Table Of ContentCurrent Research
in Ophthalmic
Electron Microscopy
4
Edited by
William R. Lee
With 88 Figures
Springer-Verlag
Berlin Heidelberg New York 1981
Transactions of the 8th Annual Meeting of the European
Club for Ophthalmic Fine Structure in West Berlin,
March 28 and 29, 1980
Editor
Professor WILLIAM R. LEE
University Departments of Ophthalmology and
Pathology, The University of Glasgow, Scotland
ISBN-13: 978-3-540-10651-7 e-ISBN-13: 978-3-642-81614-7
DOl: 10.1007/978-3-642-81614-7
Library of Congress Cataloging in Publication Data' European Club for Ophthalmic Fine
Structure. Current research in ophthalmic electron microscopy. 4. "Transactions of the 8th
annual meeting of the European Club for Ophthalmic Fine Structure in West Berlin. March
28-29. 1980"-Verso t.p. Bibliography: p. Includes index. 1. Eye-Anatomy-Con
gresses. 2. Ultrastructure (Biology)-Congresses. 3. Electron microscopy-Congresses.
4. Ophthalmology-Congresses. 1. Lee, William R., 1932- . II. Title.
QM511.E971981 599.01'823 81-1894
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Contents
Human Conjunctival Surface Mucins: A Quantitative
Study of Normal and Diseased (KCS) Tissue
W.R. LEE, S.B. MURRAY, J. WILLIAMSON, and D.L.
McKEAN. With 8 Figures . . . . . . . . . . . . .
The Basement Membrane Complex of the Human Corne
al EpithelIum
H. BREWITT and E. REALE. With 4 Figures . . . . .. 15
Shrmkage m Preparatory Steps for SEM. A Study on
Rabbit Corneal Endothelium
O.A. JENSEN, J.U. PRAUSE, and H. LAURSEN. With 6
Figures 25
Effects of Freezing on the Corneal Stroma of the Rabbit
After Keratophakia
F. HOFFMANN and J.-P. HARNISCH. With 6 Figures 35
The Post-Mortem Vacuoles of Schlemm's Canal
1. GRIERSON and N.F. JOHNSON. With 11 Figures 41
Immotile-ciha Syndrome and the Cilia of the Eye
B. SVEDBERGH, V. JONSSON, and B. AFZELIUS. With 3
Figures 57
Identification of a Basement Membrane Proteoglycan m
Exfoliation Material
J.-P. HARNISCH, H.J. BARRACH. J.R. HASSELL, and P.K.
SI~HA. With 6 Figures . . . . . . . . . . . . . . . 65
VI Contents
Cellular Composition of Post-haemorrhagic Opacities in
the Human Vitreous
J.V. FORRESTER and W.R. LEE. With 9 Figures .... 71
Experimentally Induced Lipidosis in Rat Retinal Pigment
Epithelium. A Brief Review
R. LULLMANN-RAUCH. With 4 Figures ...... . 89
Qualitative Observations on the Variation of Light In
duced Damage to the Rabbit Retina
N.M. McKECHNIE and W.S. FOULDS. With 14 Figures . 97
An Electron MicroscopIc Study of the Epiretinal Mem
brane of Human Eye~
T. HARADA, D. CHAUVAUD, and Y. POULIQUEN. With
11 Figures . 119
Regeneration of the Human Corneal Endothelium.
A S.E.M. Study
G. RENARD, Y. POULIQUEN, and M. HIRSCH. With 3 Fig-
ures .......... . 133
Indexed in Current Contents
Human Conjunctival Surface Mucins: A Quantitative Study
of Normal and Diseased (KCS) Tissue
W.R. Lee1**, S.B. Murray 2, 1. Williamson2 and D.L. McKean 3
I Tennent Instltute of Ophthalmology (Director Professor W S Foulds).
Glasgow Umversity. Scotland
2 Southern General HospItal. Glasgow. Scotland
3 PharmacologIcal Research Dept. Burton Parsons & Co . Washmgton DC, USA
Abstract. The archItecture and distribution of surface mucins were studied
qualitatively and quantitatively by transmission electron microscopy in ruthe
nium red stained biopSIes of the human conjunctiva. Six control specImens
were compared with three from early keratoconjunctivitis sicca (KCS) and
with three specimens from severe KCS cases. The area of the ruthenium
red/Os04/mucin reaction product on the conjunctival epithelial surface was
measured by image analysis and the values were expressed in 11m2 per 11m
length of epithelium. The total area of surface mucins was differentiated
from the narrow zone of mucins in close contact with the microplicae.
The value for total mucins in two cases of early KCS was higher (0.4 11m2 111m)
than the control range (0.1-0.3 11m2 111m) due to the presence of large clumps
of mucins on the surface. The values for microplical mucins in early and
severe KCS were within the control range, but were an overestimate owing
to the presence of abnormal structures, e.g. veSIcles, and abnormal clumps on
the surface. This morphometric technique has limitations, but with appro
priate material it might be a useful tool for the identification of conjunctival
surface mucins.
Introduction
The ultrastructural features of the human and animal conjunctival epithelium
are well documented (see Abdel-Khalek Williamson and Lee 1978a and Latkovic
and Nilsson 1979 for recent review articles). There is general agreement that
the numerous small processes which project from the epithelial surface (micro pi i
cae being a more accurate term than microvilli) serve as a support for the
hydrophilic mucosubstances which are essential for the stability of the tear
** Correspondmg author
W.R. Lee et al.
film. The origin and chemical nature of the mucosubstances, which hereafter
for the sake of brevity will be referred to as mucins, is not yet established
(see Holly and Lemp 1977, for review), although it has been suggested that
glycoproteins (Moore and Tiffany 1979), and in particular sialomucins (Srini
vasan, Borgul, Iwamoto and Merriam 1977) form an important component.
If the ultrastructure of surface mucins could be reliably demonstrated, their
chemical nature could be investigated by pretreatment of the tissue with e.g.
sialidase. This would however, require a quantitative form of analysis which
could differentiate between biological variation and enzyme effect. As part of
a preliminary study of the potential of this approach to the conjunctival surface/
tear film relationships, we attempted to measure surface mucins by image analy
sis (Fisher 1971) after staining with ruthenium red. Human tissue (control and
keratoconjunctivitis sicca) was used for the investigation and it serves to illustrate
the disadvantages and the potential of the method.
Material and Methods
Conjunctival tissue was obtained from 12 middle aged and elderly mdlviduals who volunteered
for the mvestigatlOn Elliptical bIOpSies (4 x 3 mm) were taken from the temporal bulbar conjunctiva
SIX mdlVlduals, four female and two male aged 58, 64, 69, 71, 76 and 78 years, did not show
any clinical evidence of tear defiCiency and the bIOpsy was taken pnor to cataract surgery. BIOpSies
were taken after applicatIOn of local anaesthetic (benoxenate hydrochloride 04%) from SIX patients
m the KCS climc. Three females, aged 48, 63 and 67 years, were conSidered to be early cases
of KCS. In three severe KCS cases, the patients, one male and two female, were 66, 67 and
68 years of age.
The conjunctival biopSies were fixed immediately after eXCISIOn m a mixture of one part glutaral
dehyde (3%), one part cacodylate buffer and one part ruthemum red solution (1,500 ppm) The
solutIOns were refngerated at 4° C Half of each specimen was processed for paraffin embeddmg
and light microscopy The remamder was processed according to the method deSCribed by Luft
(J97Ja) for Araldlte embeddmg SectIOns of 60-70 nm A (silver-gold) thickness were cut on an
LKB Ultratome III and, without counterstam, were exammed m a Philips 301 transmiSSIOn electron
microscope.
An acceleratmg voltage of 60 KV was used with standard apertures throughout the mvestigatlOn
and the ruthemum red/Os0 /reactlOn product on the epithelial surface was photographed onto
4
Kodak cut film 4463 at a magmficatlOn of X 9,100 under standard exposure conditions. Where
there was any suggestIOn that the surface epithelium had been sectIOned obliquely, the regIOn
was Ignored Thirty two evenly spaced negatives were taken from each specimen ImtJaI studies
showed that negative denSity could be vaned accordmg to the pattern of tissue and embeddmg
medIUm m relation to the light sensors on the vlewmg plate. Therefore, for the major part of
the study, the same orientatIOn was chosen whenever pOSSible. The procedure for development
(Kodak D19) of the negatives was made as umform as IS possible wlthm standard laboratory
practice.
Image Analysis
Careful attentIOn to the preparatIOn of negatives was essential for image analYSIS which reqUires
uniform stam, umform grain density and umform contrast, If valid comparisons are to be made
(Bradbury 1979). The study of human material, which was obtamed and mvestlgated during a
period of 12 months, presented many problems With regard to the fulfilment of the aforementIOned
rigid Criteria and prehmmary studies were necessary to assess the scale of potential maccuracies
The negatives were analysed With an Optomax Image Analyser MICfomeasurements Ltd., Saffron
Walden, Essex, CBII 3AQ, UK) which rapidly measures the area of a component of a given
denSity on a teleVISIOn Image The teleVISIOn camera was posItioned above the transillummated
negative to give a x 3 magmflcatlOn to the screen and a total tissue magmflcatlOn of x 27,300.
2
ConJunctival Surface M UCIns
·b
+.t
~
"
-
~
Fig. 1 a, b. ConJunctIval surface mucms stamed en bloc with collOIdal Iron and counterstamed
with uranyl acetate Note the relatIvely low density of the reaction product which has a delicate
framework (arro\\') Baboon a x 26,000 b x 90,000
The negatIve Image wa, reversed electronIcally and a frame selected and calibrated so that the
total area of reaction product m ~m2 was measured per ~m length of epithelium In the KCS
materIal, high values were obtamed as a result of the presence of large clumps of reaction product
on the surface As these serve no functIOn m tear film stability, values were obtamed (m additIOn
to total mUClns) for nlIcropiLcai mUClns. These were obtamed by restrIctmg the frame to a distance
of 0 25 ~m from the mlcrophcal surface
The output from the Image analyser was mterfaced with a Hewlett-Packard desk-top computer
(98 I 5A) which proVided a statistIcal analysIs of the data
3
W R Lee et al.
Fig. 2. Conjunctival surface muclns demonstrated by the reactIOn product of ruthellium red; no
counterstain. Note the fine strands (arrows) connecting the electron dense clumps on the surface
of the mlcroplicae. Normal female, 76 years. x 65,000
Results
Preliminary Studies
Ruthenium red is a complex and unstable substance which has, as yet, undefined
specificities (Luft 1971 a, b), so that it was not the stain of initial choice. Colloidal
thorium and colloidal iron are to be preferred, but the former is no longer
available in the U.K. owing to the health hazard. Colloidal iron-stained baboon
conjunctival tissue was available in the laboratory files and had been prepared
according to the technique recommended by Curran, Clark and Lovell (1965).
The stain demonstrated mucins on and between the microplicae (Fig. 1) as
fine strands and clumps, but the density of the crystals was such that discrimina
tion of the crystal grey level against the cytoplasmic grey level was inaccurate
when the image analyser was used to quantify the negatives. The reaction product
of ruthenium red was much denser by comparison. In the normal human con
junctiva (Figs. 2 and 3), it appeared as a dense line along the glycocalyx
of the cell membrane and as a clump on the surface of the microplicae which
in some cases were linked by fine strands. The density of the components
of the reaction product was unequal and the differences were detectable by
variation in the selector levels for grey level detection in the image analyser.
To test the importance of subjective variation in selector level adjustment, the
levels between the two extreme ranges of a) under detection (81) and b) over
4
ConjunctIval Surface M UClns
b
c d e
Fig.3a-e. IllustratIons of the vanatIOn In appearance and dlstnbutIon of surface mUCInS In the
normal conjunctIva For explanatIon see text RuthenIUm red, wIthout counterstaIn. a female,
69 years. b female, 58 years, c, d, e female 76 years. a x 25,000 b, c, d, e x 23,500
detection (85) were used to measure the area of the reaction product at one
site. The graph obtained (Fig. 4) showed a linear relationship between the area
measured and the selector setting, the highest value being some 30% greater
than the lower. The standard deviations of the means were less than I %, which
is an indicatIOn of the repeatability of the image analyser. Thus errors are
5
