Pachymetry, Specular Microscopy and Confocal Microscopy

Pachymetry, Specular Microscopy 

and Confocal Microscopy 

Ellison Bentley, DVM, Diplomate 

ACVO 

University of Wisconsin-Madison

Pachymetry 

• Why measure? 

– Indicator of corneal health 

– Reflects health of endothelium 

– Influences tonometry measurements 

– Diagnosis of disease, monitoring 

– Can be thicker or thinner (refractive surgery)

Methods of measurement 

• Historically: 

– Calipers on fresh or fixed tissue 

• Poor reflection of in vivo thickness 

• Tissue swelling after death

Optical pachymetry 

• Attachment to table 

mounted slit lamp 

• Measure oblique 

section of cornea with 

split prism 

• Split epithelial/ 

endothelial images 

aligned by user 

• Various equations used 

to determine thickness


• Variables: 

– Refractive index of 

cornea 

– Anterior radius of 

curvature 

• Sources of imprecision 

– Equation variables 

– Inter-observer variability 

• Observer variation in 

alignment of images


• Automated optical 

pachymetry: 

– Significantly decreases 

inter-observer variability 

– Increases reproducibility 

and reliability 

Non-contact automated 

optical pachymeter


• Orbscan Anterior 

Segment analysis 

system 

– Series of slit beam 

images (9000 points) 

– Anterior/posterior 

topography 

– 3-D image 

– Thinner than with other 

methods 

http://www.augenlaser-klinik.ch/images/ 

layout/grafiken_orbscan/ 

orbscan2.gif&imgrefurl

Optical technique 

• Pentacam-rotating 

Schiemfplug 

camera-can model 

the anterior chamber 

(humans) 

• Scanning slit like 

Orbscan-increased 

depth of focus


• Requires clear 

reflections of epithelial 

and endothelial surface 

– Limited: corneal edema, 

scarring, deposits, etc

Specular microscopes 

• Another form of optical 

pachymetry 

• Use electromechanical 

device 

• Central corneal 

thickness; newer 

models mid-peripheral 

From Krachmer, Cornea 2005

Pachymetry-specular microscopes 

• Measure from posterior surface of tear film to 

posterior surface of Descemet’s membrane 

– May have error of 20-30 µm 

• Contact or non-contact 

– Corneal touch-induce some compression; thinner 

measurement

Ultrasound pachymetry 

• 1980’s 

• Preferred method: 

– Independent of patient 

fixation 

– Ease of use 

– Peripheral and central 

measurement 

– Improved reproducibility 

– Improved accuracy 

– Portability


• Modern machines 

– Excellent alignment detection 

– Fast information retrieval and storage 

– Better sampling algorithms 

– Smaller probe tips (better for wider variety of 

animals, more precise measurement)


• Originally-water-filled probe tips 

• Currently-solid tips 

– Measure from anterior surface of tear film to 

posterior surface endothelium 

– Automatic gain 

– Angle sensitivity-within 5 degrees of axis 

– Most 20 MHz, but up to 65 MHz


• Hard plastic probes 

• Minimal maintenance 

• Easy to sterilize


• Electronic pulservibrates 

piezoelectric 

crystal 

• Ultrasonic pulsereflects 

at Descemet’s; 

returns to crystal 

• Reflected waveselectric 

signal goes to 

receiver


• Corneal thickness-from time it takes to pass 

from end of transducer, to Descemet’s and 

back 

Distance = velocity x time 

• Velocity-speed of sound in cornea


• Velocity 

– Human cornea-1640 m/s 

– Range-1550 m/s-1639 m/s-bovine, porcine, cat, 

human 

– 1639 m/s-best estimate for human but use 1640 

m/s-best correlate with optical pachymetry 

– Tang et al-velocity in canine: 1577+/- 10m/s 

• Since true velocity lower, most machines overestimate 

corneal thickness in dogs

Pach-Pen 

• Hand-held pachymeter 

• 20 MHz 

• Speed of sound-1640 

m/s 

• Variations in species: 

– Feline-1590 m/s 

– Pach-Pen overestimates 

thickness

Pachymeters 

• Accupach and others: 

• LCD displays 

• Printing capabilities 

• Audible read-out 

• 90-999 microns 

• + 5 microns accuracy 

• Small handheld probes

High frequency ultrasound 

• Recently reported 

• Typically 50 MHz or 

higher 

• Intraobserver 

reproducibility-high 

• Interobserver 

reproducibility-lower 

• Observer perception of 

landmarks

Confocal microscopy 

• Recently: good 

repeatibility 

• Measure corneal 

epithelial thickness 

• Bowman’s layer 

• Availability likely to 

increase-now reports in 

vet med 

www.swmed.edu/home_pages/ ophth/tscam.htm


• Instrument comparison: 

– Optical pachymetry-less reproducible, less 

reliable than ultrasound 

• Both between and within observers 

– Optical pachymeters-Systematic left eye bias 

• Central left eye thicker readings


• Studies of instrument comparison: 

– Automated optical pachymeter vs specular 

microscopy: 

• Identical reliability 

• Ultrasound pachymeters: 

– High intraobserver reproducibility but lower interobserver 

reproducibility (still higher than optical)


• Instrument comparison: 

– Specular vs ultrasound pachymetry vs ultrasound 

biomicroscopy: 

• Ultrasound (both) less variability 

• Ultrasound pachymeter-error outputs, assess probe 

placement 

• UBM-centrality and perpendicularity can be assessed 

on image 

• Cannot assess these criteria with optical devices

Sources of error 

• Instrument itself 

• Repeated measurements 

• Drying of cornea 

• Patient positioning 

• Marking of cornea

Sources of error 

• Repeated measurements: < 1.5 % variability 

– Blinking between measurements decreases 

variability 

• Recumbency increases corneal thickness in 

humans

Pachymetry in veterinary medicine 

• 1985-Chan-ling-central feline corneal 

thickness-569 + 36 µm; diurnal variation of 

49; thicker after sleeping 

– Diurnal variation due to lid closure 

– Ultrasound pachymetery; Velocity=1550 m/s


• 1986-Carrington/Woodward-feline-755 µm 

– Slit lamp based optical pachymeter 

• 1991-Gilger-canine 

– Central corneal thickness-520-597 µm 

– Peripheral corneal thicker 

– Thickness increased with weight 

– Females thinner corneas 

• Ultrasonic, Velocity=1630 m/s


• 1993-Gilger-feline 

– Central and peripheral thickness similar 

– 578 + 64 µm 

– Thickness ↑ with age up to 100 months 

– No sex difference 

– Ultrasound pachymetery; Velocity=1590 m/s


• 1995-Schoster-feline 

– 13 locations 

– Central cornea: 546 + 48 µm 

– Variations in corneal thickness 

• Temporal and sub-periaxial areas thicker 

• Superio-nasal-thinnest 

– Ultrasound; Velocity=1640 m/s


• 1995-van der Woerdt-horses 

– Central cornea: 793 + 44 µm 

– Peripheral cornea significantly thicker: 831-924 

µm 

– Auriculopalpebral nerve block, xylazine, IOP, 

age, sex-no effect 



• 1999-Ramsey-horses 

– Rocky Mountain horses with cornea globosa 

– Thickness increases with age longer than normal 

horses 



• 2003-Chandler-dogs; post transcorneal iridal 

photocoagulation (diode) 

– No significant changes in corneal thickness 

– Non-contact specular microscope


• 2003-Plummer-Miniature horses vs full sized 

– Similar values between populations 

– Ultrasound; Velocity-1640 m/s 

• 2004-Gerding-dogs post intracameral 

lidocaine 

– No significant differences 


Specular microscopy 

• Typically used for endothelium exam 

• Light → surface: reflected, transmitted or 

absorbed 

• Usually all 3 occur 

• Microscope: light transmitted THROUGH 

substance 

• Specular microscope: light reflected FROM 

optical interface


• Specular reflection = mirror like 

– Angle of refraction = angle of incidence 

– Microscope captures reflected light 

• Interfaces-2 media of different refractive 

indices 

– Reflection of some light 

– Difference in refractive indices related to amount 

of reflection


• Optical interface between 

corneal endothelium and 

aqueous humor 

– Also: corneal epithelium, 

stroma, lens 

• Equipment: contact or noncontact 

• Stationary slit, moving slit, 

moving spot 

from Krachmer, Cornea 2005


• Four zones of reflection: 

– Zone 1-interface lens, coupling fluid, epithelium 

– Zone 2-light reflected from stroma 

– Zone 3-endothelial reflection 

– Zone 4-aqueous (very little reflection-dark)


• Dark boundarybetween 

zone 3 and 4 

• Bright boundarybetween 

zone 2 and 3 

• Particularly noticableslit 

images 

http://www.djo.harvard.edu


• Increasing angle of incidence-wider slit, 

larger image area 

– ↓ image quality-increased illumination and 

increased scatter (stroma and epithelium) 

– Shortening of endothelial cells 

• Modern solution: 

– Small slits/spot-scan over tissue-↑ field of view

Specular microscopes 

• Difficult in awake veterinary 

patients 

• Patients typically fixate (no 

eye movement!) 

• Konan NonCon Robot-very 

automated 

– Chin rest, fixate, machine 

aligns by Purkinje images, 

focuses on endothelium, 

picture 

Cat endothelial cells-eyebank 

specular microscope


• Analysis: 

– Endothelial cell density (cells/mm 2 ) 

– Cell area (µm 2 /cell) 

– Pleomorphism (% of 3, 4,5, 6, 7, 8 sided cells) 

– Often automated counts


• Density 

• Polymegathism 

Krachmer, 1997, Cornea, fig 1-10


• Optical sections of living tissue 

– Magnification, resolution-differentiate cellular and 

subcellular structures 

– Based on principle of Lukosz 

• Resolution improved by decreasing field of view


• 1955-Minsky 

– Microscope condenser-focused light w/in small 

area of tissue, focused objective lens on same 

area 

– ‘Confocal’-condenser, lens same focal point 

– Now: light source focused on small volume, 

confocal point detector to collect signal, scanning 

for full field view


• Pinhole/screen aperture for viewing-eliminates 

scatter from rest of tissue-very sharp image 

• Pinhole conjugate to focal point of lens=confocal 

pinhole=confocal microscope 

http://www.physics.emory.edu/~weeks/confocal/

• Laser-provide 

high intensity 

• Mirrors-scan 

across sample 

• Light focused on 

pinholemeasured 

by 

detector 


http://www.physics.emory.edu/~weeks/confocal


• Acquire series of thin optical sections 

• 3-D reconstruction 

• Can see all corneal layers 

• Better detail than specular

Anterior epithelium 

3 µm 

8 µm 

25µm 

53µm 

3 µm 

8 µm 

(1000x Arndt, C., doct. thesis. LMU- 

München, 1999) 

Pictures 

courtesy 

C Kafarnik 

25 µm 

53 µm

Stroma and endothelium 

79 µm 

79 µm 293 µm 487 µm 

293 µm 

487 µm 

517 µm 

535 µm 

539 µm 

517 µm 535 µm 539 µm 

Pictures courtesy C Kafarnik 

(100x; Arndt, C.,doct. 

thesis-LMU- München, 

1999)

Confocal in veterinary medicine 

• Kafarnik-Vet Ophth 

2007, 2008-dogs, cats, 

birds, dogs 

– Noted lower nerve fiber 

densities in 

brachycephalics 

• Ledbetter-Vet Ophth 

2009-horsescharacterized 

normal 

Ledbetter, VO, 2009-Eq endothelium

Specular microscopy in veterinary 

medicine 

• 1979-Stapelton-7 young dogs 

– 2816 + 187 cell/mm 2 

• 1981-Peiffer-11 cats 

– 2668 + 211 cell/mm 2


medicine 

• 1981-Befanis-endothelial repair-’young’ dogs 

– Froze 90% 

– Demonstrated re-formation of intact monolayer 

by 6 weeks 

– 2,800 cells/mm 2


medicine 

• 1982-Gwin-central and peripheral cornea-dog 

– No differences cell counts centrally vs 

peripherally 

– Cell # decreased with age 

• < 1 year - > 2600 cells/mm 2 

• 1-9 years - 2300-2500 cells/mm 2 

• > 10 years - 1900-2100 cells/mm 2


medicine 

• 1983-Gwin-21 dogs, mature cataracts, phaco 

and ECCE 

– Phaco-Cell counts ↓ 22% centrally, 13% 

peripherally 

– ECCE-Cell counts ↓ 34% centrally, 31% 

peripherally 

– Cells enlarged, pleomorphic with both surgeries


medicine 

• 1985-Glasser-cats, effect of BSS vs BSS plus 

irrigation of anterior chamber 

– No difference in cell density 

– Increased polymegathism, pleomorphism in BSS 

group 

– No change cell morphology with BSS plus


medicine 

• 1986-Nassise-Saline, BSS, BSS plus 

glutiathione-AC irrigation in dogs 

– 22 minute irrigation with 100mls 

– No changes endothelial cell density 

• Mild corneal thickness increase with saline group


medicine 

• 1990-Gerding-dogs-replace aqueous with: 

BSS, sodium hyaluronate, sodium 

chondroitin-sulfate/sodium hyaluronate, 

hydroxypropyl methylcellulose 

– No significant differences in cell density or 

morphology or thickness


medicine 

• 1992-Gerding-intracameral tPA in dogs 

– High dose group (50 µg/100 ml)-18% decrease in 

hexagonal cells 

– No other changes in cell density, morphology or 

thickness noted


• 2001-Andrew-equine 

– 3155 cell/mm 2 

medicine 

– Decreased cell density in ventral quadrant vs 

medial/temporal 

– Cell density dereased with age


medicine 

• 2002-Andrew-36 llamas, 20 alpacas 

– Llamas-2668 cells/mm 2 

– Cell density decreased with age 

– Alpacas-2275 cells/mm 2 

– Polymegathism frequent


medicine 

• 2003-Chandler-post trancorneal iridal 

photocoagulation in dogs 

– No significant difference in endothelial cell counts 

or morphology 

• 2004-Gerding-post lidocaine in AC in dogs 

– No significant effects on endothelial cell counts or 

morphology

Questions??

Pachymetry, Specular Microscopy and Confocal Microscopy

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