Journal of = TNOA

History

=20 Observation of the corneal endothelium by specular reflection = dates back=20 to the early part of this century; first by Vogt in 1918 with slit lamp=20 biomicroscope. But he = faced=20 problems due to fine continuous eye movements, limited magnification and = annoying light reflexes. = Specular=20 microscope was first described by Maurice in 1968. But it was not until a = suitable=20 modification of the instrument was done by Laing et al in = 1975¹ that=20 clinical specular microscopy became a viable method. It was later modified by Boume = &=20 Kaufman². The = initial=20 specular microscopes were narrow field microscopes with a field of view = of only=20 0.04mm². The = current=20 wide field specular microscope has a field of view of 1 mm². = There=20 are contact microscopes (in which the objective lens comes in contact = with the=20 cornea) and non-contact specular microscopes.

Optical principles

=20 To interpret accurately endothelial photomicrographs obtained = clinically, it is helpful to understand the optical principles of = specular=20 microscope^{3. =}It is a=20 reflected light microscope (epi-illuminated) that projects light onto = the cornea=20 at near normal incidence and images the light reflected from an optical=20 interface of the corneal tissue-most typically the interface between the = corneal=20 endothelium and the aqueous humour. Of primary importance in clinical = specular=20 microscopy is the light that is reflected specularly i.e. mirror-like, = where the=20 angle of incidence is equal to the angle of reflection. Light passes through the slit = of the=20 specular microscope and illuminates the cornea. Most of the visible light = incident on=20 the normal transparent cornea is transmitted through it. The illumination beam of the = clinical=20 specular microscope encounters a series of interfaces between optically = distinct=20 regions. Some light is = reflected=20 from the posterior corneal surface. =20 Some light is scattered and some absorbed by various corneal = tissue=20 layers. A portion of the = reflected=20 and scattered light is collected by the objective of the photomicroscope = producing an image at the film plane. =20 The larger the difference in refractive index between the two = regions,=20 the more intense will be the reflected light beam.

The fraction of incident light reflected (R) is = given by the=20 formula

R =3D = n₁-n₂

=20 n₁+n₂

=20 Where n₁& n₂are the indices of = refraction of=20 the two regions considered.

Considering refractive indices of

Lens &nbs= p; = ; =20 =3D =20 1.517

Saline &nbs= p; = ; =20 =3D =20 1.330

Cornea &nbs= p; = ;=20 =3D =20 1.376

Aqueous = humour =20 =3D =20 1.336

And using the = above relation,=20 the following are calculated:

Reflection from = lens-saline=20 interface=3D0.36%

Reflection from = saline-cornea=20 interface=3D0.025%

Reflection from = cornea-aqueous=20 humour interface=3D0.022%

Reflection from = the=20 intracorneal interfaces cannot be calculated, as the refractive indices = of the=20 separate layers of the cornea have never been measured.

Instrument=20 description

Light passes through a slit aperture into a = system of=20 mirrors that directs light out through the objective lens and its = attached=20 dipping cone into the cornea. = The=20 dipping cone lens is a flat surfaced extension on the water immersion = objective=20 which applanates the cornea (as in applanation tonometry). The focusing = knob=20 adjusts the excursion of this dipping cone to focus the image for = corneas of=20 different thicknesses. = The light is=20 reflected form the endothelium back through the objective lens and the = eye-piece=20 lens thus forming an image on the viewing screen (of the still/video = camera)=20

Improvements in instrumentation and methods

Improvement in image quality and ease of use of = the=20 original instrument was achieved with the design of an improved = objective lens=20 and the resulting images were of sufficient quality (even in edematous = cornea)=20 for morphometric analysis using computerized image processing systems by = Laing=20 et al. Sherrard et = al developed a fluorite cone = for objective lens that reduced = the=20 interfering objective lens-epithelial reflection and enabled a wider = field of=20 cells to be imaged (using a wider slit) at least in clear corneas. This type of objective = lens=20 enables corneal epithelial photograph to be taken^{4 .}

&n= bsp; =20 Konan non-con Robo instrument is the fully automated instrument = where=20 tracking the cornea and imaging the endothelium are fully = automatic. The objective lens first = positions=20 itself relative to the cornea by using Purkinje images till proper = specular=20 reflection is acknowledged. = Then it=20 objectively focuses back to the endothelial surface and the resulting=20 endothelial photograph is displayed on the video-monitor.

The=20 current specular microscopes have many advantages.

= ; =20 i. = ;=20 Wider field of view and minimizes annoying reflection = from=20 incident light⁶.

= ; =20 ii. = ;=20 Wide field microscope offers a wider view providing = better=20 examination of the endothelial mosaic.

= ; =20 iii. = ;=20 Addition of highly sensitive video camera and recording = systems⁷ as well as the scanning system introduced by=20 Koester⁸ have = increased=20 the resolution of the micrographs

Patient set-up

=20 First the procedure is explained to the patient especially that = they will=20 see a light flash and the whirring of the motor drive. Patient is = advised not be=20 frightened and not to move during the actual procedure. Local anesthetic drops are = used=20 topically. When corneas = are=20 contacted with the dipping cone a few epithelial irregularities may be = seen=20 which disappear in a few hours. =20 Positioning the eye in a straight ahead position is best achieved = with a=20 fixation light. Optical = photographs=20 are got when the cornea is thin, clear, with minimal scarring or edema = and with=20 an intact epithelium. = Light=20 reflexes from the iris can obscure the endothelial mosaic and are best=20 eliminated by dilating the pupil. =20 Systematically scanning superiorly, inferiorly, nasally and = temporally=20 will ensure a thorough evaluation of the endothelial monolayer.

Clinical specular=20 microscopy

It could be:

1. = By using contact objective lenses that touch the cornea = and=20 inhibit eye movement which gives higher magnification and = resolution.

2. = By using non-contact objective lenses which gives lower = magnification and resolution.

Of clinical importance = is the width=20 of the slit of light projected onto the cornea by the specular = microscope.

a .Using a narrow = slit of=20 light⁹

When a narrow=20 slit of light is focused onto the endothelial cell-aqueous humour = interface,=20 considerable amount of light is reflected specularly back towards the = film by=20 the

- =20 Lens-coupling fluid interface

- =20 Coupling fluid - epithelium interface

- =20 Endothelium =96 aqueous humour interface

At the film plane, = light from=20 various regions overlap. = When a=20 bright region overlaps a dark region, the dark region is not seen. So not all regions are seen in = the=20 photograph.

Four zones are = described.

Zone 1 -=20 =20 Bright region-formed by light reflected from the lens-coupling = fluid

&nbs= p; =20 interface or coupling fluid-epithelium interface or both.

Zone II - =20 Shows part of the stroma.

Zone III - =20 Shows the endothelial layer.

Zone IV- =20 Shows part of the aqueous region.

Interface between zones = III and IV=20 is called the dark boundary. = It=20 separates the illuminate cornea from the non-illuminated area. One side of the boundary is = dark because=20 negligible light is scattered from the aqueous humour.

The interface between = zones II and=20 III is called the bright boundary. =20 It separates the endothelial reflection from the overlying = illuminated=20 corneal stoma, neither side of this boundary is dark. It is less distinct than the = dark=20 boundary.

b. Using a wide slit = of=20 light

=20 When a wider slit of light is used, a larger field of = endothelial=20 cells result. But here a = wider beam=20 illuminates more of the corneal tissue anterior to the endothelium. As a result the volume of = interfering=20 stroma increases and a greater amount of scattered light reaches the = film plane.=20 The net result is reduced contrast of the endothelial image and a loss = of=20 cellular definition. Here = the=20 region produced by scattered light which is exclusively of stromal = origin, as=20 seen with a narrow slit light beam, progressively disappears. So a greater number of cells = are visible=20 with a wider slit. But = there is=20 masking of information about abnormalities confined to the corneal = stroma. Therefore it is apparent that = a =91trade=20 off=92 between the contrast of the photographs and the number of cells = able to be=20 photographed must be made. = This=20 limitation is not due to the instrument but rather to the light = scattering=20 properties of the corneal tissues overlying the endothelium.

Qualitative = evaluation of the=20 endothelium

=20 Qualitative analysis identifies abnormal endothelial = structures and=20 grades the endothelium either according to the number or size of the = abnormal=20 structures present or on the basis of an overall assessment of = endothelial=20 appearance.

=20 This also provides a rapid clinical evaluation of the endothelium = to=20 assess the risks of intraocular surgery, to establish a diagnosis or to = decide=20 on treatment.

The parameters = evaluated are

- =20 cell conformation

- =20 cell boundaries and their intersections

- =20 Posterior corneal surface (configuration of the dark=20 boundary)

- =20 Presence of a cellular structures

i. Cell conformation =

=20 Clinical specular microscope resolves the endothelial mosaic = of the=20 normal cornea into quasiregular pattern of contiguous cells having well = defined=20 boundaries. The central = endothelial=20 cells of young normal endothelium are approximately of the same = size-between 200=20 to 400 =B5m²⁼²⁰(according to age) and the distribution of cell area is approximately = normal=20 (Gaussian). In young = normal=20 endothelium, cell side lengths are all roughly equal. Angles of intersection are = approximately=20 60⁰at all ages.

ii. Cell boundaries = and=20 intersections

=20 Light incident on the cell boundaries is scattered so that the = reflected=20 rays do not return to the collection optic. So cell boundaries are dark in = the=20 endothelial photomicrograph. Commonly they appear as straight narrow = lines. They intersect in such a way = that three=20 angles of intersection are formed of approximately 60⁰ each. Angles of intersection which = vary=20 considerably from these are thermodynamically unstable intersections = suggesting=20 that they are formed by abnormal cells.

iii. Posterior = corneal=20 surface

=20 Information regarding posterior corneal surface is obtained by = observing=20 the appearance of the dark boundary i.e. the interface between the = endothelial=20 cell pattern and the adjacent dark zone produced by the aqueous = humor. If the endothelial surface is = smooth,=20 the dark boundary will be straight. =20 If it is irregular, the dark boundary will be irregular.

The four basic possible = types of=20 dark boundaries are

- =20 smooth and straight

- =20 rough

- =20 wavy

- =20 endothelial excrescence being=20 reflected.

- =20 A combination of any of the above can also be = seen.=20

iv. Presence of = acellular=20 structures: (Miscellaneous structures)

Both=20 inter-endothelial and intra-endothelial cell structures are seen which = may be=20 either dark or bright in appearance.

=20 One type of dark structure which disrupts the endothelial cell = pattern is=20 seen in varying sizes. = Each has=20 dark sides and a central bright spot. =20 They represent corneal guttata and are surrounded by a ring of = abnormally=20 shaped cells. Corneal = guttata are=20 seen much earlier with specular microscope than with slit lamp.

a.=20 Intracellular structures

= ; =20 i. = ;=20 Bright structures, some of which may be only the = cell=20 nucleus, vary in size and are typically contained in a single = endothelial=20 cell. They appear to be = associated=20 with stressed cells explaining why they commonly are seen in enlarged=20 cells. The larger the = endothelial=20 cell the bigger the bright structures, which can be multiple too.=20

= ; =20 ii. = ;=20 Bright structure spanning several endothelial = cells=20 appears white or orange in colour and sparkles. They appear to correspond to = the=20 pigmented endothelial deposits seen with slit lamp=20 biomicroscope.

= ; =20 iii. = ;=20 Two additional type of dark bodies (both intracellular) = have=20 also been noted

First type - small, central or Para central = in the=20 cell with sharp well defined

&n= bsp; =20 edges.=20 Presumably represent the base of endothelial cilia.

Second type - larger with = indistinct=20 edges. They represent = intracellular=20 vacuoles

&n= bsp; =20 or blebs.

=20 .

b.=20 Intercellular structures

=20 Dark structures, mostly lying at the endothelial cell = intersections,=20 tend to be of uniform size, randomly positioned across the endothelial = cell=20 pattern. They are seen in = patients=20 with anterior uveitis and they are believed to represent invading = inflammatory=20 cells.

=20 Although the nature and significance of the abnormalities = detected in the=20 qualitative analysis of the endothelium are not presently known, their=20 recognition represents an initial step in the elucidation of their=20 patho-physiologic significance.

Quantitative = morphometric=20 analysis

Here the aim is to = assign a number=20 or a set of numbers to the specular photomicrograph that can provide a = measure=20 of the endothelial status.

Various morphologic = parameters can=20 be quantified

1. = Cell size (cell area and cell density)

2. = Polymegathism (variation of cell size such as = coefficient of=20 variation of mean cell area)

3. = Plemorphism (variation of cell shape such as percentage = of=20 hexagonal cells or coefficient variation of cell shape)

4. = Cell perimeter

5. = Average cell side length

6. = Cell shape

But to date only = polymegathism and=20 pleomorphism and several other variables related to these parameters = have proved=20 useful in determining the endothelial status

=20 Two equivalent parameters have been used to quantify endothelial = cell=20 size

a) = Mean cell area-expressed in units of square microns (u²)

^{b) =20}Cell density (cell count)- obtained by dividing = the=20 number of cells by the area of that frame expressed in units of cells = per square=20 millimeters (cells/mm²).

These two quantities = are related by=20 the following equations:

&nbs= p; = ; = &= nbsp; =20 10⁶

Mean=20 cell area (u^=
2/cell)=3D &n= bsp; &nb= sp; =20

&nbs= p; = ; = =20 Cell density (cell/mm²)

Two different methods = can be used=20 to measure either of these two parameters of cell size.

1. Fixed frame = analysis=20

=20 In this method one counts the number of cells within a frame or = window of=20 constant area. All cells = lying=20 completely within the frame are counted as whole cells. Each cell that is only partly = within the=20 frame is counted as half cell regardless of the fractional area of that = cell=20 located within the frame

=B7 =20 Cell count =3D Sum of the number of whole and = half cells=20 within the frame.

=B7 =20

(cells/mm²)

Cell density = =3D =20 Cell count

&nbs= p; =20 A= rea=20 of the frame

The = area of the=20 frame must be referred to the endothelium.

=20 Actual area of the frame

i.e.

&nbs= p; =20 (Linear magnification of specular microscope)^=
2

Mean cell area =3D Area = / Cell=20 count

In practice, a minimum = of 35=20 contiguous cells should be within the counting frame (preferably 50-100 = cells)=20 otherwise the error associated with counting will be too large.

2. Variable frame=20 analysis

It is most conveniently = done by=20 using a computer based analysis. = It=20 eliminates the problem of counting fractional cells along the boundary = and so a=20 more accurate determination of mean cell size than by fixed frame = analysis is=20 possible. Here one = measures the=20 variable area occupied by an integral number of cells by tracing around = a=20 contiguous group of cells. = Each=20 cell is marked and then the computer calculates the cell density by = diving=20 marked cells by the area of the frame. =20 The error resulting from the counting method here is very small = as=20 compared to fixed frame analysis. =20 Also additional information regarding the range of cell densities = or=20 range of cell areas is got.

Individual cell=20 analysis

In fixed frame analysis = only a=20 average cell size can be determined. =20 But invariable frame analysis single cells can be traced with the = stylus=20 of the plan meter or digitizer and this then permits individual cell=20 analysis. It provides = more=20 information about endothelial cell pattern than those which give only = cell=20 density or average cell area. =20 Individual cell analysis is probably the most accurate method for = the=20 determination of cell density. = It=20 can be performed manually¹¹, semi-automatically or fully=20 automatically. Fully = automated=20 systems have been developed by Lester et al¹², Nishi et al, = Hartmann=20 et al¹³ and companies like Alcon, Topcon etc.

Computerized = morphometric=20 analysis¹⁴

A⁼²⁰specular photograph is chosen on the basis of cell boundary = clarity and is=20 enlarged to 400 times the original magnification. Before the specular photograph = is=20 printed, a calibration negative of a micrometer scale is taken with the = same=20 specular camera and placed in an enlarger to ensure accurate cornea to = print=20 magnification. A hundred = individual=20 cells are numbered consecutively to minimize sampling and analysis = error. Individual cells are digitized = by=20 marking each cell apex with a graphic tablet pen. The coordinates are entered = into a=20 digitizing tablet and analyzed by endothelial analysis software. Polymegathism is calculated by = dividing=20 the standard deviation of the mean cell area by the mean cell area. The coefficient of variation = polymegathism) is a dimensionless index = and provides=20 quantitative measurement of cell variation.¹⁵. The coefficient of variation = of normal=20 cornea is about 0.25. = Cell shapes=20 are described by the number of apices of each cell and the variation is = analyzed=20 automatically by digitizing the apex of each cell (pleomorphism). Further studies are in = progress to=20 describe better the relationship between anatomic form and functional=20 capability.¹⁶

In vivo findings of = specular=20 microscopy and clinical indications

=20 Specular microscopy has helped the most in the clinical = evaluation of=20 corneal endothelium. In = normal=20 cornea, a single layer of endothelial cells cover the posterior surface = of=20 Descemet=92s membrane in a well arranged mosaic pattern. Each endothelial cell is = approximately 5=20 mm thick and 20 mm wide. = They are=20 polygonal, mostly hexagonal (70-80%) in shape. Dimensions are quite = uniform. The cell density is about = 35000=20 (cells/mm²⁾in young adults. =20 Endothelial cells have a large nucleus and abundant cytoplasmic=20 organelles like mitochondria, endoplasmic reticulum, free ribosomes and = golgi=20 apparatus. They are = closely=20 interdigitated with several functional complex structures. The most important = physiological=20 function of corneal endothelial is to regulate the water content of the = corneal=20 stroma.

1. = Aging

It is established that = the central=20 endothelium changes as a function of age^{17,18, 19.} In most=20 individuals, cell density decreases or mean cell area increases) from = birth to=20 death. From birth to = adolescence,=20 the cell density rapidly decreases^20. From 20-50 years it seem to be = relatively stable and after 60 years, cell density decreases = significantly in=20 most people. Since at = this age the=20 globe does not change in size this observation seems to represent the = true loss=20 of endothelial cells. = With=20 increasing age some people display a significant difference between the = two eyes=20 that is currently not understood. =20 Polymegathism and pleomorphism have been found to correlate with=20 age.

2. Irodo corneal = endothelial=20 syndrome

=20 It is believed to result from a defect of corneal=20 endothelium²¹(early cases may be confused with corneal = guttata). It demonstrates a = characteristic=20 appearance in specular micorscopy^{22, 23} . In early cases there is = rounding-up of=20 cells, loss of cell border clarity, increased granularity of the = intercellular=20 details and appearance of small dark areas which may enlarge and become=20 completely blacked out areas within the cells. As it progresses, the = endothelial=20 monolayer mosaic is no longer recognizable as a mosaic of cells and = there may be=20 a =93reversal appearance=94 =96 black central areas with white border as = the cells=20 overlap each other.

3. Endothelial=20 dystrophies

=B7=20 Fuch=92s endothelial dystrophy

In this condition guttata are = easily seen=20 by specular microscopy²⁴ (but corneal guttae can also occur = as a=20 result of ageing and corneal inflammation). Individual excrescence begins = as a tiny=20 structure, which grows in size to obscure the view of the endothelial = cell lying=20 directly behind it. = Surrounding=20 cells appear abnormal. = There are=20 two types of guttae identifiable in vivo-one has a smooth, regular = posterior=20 surface and the other has an irregular posterior contour.

=B7Posterior=20 polymorphous dystrophy

It = is similar=20 to ICE syndrome, but can be distinguished using specular microscopy by=20 observing

-- = Dark=20 structures which have thick dark borders and lie anterior to = recognizable=20 endothelial cells that have an undistorted appearance and almost always = larger=20 than normal

-- = Snail track=20 (irregular) borders are seen as compared to rail track (parallel) = borders seen=20 at Descemet=92s membrane tears.

4. Endothelial wound = healing=20 mechanism

Before the advent of = specular=20 microscopy sloughing and sliding was the presumed mechanism of = endothelial wound=20 healing. But with = specular=20 microscopy, two additional healing mechanisms have been described.

a) = Endothelial cell coalescence (cell fusion) where the = common=20 cell membrane between two cells degenerates to result in a larger cell=20 containing two nuclei and all the cellular organelle of the two = individual=20 cells^25,26

b) = Endothelial cell mitosis, once generally believed to be = impossible for adult human endothelial cells, has also been demonstrated = in=20 adult human cornea after successful treatment for graft rejection. But the trigger for = endothelial cell=20 mitosis has not been identified.

5. Keratoconus=20

Here there is an increase in = cellular=20 pleomorphism and two populations of cells are seen =96 small and large = cells. Directional enlargement of = many=20 endothelial cells occurs²⁸ with the long axis oriented = towards the=20 apex of the cone and the cells appear stretched. Many have a dark structure = completely=20 within them. Corneas with = history=20 of acute hydrops have a localized area of endothelium in which the cells = are=20 7-10 times larger than normal.^{28, 29.}

6. Glaucoma =

=20 Persistent elevation of intraocular pressure produces a gradual = loss of=20 endothelial cells and a progressive loss of endothelial=20 function³⁰. It = results=20 from metabolic disturbances such as prolonged low oxygen concentration = in=20 aqueous humour.

7. Intraocular=20 inflammation

=20 During an episode of acute anterior uveitis mono-nuclear = inflammatory=20 cells penetrate and infiltrate both between endothelial cells and also = between=20 endothelial cells and Descemet=92s membrane. =20 In advanced cases the inflammatory cells can dislodge individual=20 endothelial cells and cause them to float in the aqueous humour.

8. Preoperative = evaluation

A clear cornea with a = normal=20 pachymetry reading is no assurance of a normal endothelial morphology or = cell=20 density. It is important to assess cell density as well as polymegathism = and=20 pleomorphism. Cell = density=20 threshold for development of corneal edema and bullous keratopathy has = been=20 estimated to be between 400-700 cells/mm^{2 (31).}So assuming a = cell=20 loss of 0-30%, any patient before most anterior segment surgeries should = have at=20 least 1000-1200 cells/mm^2.

=20 Age cannot be used to predict endothelial cell counts. Most patients including those = over 70=20 years should have a cell count of at least 2000 per square = mm³¹. Cell density difference = between a pair=20 of eyes must be more than 280 cells/mm²to be meaningful = (ideally no=20 difference). A = polymegathic and=20 pleomorphic endothelium does not tolerate surgery as well as a more = uniform=20 endothelial monolayer. A = monolayer=20 with a coefficient of variation greater than 0.4 or with less than 50% = hexagonal=20 cells, should be considered abnormal and possibly at a greater risk for=20 post-operative edema.

=20 Age cannot be used to predict the endothelial morphologic=20 appearance. A difference = in cell=20 polymegathism and pleomorphism between a pair of eyes should be greater = than=20 15-20% to be meaningful.

=20 If slit lamp biomicroscopy shows the following, the use of = specular=20 microscopy helps further

=B7 =20 Guttata

=B7 =20 Pigmented and inflammatory cells

=B7 =20 Keratic precipitates

=B7 =20 Endothelial surface or Descemet=92s membrane=20 irregularities

=B7 =20 Increased corneal thickness

9. Donor=20 corneas

Specular microscopic = evaluation can=20 be done of whole globes³² as well as corneas stored in tissue = culutre^{33,34, 35}. = For=20 transplant surgeries, endothelial cell count required is at least = 200-2055=20 cells/mm².Cell loss after routine penetrating=20 keratoplasty will range from 30-60% with stabilization of endothelial = monolayer=20 in three to four years. = Giant=20 endothelial cells indicate extensive cell loss. Survival of the graft = depends on=20 the initial cell count as well as on the degree of endothelial trauma = during the=20 intra-operative and post-operative course.

10. Penetrating = keratoplasty=20 (PKP)

Specular microscopy on = successful=20 corneal grafts indicates that substantial endothelial cell loss occurs = during or=20 immediately after surgery which varies from 5% to 80% after PKP. Giant=20 endothelial cells indicating extensive cell loss have been = observed. A grafted cornea can remain = transparent=20 and support 6/6 vision with less than 20% of its normal endothelial cell = denisty^37. Specular=20 microscopy is of value in following high risk patients since early = evidence of=20 graft rejection can be seen. = During=20 rejection episodes, one sees intercellular bright bodies, black = inflammatory=20 cells and generally recognizable keratic precipitates on the endothelial = surface.

11. Cataract = extraction & IOL=20 implantation

=20 In uncomplicated cases of cataract extraction the mean cell loss = can=20 range from virtually nil to levels that exceed 40%. Post-operative endothelial = cell loss is=20 somewhat more in intra-capsular than in extra-capsular cataract=20 extraction^{38, 39}.ECCE without intraocular lens=20 implantation is less traumatic to the endothelium than ECCE with IOL=20 implantation^{40, 41.} Anterior chamber lens causes = more damage=20 than a posterior chamber lens^42. If the IOL touches the = endothelium, even=20 momentarily, there is significant endothelial damage because of strong = adhesive=20 force between the methacrylate lens and the endothelial cell membrane = and the=20 cell membrane gets torn from the endothelial cells, irreversibly = damaging the=20 cells when the two surfaces are separated. =20 In phacoemulsification endothelial damage occurs due to the = mechanical=20 injury caused by anterior chamber instrumentation and anterior chamber=20 manipulation of a hard lens nucleus, ultrasonic vibrations, heat = generated at=20 the ultrasonic tip and prolonged intraocular=20 irrigation^43.

^REFRENCES

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3. = Laing R.Sandstrom M, Leibowitz H: Clinical = specular=20 microscopy I-Optical principles, = Arch Ophthalmol 97: 1717, 1979.

4. = Lemp M. Guimaraes R, Mahmood Me: Invo morphology = of the=20 human corneal endothelium- color microscopy, CORNEA 2: 295,=20 1983.

5. = Lemp M, Fold J: The effects of extebnded wear=20 hydrophilic contract lenses on the human ** corneal epithelium. Am J = Ophthalmol=20 101: 27, 1986.

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10. = Waring III=20 Go. Krohn MA, Ford et al: Four methods of measuring human endothelial = cells from specular = photomicrographs: Arch=20 Ophthalmol 97: 848-855.1980.

11. = Laing R,=20 Sandstrom M, Leibowitz H: Quantitative evaluation of corneal endothelial = micrographs. Arch Ophthalmolol 97:1720, 1979

12. = Nishi O.=20 Automated morphometry of corneal endothelial cell. Use of video camera and = vediotape=20 recorder. BJO 72: 68, 1988.

13. = Hartmann C,=20 Koditz W:Automated morphometric endothelial analysis

14. Yee = RW.Widefield clinical specular microscopy and computerized morphometric = analysis=20 Podos. SM, Yanoff M eds. = Text book=20 of ophthalmology Vol 8:External diseases, London , Mosby 1994,=20 7.10-7.12.

15. Rao = GN.=20 Waldron W.Aquavella J; Morpholoogy of graft endothelium and donor age. = Arch Ophthalmol 64: 523-527,=20 1980.

16. Rao = GN: Morphometary of coneal=20 endothelium.CORNEA 3: 153-154, 1984/1985.

17. = Bourne W,=20 Kaufman H: Specular microscopy of human corneal endothelium in vivi. Am = J=20 Ophthalmol 81:319, 1976.

18. = Lauk A et al:=20 Endothelial cell population changes of human cornea during life. Arch Ophtalmol 96:2031,=20 1978.

19. = Sturrock G,=20 Sherrard E, Rice N: Specular microscopy of the corneal endothelium. Br J Ophthalmol 62:809,=20 1978

20. = Sherrard E,=20 Novakovic O, Speedwell L:Age related changes of the corneal endothelium = ad=20 stroma as seen in vivo by specular microscopy. EYE 1:197, = 1987.

21. = Campbell D,=20 Shields M, Smith D: The corneal endothelium in the spectrum of essential iris = atrophy. Am j Ophthalmol = 86: 317,=20 1978.

22. = Hirst LW,=20 Quigley HA, Stark WJ, Shields MB specular microscopy of iridocorneal = endothelial=20 syndrome. Am J Ophthalmol = 89:11-21,=20 1980.

23. = Sherrard Es,=20 Frangooulis MA, Kerr Muir MG: On the morphology of cells of posterior = cornea in=20 iridocorneal endothelial syndrome. CORNEA 10:233-243, = 1991.

24. = Laing R et=20 al: The endothelial mosaic in Fuch=92s dystrophy- A qualitative = evaluation with=20 specular microscope. Arch = Ophthalmol 99:80, 1981.

25. = Neubauer L et=20 al: Coalescence of endothelial cells in the traumatized cornea = 1.Experimental=20 observation in cry0-preserved tissue. Arch Ophthalmol 101: 1787,=20 1983.

26. = Laing R et=20 al. Coalescence of endothelial cells in traumatized cornea II. Clinical observation. Arch Ophthalmol 101: 1712,=20 1983.

27. = Laing R et=20 al: Evidence of mitosis in the adult corneal endothelium. Ophthalmology 10: 1129,=20 1984.

28. = Laing R et=20 al: The human endothelium in keratoconus-A specular microscopic = study. Arch Ophthalmol 97: 1867,=20 1979.

29. = Skuta G,=20 Sugar J Erickson E: Corneal endothelial cell measurements in = megalocornea. Am J=20 Ophthalmol 101:51, 1983.

30. = Irvine A: The=20 role of endothelium in bullous keratopathy. Arch Ophthalmol 56: 338,=20 1956.

31. = Mishima S:=20 Clinical investigations in the corneal endothelium. Ophthalmology 89: 525-530,=20 1982.

32. = Matsuda M,=20 Yee RW, Glasser DB et al. Specular microscopic evaluation of donor = corneal=20 endothelium. Arch = Ophthalmol 104:=20 259-266, 1986.

33. = Bourne WM:=20 Examination and photography of donor corneal endothelium. Arch Ophthalmol 94: 1799-1800, = 1976.

34. = Roberts CW,=20 Rosskothen HD, Koester CJ: Wide field Specular microscopy of excised = donor=20 corneas. Arch Ophthalmol = 99:=20 881-883, 1981.

35. = Nesburn AB,=20 Mandelbaum S, Willey DE et al. = A=20 specular microscopic viewing system for donor corneas: Ophthalmology 90: = 686,=20 1983.

36. = Matsuda M,=20 Bourne WM: Long-term morphologic changes in the endothelium of = transplanted=20 corneas, Arch Ophthalmol 103: 1343-146, 1985.

37. = Bourne W,=20 Kaufman H. The endothelium of clear corneal transplants. Arch Ophthalmol 94:1730,=20 1976.

38. = Liesegang=20 T.Bourne W, Ilstrup D: Short and long-term endothelial cell loss = associated with=20 cataract extracion and IOL implantation. =20 Am J Ophthalmol 97: 32, 1984.

39. = Schultz R et=20 al: Response of the corneal endothelium to cataract surgey. Aarch Ophthalmol 10: 1164,=20 1986.

40. = Bourne W,=20 Kaufman H.Endothelial damage associated with IOLs. Am J Ophthalmol 81: 482,=20 1976.

41. = Sugar A et=20 al.Endothelial cell loss from IOL insertion. Ophthalmology 85: 394,=20 1978.

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43. = Binder P et=20 al: Corneal endothelial damage associated with phacoemulsification. Am J Ophthalmol 82: 48, 1976.=20

SPECULAR = MICROSCOPY: AN=20 OVERVIEW

History

Optical principles

Improvements in instrumentation and methods

Patient set-up