The cornea is a normally transparent and avascular
multilayered tissue forming the anterior part of the eyeball and is the
main refractive element of the eye. The structure of the cornea is illustrated
in Fig. 1. The great bulk (90% of thickness) is made up of the stroma that
is bounded externally by Bowman's membrane and epithelium and internally
by Descemet membrane and endothelium. In order to keep maintaining the
normal transparency of the cornea, various physiological processes must
be active. The corneal stroma is normally in a state of relative deturgescence
and if it is allowed to imbibe water it swells and loses its transparency.
Both epithelium and endothelium layers represent semipermeable membranes
that inhibit the leakage of fluid and ions in the stroma. The epithelium
consists of 5-6 layers of cells and therefore it is an effective barrier
to the diffusion of tear film fluid into the stroma. Conversely, the endothelium
is a single layer of closely packed hexagonal cells whose permeability
to small ions is 100 times higher than epithelium. The barrier function
of the endothelium is weak. Nevertheless, due to the presence of an active
pump mechanism that removes fluid from the stroma, it assumes a critical
importance in regulating the optimum level of stromal hydration to maintain
corneal transparency. Because of the lack of proliferative capacity of
the corneal endothelium, cell damage may be healed by migration and enlargement
of remaining cells. This results in a decrease of the cell density, an
increase of variation between the cell sizes and a disruption of the normal
regular hexagonal pattern. The development of improved systems for the
clinical specular microscopy in vivo has strongly contributed to the study
and to the comprehension of the physiopathology of the corneal endothelium
[1-2]. The commonly employed parameter in the evaluation of the endothelial
pattern is the mean cell density expressed as cells/mm2.

The more recent systems allow both the direct
acquisition of the images and automated morphometric analysis. The automated
reconstruction of the endothelium cell borders is not always precise, and
although it allows to evaluate in a reliable way the Endothelial Cell Density
(ECD) and the Coefficient of Variation (CV), it does not allow the correct
recognition of the cell shapes, if only in terms of a coefficient of roundness
coefficient (approximation to the circle), whose correlation with the cell
shape is questionable. The semiautomated devices for morphometric analysis
are time consuming and provide results that could be influenced from the
operator criterion used in the cell apex definition. The main advantage
of such devices is the capability to evaluate the cell shape.
[1] RA.Laing, MM.Sandstrom, HM.
Liebowitz, "In vivo photomicrography of the corneal endothelium", Arch.
Ophthalmol. 93: 143, 1975
[2] WM Bourne, HE Kaufman, "Specular
microscopy of human corneal endothelium in vivo", Am. J Ophthalmol. 81:
319, 1976
MORPHY has been developed to give a real-time
easy-to-use tool to the doctor in order to deal with an useful support
in clinical diagnostic. The program consists of two main section relative
to the digital acquisition of the microscopic images and their processing
respectively.
The acquisition section of the program makes
use of a MATROX "Frame Grabber" board
and its relative library. It has to acquire the corneal endothelium microscopic
images coming form a specular microscopy (KONAN - 5500 SP), with on board
a 40x lens. Moreover, the microscopy has been interfaced with an analogue
camera (Sony Hi Resolution SSC M370 CE).
A simple hardware interface has been designed
and manufactured to enable the user to control the camera focus and the
grabbing procedure directly from the console.
The second section of the program has to process
the acquired images following the above described algorithm. The image
processing steps can be controlled on-line by the doctor who can modify
some of the algorithm’s parameters (e.g. the threshold value in the "Threshold"
operation). The last processing step will give to the doctor a final report
containing both the items of the detected cells and their area. In particular,
this step, making use of both the information coming from the skeletonized
and branchpoint images by way of a tracking procedure, will be able to
detect the cell's shape and calculate the respective areas.
The MORPHY’s output data are:

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