
vides three-axis control. Immediatelv
before the corneal examination, the eye
was anaesthetized. The subject was
seated at the ocular examination table
and a drop of methylcellulose gel was
applied to the tip of the microscope
objective; the microscope was slowly
positioned to make contact between the
gel and the cornea. The tip of the micro­
scope objective never touches or flattens
the corneal surface; a layer of gel is
always present between the objective
and the eye. The z-axis position of the
confocal microscope was controlled by
manual movement of the microscope
stage with a joystick. Alternatively, a
computer-controlled stepping motor
 A new real-time scanning slit system for clinical examination of the cornea 399

Figure 18.48 In vivo confocal microscopic en face image of human cornea. This optical section shows the
polygonal corneal endothelial cells. This image is 500 microns below the surface of the cornea (Figure
18.39). The numerous small black particles are due to inflammation of the eye. The focal plane is at the
interface between the cornea and the aqueous humor. Objective 25x, NA 0.6.

scanned the focal plane Of the confocal
microscope across the full thickness of
the cornea.
7. The microscope is built from a modular
system. This design concept permits
rapid modification of the system to suit
different applications. The confocal
microscope can be used in several
modes.
The first configuration is capable of obtain­
ing real-time (video rates) en face optical
sections across the full-thickness of the in
vivo human cornea. The thickness of the
optical section can be modified by adjust­
ing the width of the two confocal slits in
the microscope. The depth of the optical
section can be manually adjusted, or auto­
mane continuous scanning (Z-Scan attach­
ment) oi the depth of the focal plane can be
initiated. Both scan time and scan depth
can be adjusted.
The second configuration provides a quan­
titative measure of the reflected and back­
scattered light in a given focal plane. This
Z-Scan attachment is used for the confocal
photometric recording of scattered light and
fluorescence intensity profiles through the
full thickness of the cornea. The Z-Scan can
also be used for quantitative photometric
measurements of fluorescence and/or scatter­
ing in the anterior chamber.
This configuration can also be used for
fluorescence measurements. Both autofluo­
400 Anterior ocular microscopy

Figure 18.49 In vivo confocal microscopic en face image of human cornea. This optical section shows the
polygonal corneal endothelial cells. This image is 500 microns below the surface of the cornea. The
numerous small black particles are due to inflammation of the eye. The focal plane is at the interface
between the cornea and the aqueous humor. This image shows a different field of view than in the.
previous figure. Objective 25X, NA 0.6.

rescence and fluorescence from externally
applied fluorescent dyes or fluorogenic sub­
strates can be measured (Thaer et al., 1990).
This configuration is suitable for the in vivo
measurements of cellular metabolism based
on the principles of non-invasive redox fluo­
rometry.
18.11 RESULTS
The following in vivo human corneal confocal
microscopic images were made by photo­
graphing the single still video frames on the
monitor with a camera. These figures are
photographs of single video frames. There
was no image processing or frame averaging.
The figures are typical raw data. The figures
show the superficial cells (Fig. 18.39), the
wing cells (Fig. 18.40) and two fields of the
normal human basal epithelium (Figs. 18.41
and 18.42). High contrast photographs of
corneal nerves are shown in Figs. 18.43­
18.46. The corneal endothelium is shown in
Figs. 18.47-18.50.
The full three-dimensional reconstruction
of the ex vivo rabbit cornea is the only figure
in this chapter which is not from the in vivo
human cornea (Fig. 18.51). The optical sec­
tions which were used for the reconstruction
were obtained with a laser scanning confocal
microscope. The inclusion of this figure in
the chapter is to present the future of in vivo
 Discussion 401

FiSU'" 18.50 In viv. eonfocel microKOpic ... f.<e image 01 human come" The image shows a ..pon of
the comeal endothelium with endothelial defects. The three black regions represent endothelial defects.

sufficient light collection efficiency so that

three-dimensional visualization of ocular single video frames show high contrast,
imaging. sharp images of the living in vivo human eye.
There is no need for video frame averaging
or digital image processing techniques to
18.11 DISCUSSION
enhance the contrast of the single video
A new, non-applanating, non-invasive real­ images. This is not the case for confocal
time, slit-scanning confocal microscope has microscopes designed on Nipkow disk (pin­
been developed for the observation of the in hole) configuration - they pass such low light
vivo human eye. This chapter illustrates the from the eye to the camera that frame averag­
use of the instrument for the observation of ing and digital image processing enhance­
the cornea. The high contrast and large light ments are required.
collection efficiency of the microscope are The light collection efficiency of this flying
due to the use of confocal slits in conjunction slit in vivo ocular confocal microscope is due
with a high numerical aperture objective. to the use of slits (instead of pinholes) and a
The microscope does not contact the cor­ microscope objective with a numerical aper­
nea nor does it applanate the surface of the ture of 1.0. These two design implementation
cornea: a polymer gel is applied in a layer results are bright, sharp, high-contrast, high­
between the ocular surface and the tip of the resolution confocal images of the in vivo
microscope objective. This instrument has
402 Anterior ocular microscopy

Figure 18.51 Three-dimensional volume reconstruction of the full thickness of the ex vivo rabbit cornea
shown in an isometric view. The endothelium is on the top of the figure and the epithelium is on the
bottom. The linear bright feature on the front face is a nerve fiber. The thickness of the reconstruction is
400 microns and the epithelial thickness is 40 microns. This figure demonstrates that thin optical
sections of living cornea can be reconstructed in a computer to yield a three-dimensional volume
visualization of the cornea.

human cornea. The images are obtained in
real time and are stored on magnetic video
tape (Sony V-Matic).
The advantages of slits over confocal
microscope systems based on Nipkow disks
containing pinholes are the following. Slits
have the possibility of continually adjusting
the depth of focus on the z-axis. In addition,
a slit confocal system passes many times
more light from the subject's eye to the
detector as compared to pinhole type confo­
cal systems. Another advantage of adjustable
slits is that the scanned field of view and the
light intensity, incident on the field of view,

can be easily varied. Finally, the light inten­
sity incident on the subject's eye is signifi­
cantly less with a slit confocal microscope as
compared to a Nipkow disk pinhole system.
This new in vivo confocal instrument has
unique advantages over other confocal sys­
tems. The bright, high contrast confocal
images of the wing cells and the basal cells
demonstrate its unique optical characteris­
tics. The low reflectivity of the wing and
basal epithelial cells in the normal human
cornea presents a low contrast object for
confocal microscopy. The high rejection of
stray light and narrow depth of field, coupled
with the high numerical aperture microscope
objective (NA 1.0), results in the ability of the
instrument to clearly image these cell layers
in the live, normal human cornea.
The clear advantage of slit-scanning confo­
cal microscopes (the new microscope dis­
cussed here, and the Koester wide field
specular microscope) for ophthalmic diag­
nostics and basic eye research is best appre­
ciated when imaging the in vivo human basal
epithelium in the anterior cornea. The real­
time confocal microscope described here pro­
vides high-contrast, high-resolution images of
both the wing and basal epithelial cells in the
normal in vivo human eye. The modified wide­
field specular microscope of Koester has the
ability to image basal epithelial cells in the
normal in vivo human eye. however, the
Koester microscope uses a 35 mm film camera
as the detector and is therefore not real-time.
Ocular confocal microscopes designed on
the slit-scanning principle provide sensitiv­
ity and resolution that has not been possible
with Nipkow disk based microscopes. The
advantages of a slit-scanning confocal system
over a Nipkow disk pinhole system are illus­
trated below.
A Nipkow disk (pinhole) real-time confo­
cal microscope (Tandem Scanning Corpora­
tion, Inc.) did not have the capability to
image basal epithelial cells in vivo in the
normal eye; however, in swollen or patho­
logical specimens it was capable of imaging
Discussion 403
the basal epithelial cells. Jester et al. have
written several papers in which they cat­
egorically state their inability to image basal
epithelial cells in the normal in vivo cornea
(jester et al., 1990; 1991; 1992). They state,
'Reflections from cells below the surface are
not normally resolved but can be imaged in
certain disease states.' In another paper
Jester et al. state, 'By in vivo confocal micros­
copy, only the first layer of superficial cells
can normally be imaged in the intact, living
eye.' They then state, 'In vivo confocal optical
sections do not detect any reflections from
the wing and basal cell layers in the intact,
normal living eye.' Finally, in a recent paper
published in Investigative Ophthalmology and
Visual Science, Jester et al. wrote on their in
vivo confocal observations of the normal cor­
nea in the live rabbit, 'Below the superficial
epithelium, wing and basal epithelial cells do
not appear to reflect light above background
levels. Because the cornea is a transparent
tissue and optically configured not to scatter
light, it is to be expected that some structures
of the cornea may not be visualized. . .
under pathologic and ex vivo conditions,
images of the wing and basal epithelial cells
can be "detected.' Although they averaged
video frames they were still unable to image
the normal in vivo basal epithelial cells. They
used a Nipkow disk confocal microscope
produced by the Tandem Scanning Corpora­
tion, Inc. Furthermore, they 'explain' their
inability to image basal epithelial cells in the
normal in vivo eye by stating that the normal
epithelium of the cornea is transparent and
should not reflect light. Our results on many
human subjects, all made on the normal
living cornea, are at variance with the results
of Jester et al. Koester has also published
results indicating the ability of his wide-field
specular microscope (slit confocal micro­
scope) to image the basal epithelial cells in
the normal in vivo eye. Our results, and those
of Koester, demonstrate the advantage of a
slit-system-based confocal microscope for
the in vivo examination of the eye.
404 Anterior ocular microscopy
The in vivo real-time scanning slit confocal
microscope can be utilized for two other
types of imaging. The set of optical sections
of the full thickness of the cornea provides a
new way to visualize the cornea. The set of
optical sections can be constructed into a full
three-dimensional visualization (Masters,
1991a,b,c; Masters and Paddock, 1990; Mas­
ters, 1993; Masters and Farmer, 1993). This is
illustrated in Fig. 18.51. Once the two­
dimensional optical sections are formed into
the three-dimensional image in the com­
puter, it can be viewed from any angle and
the transparency of the view varied. There­
fore the observer could look inside the cor­
nea from any angle.
The second type of confocal imaging is
redox imaging. This method is based on the
intrinsic fluorescence from the reduced pyri­
dine nucleotides. Several papers illustrate
this method of non-invasive metabolic imag­
ing of the cornea (Masters, 1990b; Masters et
al.,1993).
18.13 APPLICATIONS OF CONFOCAL
MICROSCOPY TO CONTACT LENS SCIENCE
What are some of the applications of this
new real-time slit scanning confocal micro­
scope in basic eye research and in clinical
ophthalmology?
The instrument is currently configured for
real-time confocal microscopy of the cornea.
With the introduction of a microscope objec­
tive with a longer free-working distance it
would be possible to image the in vivo
human lens. Applications to the ocular lens
include the investigation of the natural pro­
gression of cataracts in animal models and in
human subjects. The investigation of drugs
which cause retardation of cataract develop­
ment could be monitored in vivo with the use
of the confocal microscope.
The in vivo study of intraocular lenses in
humans could be performed. The effects of
laser surgery on the physical integrity and the
optical quality of the intraocular lens could be
studied. Also, the migration and adhesion of
cells to the intraocular lens could be investi­
gated with this real-time confocal microscope.
In vivo studies of the cornea and the tear
film are current themes of investigation. A
major use of this new in vivo confocal micro­
scope is the investigation of the etiology and
treatment of the dry eye. The new imaging
capability permits new studies of tear secre­
tion, the renewal of the ocular surface and
the pathophysiologic conditions which
result in the dry eye.
Another area of investigation is the
anatomy and physiology as related to contact
lens wear. The real-time in vivo confocal
microscope could follow the time-dependent
changes on both the contact lens and the
cornea. The real-time system could follow
changes in corneal innervation, renewal of
the corneal epithelium, changes in the cor­
neal stroma and changes which occur in the
corneal endothelium.
The effects of corneal laser surgery on the
migration and distribution of stromal kerato­
cytes is currently under investigation. The
real-time in vivo confocal microscope is
capable of imaging the keratocyte nuclei, and
therefore keratocyte densities prior to and
after laser refractive surgery. The investiga­
tion of the time dependence of the post
surgery stromal haze, as well as its quantita­
tive analysis with respect to size and light
scattering ability, is currently under way on
human subjects.
The use of the real-time in vivo confocal
microscope for the diagnosis and examina­
tion of corneal dystrophies, corneal erosions,
superficial punctate keratopathy, corneal
inflammation and corneal infection are clini­
cal applications in which new technology
will lead to better diagnosis, treatment and
understanding of ocular disease.
ACKNO~EDGEMENTS
This work was supported by a grant from
NIH, National Eye Institute, EY-06958
(BRM). The authors acknowledge the

co-operation from The Institute for Medical
Vision Aid and Helmut Hund GmbH.
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