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Proceedings Paper

Detecting thermal phase transitions in corneal stroma by fluorescence micro-imaging analysis
Author(s): P. Matteini; F. Rossi; F. Ratto; I. Bruno; P. Nesi; R. Pini
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Paper Abstract

Thermal modifications induced in corneal stroma were investigated by the use of fluorescence microscopy. Freshly extracted porcine corneas were immersed for 5 minutes in a water bath at temperatures in the 35-90°C range and stored in formalin. The samples were then sliced in 200-μm-thick transversal sections and analyzed under a stereomicroscope to assess corneal shrinkage. Fluorescence images of the thermally treated corneal samples were acquired using a slow-scan cooled CCD camera, after staining the slices with Indocyanine Green (ICG) fluorescent dye which allowed to detect fluorescence signal from the whole tissue. All measurements were performed using an inverted epifluorescence microscope equipped with a mercury lamp. The thermally-induced modifications to the corneal specimens were evaluated by studying the grey level distribution in the fluorescence images. For each acquired image, Discrete Fourier Transform (DFT) and entropy analyses were performed. The spatial distribution of DFT absolute value indicated the spatial orientation of the lamellar planes, while entropy was used to study the image texture, correlated to the stromal structural transitions. As a result, it was possible to indicate a temperature threshold value (62°C) for high thermal damage, resulting in a disorganization of the lamellar planes and in full agreement with the measured temperature for corneal shrinkage onset. Analysis of the image entropy evidenced five strong modifications in stromal architecture at temperatures of ~45°C, 53°C, 57°C, 66°C, 75°C. The proposed procedure proved to be an effective micro-imaging method capable of detecting subtle changes in corneal tissue subjected to thermal treatment.

Paper Details

Date Published: 11 February 2008
PDF: 5 pages
Proc. SPIE 6844, Ophthalmic Technologies XVIII, 68441U (11 February 2008); doi: 10.1117/12.761540
Show Author Affiliations
P. Matteini, Istituto di Fisica Applicata (Italy)
F. Rossi, Istituto di Fisica Applicata (Italy)
F. Ratto, Istituto di Fisica Applicata (Italy)
I. Bruno, Univ. di Firenze (Italy)
P. Nesi, Univ. di Firenze (Italy)
R. Pini, Istituto di Fisica Applicata (Italy)

Published in SPIE Proceedings Vol. 6844:
Ophthalmic Technologies XVIII
Bruce E. Stuck; Fabrice Manns; Per G. Söderberg; Michael Belkin M.D.; Arthur Ho, Editor(s)

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