This document discusses techniques for measuring corneal thickness and examining the cornea using confocal microscopy. It describes pachymetry methods including optical, ultrasonic, and specular microscopy pachymetry. Ultrasonic pachymetry is now the preferred method due to its ease of use, precision and ability to measure thickness eccentrically. Confocal microscopy allows high magnification examination of the living cornea and measurement of thickness for each layer.

1. Corneal Pachymetry &
confocal microscopy
Dr. Ram Singh
(Department of Ophthalmology)
S.P. Medical College, Bikaner

2. Pachymetry: - Literally meaning of this word– Thickness
• Measurement of corneal thickness is important factor in
deciding different kind of refractive surgeries.
• It is can be performed with optical pachometer or an ultra
sonic pachometer.
• Certain specular's microscopes are calibrated in such a
way so that they can measure corneal thickness while
focusing corneal endothelium.

3. OPTICAL PACHYMETRY :- This was the
original method to measure corneal
thickness.
• - It is used with slit lamp. Non Contact
Method
• - optical pachymentry has advantage -
• (1) It does not touch the cornea so does
not damage epithelium which
sometimes happen with contact
methods eg. ultrasonic pachymetry and
specular microscopy.
• Major problem in clinical, practical use
of this instrument is repeatability of
measurements, particularly among
observers.

4. Major sources for these problems.
(1) Lack of small fixation target for the pt, which is located in a fixed
position to the instrument.
(2) Lack of known alignment of the pachymeter with the cornea in a
reproducible position so that the slit beam intersects the cornea of
the same angle for consistent thickness reading.
• Location on the cornea that is being measured can be identified
visually.
• It is not easy to return to that specific point without auxiliary fixation
devices.
• Errors in accuracy and precision inherent in this method are
minimized when the instrument is used by single observer & errors
are <10µm, an acceptable error for practical a clinical refractive
keratotomy.
• Errors can increases up to 20µm or more when a multiple users and
this is unacceptable.

5. There are two criteria for measurement of
corneal
thickness.
(1) "Just touch" criterion: - Alignment is
made in such a way that an imaginary line
extends from the endothelial border of upper
image to the epithelial border of lower
image.
(2) Overlap – Method: - imaginary extension of
the bright portion of endothelial image is
over lapped with bright portion of epithelial
image. Because bright portion is actually
produced by the finite width of the slit lamp
as it passes through each surface of cornea.

6. JUST TOUCH METHOD IS EASIER, POPULAR A PRACTICAL
AMONG MOST OBSERVERS.
• Five different studies using five different optical pachometers all
showed mean central corneal thickness values 0.51 to 0.52 mm
(S.D. 0.02 to 0.04mm)
• Kremer – Ultrasonic pachometer – (sound speed in cornea 1640m/
sec.) – central average corneal thickness – 0.512+ 0.035mm in 175
eyes.
• Novak et al. compared optical (Haag streit 900 Ĉ mishima Hedbys
attachments).
• Specular microscope (Pro-koster) and
• Ultrasonic (Accutome, 1630 ± 10m/sec.) Pachymetry in 93 pts in
study – using mean value of 3 method corneal thickness
measurements reading for each instrument on each eye.
• Optical – 0.554 ± 0.028mm
• Specular microscopy 0.551 ± 0.37mm
• Ultrasonic 0.542 ± 0.035mm.

7. • ULTRASONIC PACHYMETRY
• Developed by Henderson and Kremer in 1980
• Currently Preferred Method for corneal thickness measurement due to ease of use,
precision, portability and ability to measure corneal thickness eccentrically.
• Principle: - Instruments functions by measuring the amount of time (transit time)
needed for ultrasound pulse pass from the one end of Transducer to descemet's
membrane and back to the transducer.
• C→ speeds of ultra sound wave in cornea.
• Determined by density and compressibility of cornea.

8. • Cornea is made of 78% mater.
• Propagation Velocity → Water 1524m/sec.
→Steel – 6000m/sec.
• Thus it is imp. that propagation velocity of cornea be known
accurately because this variable can be set on many ultrasonic
pachometer and different setting will change than thickness of
cornea.
• = Speed of sound in cornea: - Current standard is 1640 m/sec.
• Kremer selected 1640 m/sec., because that measurement gave
corneal thickness of 0.512 ± 0.035 in 175 Eyes.

9. • Components of ultrasonic pachometers
• Probe handle with its transducer and tip
• Housing of instrument
• Accessories and convenience features.
• Pachometer probe handle: - it has piezoelectric crystal that emits an
ultrasonic beam of ≅ 20 MHz
• - All Probes are hand held.
• - Visualization of tip straight probe is sometimes difficult under the
operative micro scope in comparison to angled probe handle.

10. • Transducers: - Transducer sends the beam of
ultrasound wave through the probe tips into the cornea
and receives them on return.
• Width of transducer beam is related to the size of the
emitting crystal and of the width and configuration of the
probe through which it passes.
# #:- A wide probe tip and wide transducer beam reduces the
accuracy of the corneal thickness reading at a single point.
• Transducer has limited lifetime approximately 150-200
cases. It loses its accuracy and precision.
• The reading becomes increasingly variable on the
calibration block. The probe should be changed.

11. • Probe tip: - it is interface between the cornea and
transducer.
• Material in the probe should not attenuate the ultrasound
beam and the geometric design of the probe tip should
facilitate its optimal transmission.
• Diameter of probe should be <2mm. to diminish the area
over which the ultrasound beam is spread (to allow)
observer to see exactly where the tip is placed on the
cornea.
• Surface of tip should be smooth to avoid any injury to
corneal epithelium.

12. TYPES OF PROBE: -
(1) Open, water filled type requires
frequent refilling.
(2) Solid tipped probes containing
an internal fluid
reservoir that is refilled
periodically.
(3) All solid tip probe with no
internal reservoir and no refilling.
• Each can provide accurate and
precise measurement, but
convenience and practicality
vary.

13. (1) Open water filled tips: - Used Earlier
Inconvenient to use:-
• As fluid pulled out of the tip by surface tension & capillary action, air
bubble enter and give erroneons readings.
(2) The solid tipped probe – have replaceable couplet (glue or oil)
• and are more convenient.
• - Frequency of refilling varies from once a week to once a year,
depending on design.
• Tekner optha sonic pachometer has oil interface and has to
changes once a year.
(3) All Solid tipped – No replaceable couplants
• Tips are made of polystyrene.
• More convenient to handle a requires less maintenance eg.
accutome corneometer, pach – pen, sonogaga and Humphreys
Pachometers

14. - All pachomaters average a series of thickness measurements
to give the single, final read out display of corneal thickness.
- Instruments take 30-500 reading in a fraction of second.
There are two methods by which pachometer create an average
reading.
1. Pulse locked method: - Unit will record all readings that are
within 5 to 10 0 of perpendicularity or within 5 to 10µ of each
other, rejecting those outside the range.
2. Fixed no. of consecutive reading must be within 5 to 100 of
perpendicularity on 5 to 10µ of each other, before there are
averaged. If the probe is not perpendicular or the readings are
too disparate, the series is rejected and must be began again.

15.  Resolution of instrument is smallest unit
 measurable by machine - 1µ
 Clinical accuracy of most instrument ± 5 to 10µ
 Ultrasonic corneal pachometer can measure thickness
range 200 to 2000 µ
 Most pachometers have a selected speed of 1640m/sec.
Some units allow adjustments of sound speed, so
operators can select faster or slower speed.
 ** Selection of faster speed will produced a thicker
corneal reading

16.  Other methods for corneal thickness measurement.
 High frequency ultrasound corneal pachymetry
 70MHz is used frequency
 This technique produces B-scan images in real time, by on the fly
analog processing, involving rectifying averaging reflected
ultrasound waves.
 Pachymetry using the ORBSCAN topography system
 Orbscan technique results in pictorial representation of corneal
topography in true as opposed to derivative terms.
 The creation of a surface of orbascan topographic measurement
provides the basis for the derivation of pachymetric & radius of
curvature maps.
 Pachymetry by laser Doppler interferometry (LDI)

17.  Penta Cam – trade name of
comprehensive anterior segment
analyzer (five in one innovation)
 It is 3-Dimensional (3D) rotating
scheimpflug camera.
 It can perform five functions in 2
sec.
1. Scheimpflug image of anterior
segment
2. 3-D anterior chamber analyser
3. Pachymetry
4. Corneal topography
5. Cataract analyser

18.  Pachymetry by pentacam is displayed as a color image
over its entire area from limbus to limbus.
 Actual thickness can be measured individually by a
mouse click at any locations.
 Thickness in the pupil centre
 Thickness in the apex
 Thinnest location
 Corneal volume
 Applications –
1. Preoperative planning for corneal refractive surgery
2. Glaucoma screening
3. IOP modification with regard to corneal thickness
4. Keratoconus detection & quantification.

19. CORNEAL CONFOCAL MICROSCOPY
 This unique method offers the ability to
examine objects at high magnification.
 This revolutionary new tool permits real time
observation of living corneal (in vivo) in
patients at magnification ranging from 20X to
500X.
 It also measure thickness of each layer by
using computerized scanning system
providing the total corneal thickness in
studied area.
 Beside endothelium examination also
measure endothelial cell count (density)
which is comparable to specular microscopy.
 It offers the possibility to visualize structures
posterior to haze, scars or edema with in the
cornea.

20.  Principles – In a normal microscope
image formation is composed of a single
sharp image in addition to superimposed
blurry images. Depth of field is inversely
proportional to magnitude of
magnification.
 Unique property of confocal microscope
that it eliminates the super imposed
blurred images that normally occurs with
relatively high magnifications it can
exceed the final resolution of the
ordinary light microscope by > 50% of
image sharpness.
 This unique property is d/t its ability to
project intense illumination & capture its
reflected light through a narrow focal
plane; blocking the out of focus rays.

21.  first study with clinical approach using this
instrument was done by Ichijima in 1992 to
document the changes in superficial oepithelial
cells after extended wear rigid contact lenses.
 Later Cavanagh used a tendon scanning
confocal microscope & examined various
corneal diseases.
 Confocal microscope uses white light or a
focused laser beam but clinical white light is safe
becuase laser having risk for damaging living
tissue.

22.  Procedure – After topical anaesthesia patient is
guided to the chin rest.
 A clear visoelastic solution is applied to the cover tip
of the microscope to avoid corneal abrasion.
 Machine is approximated until a bright image is
seen & the then the corneal scanning is done.
 Once the epithelium is well focused, the zooming
examination of all corneal layers can be fulfilled in
only 30 sec. examination is stored in computer
software.

23. NORMAL CORNEA -
 Epithelium – Superficial
layers – large surface cells
arranged in irregular
polygonal mosaic. 1. At the superficial epithelium, poorly demarcated roundish cells
demonstrate hyperreflective nuclei (arrows) on confocal
These cells demonstrate microscopy (original magnification 210).
hyper reflective nuclei.
Basal epithelial cells –
Immature cells appear without
nuclei reflectivity or faint.
2. Bowman's Layer – Acellular
Bowman’s layer is an acellular hyperreflective structure, where subepithelial nerve
hyper reflective structure plexus (arrows) may be identified easily (original magnification 210).
subepithelial nerve plexus
may be seen.
In normal cornea, vessels are
not present in epithelium &
Bowman's layer.
Basal epithelial cells appear hyporeflective and have hyperreflective borders
(original magnification 250).

24. 3. Stroma – Hyper reflective keratocyte nuclei are scattered against a dark
background.
Ketatocyte density is maximum (800 cells/mm2) immediately under
Bowman's membrane and decreased (65 cells/mm2)sharply towards
posterior cornea.
Nerves – Which may present branching images, are found in the stroma
and are thicker than at the subepithelial level.
In normal eyes vessles are not found in stroma.
4. Descemet's Membrane – Acellular layer of moderate reflectivity : however
nerve plexus is absent.
This layer is rather difficult to see under normal circumstances.
In the stroma, hyperreflective keratocyte nuclei (arrows) are scattered against a dark
background (original magnification 250).

25. 5. Endothelium – Regular, hexaogonal, hyperreflective
shape surrounded by hyporeflective borders and the
absence of any nuclei reflection.
Endothelial count with confocal & specular microscopy
are comparable.
Negative correlation b/w age & endothelial count.
No vessels or nerves are present in this layer.
The physiologic responses of the corneal to different
stimuli may by analyzed by confocal microscopy.
Activated keratocytes presenting as cells with increased
reflectivity in the stroma, are seen when cellular
metabolic activity is increased.
Scar tissue, infection, inflammation – Hyperreflective
images.
Vessels – Lumen – Hyporeflective
Wall – Hyperreflective

26.  Stroma – Hyper reflective keratocyte nuclei are scattered
against a dark background.
 Ketatocyte density is maximum (800 cells/mm2)
immediately under Bowman's membrane and decreased
(65 cells/mm2)sharply towards posterior cornea.
 Nerves – Which may present branching images, are
found in the stroma and are thicker than at the
subepithelial level.
 In normal eyes vessles are not found in stroma.

27. Stromal scar appears hyperreflective on confocal microscopy (original
magnification 210).

28. Vessel lumen appears hyporeflective on confocal microscopy,
whereas vessel wall demonstrates well-delineated
hyperreflectivity (arrows) on each side of the lumen (original
magnification 210).

29. Cotton candy-like hyperreflective material may be found at the subepithelial level in
amyloidosis with corneal deposits (original magnification 210).

30. Cystic epithelial lesions are demonstrated in a patient with Fuchs’ dystrophy.
Horizontal field width 5 610 mm. (Reprinted from Ophthalmology
Hernandez- Quintela et al82, Copyright 1998, with permission of American
Academy of Ophthalmology.)

31. Hyperreflective deposits (arrows) are found in area devoid of epithelium in an
eye treated with topical ciprofloxacin (original magnification 240).
(Reprinted from Essepian et al60 with permission of Cornea.)

32. Confocal microscopy in a case of corneal lattice dystrophy disclosed hyperreflective, linear, and
branching images (black arrows) in the stroma. The white arrows indicate some hyperreflective
keratocytes (original magnification 210). (Reprinted from Graefes Arch Clin Exp Ophthalmol. Chiou
AG, Beuerman RW, Kaufman SC, Kaufman HE: Confocal microscopy in lattice corneal dystrophy.
237:697--701, 1999, with kind permission of Springer Science and Business Media.)

33. Highly reflective and irregular material (*) is found at the level of Bowman’s
layer region and anterior stroma in Reis-Bu¨ckler dystrophy. Bar 5 50 mm.
(Reprinted from Ophthalmology Werner et al,222 Copyright 1999 with
permission of American Academy of Ophthalmology.)

34. In granular dystrophy, reflective diffuse deposits (arrows) may be found
in the stroma. Bar 5 50 mm. (Reprinted from Ophthalmology Werner
et al222 Copyright 1999, with permission of American Academy of
Ophthalmology.)

35. Stromal intracellular hyperreflective material is the hallmark of fleck
dystrophy. In the mid-stroma a cluster of hyperreflective dots is enclosed in
a cyst-like structure. Calibration bar 5 50 mm. (Reprinted from Frueh and
Bo¨hnke62 with permission of Cornea.)

36. Stromal crystalline accumulation is associated with Schnyder’s dystrophy
and is readily revealed by confocal microscopy (image is 250 170 mm).
(Reprinted from Ophthalmology Vesaluoma et al215 Copyright 1999, with
permission of American Academy of Ophthalmology.)

37. Subbasal nerve plexus presenting beads in a case of cornea plana (image is
390 290 mm). (Reprinted from Vesaluoma et al217 with permission of
Investigative Ophthalmology and Visual Science.)

38. Confocal microscopy (original magnification 210). Areas of highly abnormal cells characterized by marked
epithelial-like appearance and loss of regularity in size and shape were found. Hyperreflective structures
were found within and adjacent to these abnormal areas. Relatively normal appearing endothelial cells
were also detected (upper right corner of the photograph). (Reprinted from Chiou et al33 with permission of
British Journal of Ophthalmology.)

39. Cornea guttae (arrows) appear as roundish hyporeflective images with an
occasional central highlight at the level of the endothelium. Cell
pleomorphism shown is this picture is also a common feature (original
magnification 210).

40. Epithelial cells at the level of the corneal endothelium is pathognomonic of
epithelial downgrowth (original magnification 230). (Reprinted from Journal
of Cataract and Refractive Surgery Chiou et al36 Copyright 1999, with
permission of ASCRS & ESCRS.)

41. Confocal microscopy in a case of fibrous retrocorneal membrane after
penetrating keratoplasty (original magnification 210). At the endothelial cell
layer, a hyperreflective fibrous-appearing layer was demonstrated at the
periphery of the graft. (Reprinted from Chiou et al30 with permission of
Cornea.)

42. Subepithelial extracellular deposits (D) may be found 3 months after
photorefractive keratectomy at the epithelial-stromal interface (image is 382
382 mm). N 5 stromal nerve. (Reprinted from Ophthalmology, Corbett et al40
Copyright 1996 with permission of American Academy of Ophthalmology.)

43. Linear structures may be detected in the stroma years after photorefractive
keratectomy. Bar indicates 50 mm. (Reprinted from Archives of
Ophthalmology, Frueh et al63, Copyright 1998 with permission of
American Medical Association.)

44. Confocal microscopy of the cornea performed 10 days after the surgery.
Hyperreflective interface debris could be detected (arrows). A superficial
stromal nerve was also visualized (arrow heads) (original magnification
210).

45. Acanthamoeba cysts (black arrows) and trophozoite (white arrows) may be
visualized by confocal microscopy (marker 5 100 mm). (Reprinted from
Pfister et al177 with permission of Cornea.)

46. At a depth of 115 mm from the anterior corneal surface, linear and
branching structures are detected in a case of aspergillus keratitis
(original magnification 135).

47. Confocal microscopy can also resolve the double-walled structure of the
acanthamoeba ectocyst surrounding theendocyst (white arrow). Several
pear-shaped cysts are shown by the black arrows (marker 5 100 mm).
(Reprinted from Pfister et al177 with permission of American Journal of
Ophthalmology.)

48. Acanthamoeba radial keratoneuritis presents as swollen nerve (arrowheads). A bright
irregular body on the nerve (black arrow) is consistent with an acanthamoeba
trophozoite. A stromal keratocytes is shown by the white arrow. Inset: normal human
stromal nerve (arrowheads) and stromal keratocyte (white arrow). Arrows 5
keratocytes (marker 5 100 mm). (Reprinted from Pfister DR et al177 with permission of
American Journal of Ophthalmology.)

49. Refractive objects showing eight consecutive spots arranged in a straight
row in a case of Borrelia keratitis (horizontal field width 5 275 mm).
(Reprinted from Linna et al125 with permission of Cornea.)

50. Microsporidial keratitis has been reported to demonstrate images of epithelial cells of the
corneal surface containing intracellular spores upon confocal microscopic examination. An
enlargement of the two cells outlined shows numerous small, discrete, high-contrast,
intracellular microsporidial spores (white arrows), and an aggregate of tightly packed
microsporidial spores (gray arrows) (marker 5 100 mm). (Reprinted from Shah et al194 with
permission of American Journal of Ophthalmology.)

51. Degenerated fine stromal hyperreflective dots may be detected in long-term
contact lens wearers. Bar550 mm. (Reprinted from Ophthalmology, Bohnke
and Masters,11 Copyright 1997 with permission of American Academy of
Ophthalmology.)

52. Thanks