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Specular Microscopy : Specular Microscopy Basics


first pg graphic

The first laboratory specular microscope (courtesy of David Maurice)
(from photo Museum, Rosenwasser, G.O.D., in Mannis, M.J. and Mannis, A.A., eds. Corneal Transplantation: A History in Profiles, J.P. Wayenborgh, Belgium, 1999)

Since the endothelial cells are one of the most important structures in a donor cornea, their morphology and concentration must be carefully evaluated.   This can be done at the slit lamp by a technique called specular reflection or with a specular microscope.  Specular reflection refers to the viewing of objects that occurs when light is reflected from the interfaces of materials with different indices of refraction. This occurs in a mirror like fashion where the angle of incidence is equal to the angle of reflection.  An endothelial cell is different in refractive index than the aqueous and also Descemet's. while most of the light goes through these two transparent layers, about 0.02% is reflected backward to form an image of these structures. The more regular and numerous the cells are, the better their function is thought to be. Tightly packed, hexagonal-shaped cells with little variation in shape and size are considered normal. Cell densities greater than 2000/mm are generally accepted as suitable for transportation.

The optics of specular microscopy appear unfathomable if viewed as a whole picture (below).  By breaking down the optics to simple geometric surfaces, however, one can easily understand the principles.

fig 17

(Figure 17)

The flat surface which acts like a mirror is the easiest to understand.  Light which strikes the surface (bold lines) at a particular angle is reflected from the surface (thin lines) at the same angle (angle a = angle b). 

fig 18

(Figure 18)

The light is reflected from concave and convex surfaces in a similar fashion.  The difference is that much of the light is lost by reflecting in directions away from the viewing angle (dotted lines).  The techniques of specular reflection and specular microscopes collect a narrow beam portion of the reflected light to avoid unwanted bright reflection, which would wash out the image.  Besides the top of convex surfaces, the bottom of concave surfaces, and optically flat (mirror-like) surfaces,  the structures appear dark.  The top and bottom points may give rise to a central bright spot in these types of structures.  If the surface is very irregular, the light reflections are relatively randomly distributed, making the surface appear gray.  This is the average of many flat, concave and convex elements which make up the irregular surface.

 

fig 19

(Figure 19)

fig 20

(Figure 20)

Putting all the components together we get a complete view of the surface. Note that many rays of light get reflected away from the observer by the irregularities in the surface, while a few from the flat, mirror-like areas, still reach the observer.  This is what causes the light and dark areas in the specular microscopy images.

fig 21

(Figure 21)

Viewing the cells by specular microscopy allows the evaluation for cell density, variation in size (polymegathism), variation in shape (pleomorphism) and other factors such as injury, inherent disease, and inflammatory or foreign material.  Specular microscopic appearance of the cells varies with temperature, time of preservation, and media.  Freshly preserved tissue at room temperature is most easily evaluated.  Refrigerated tissue must be allowed to warm to room temperature to avoid condensation on the container and to allow cells to resume a near normal shape.  Warming should not be rushed.  The tissue should return to refrigeration as soon as possible.

The cornea should be evaluated as centrally as possible, since this is the area that will occupy the visual axis.  Several systems are available to perform specular microscopy.  A "back-up" method is to perform it with a slit lamp.  Estimates are based on counting 4-5 fixed 0.01mm2 frames or approximately 100 cells.

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