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Steven J. Gardner, Brendan E. Allman, Christina S. Kamma-Lorger, Carlo Knupp, Nick White, Keith M. Meek; Measuring The Refractive Index Of Activated Bovine Corneal Fibroblasts, Using Quantitative Phase Imaging. Invest. Ophthalmol. Vis. Sci. 2012;53(14):129. doi: https://doi.org/.
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Recent studies have suggested that the development of post operative corneal haze could be due to an increase in the amount of light scattering from corneal keratocytes. Changes in the refractive index after the activation of keratocytes could be responsible as the underlying mechanism for this manifestation. Studies have hypothesised that quiescent keratocytes produce crystallin molecules in order to change the refractive index of their cytoplasm to match the surrounding extra-cellular material, and hence reduce the amount of light scattering. In order to test this hypothesis, we have measured the refractive index of bovine corneal fibroblasts, using the technique of quantitative phase imaging of live cells in-vitro.
Bovine myofibroblasts were cultured in Dulbecco's modified Eagle's medium. These cells were then plated onto glass-bottomed Petri dishes, and imaged under bright-field Kohler illumination conditions. Images were taken in stacks separated by one micron in the z-direction. A phase image was then produced from the in-focus image and one each of positively and negatively out-of-focus images, using software obtained from Iatia Ltd. Measurements of the cell’s thickness was obtained using confocal imaging of live cells suspended in fluorescent isothiocyanate-dextran. Phase and thickness measurements were then used to calculate the refractive index of the fibroblasts for both their nucleus and the surrounding cytoplasm.
The calculated refractive index for fibroblast cells (n_cell=1.365) was found to be lower than the overall refractive index of the cornea that has been shown in previous studies (n_cornea=1.375). n_cell did not significantly differ from the nucleus to the cytoplasm.
The observed lower value of n_cell when compared to the surrounding material would result in an increase in scattering within the corneal tissue, and hence a loss of transparency. Further work will focus on obtaining a value of n for quiescent keratocytes, in order to model changes in light scattering and to test the cytoplasmic crystallin theory of corneal cell light scattering.
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