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J. Davis, P.D. Calvert, E.N. Pugh, Jr.; Multiphoton Imaging of Live Mouse Retinal Pigment Epithelium Cells and Measurement of Small Molecule Diffusion with a Vital Fluorescent Dye . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2028.
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© ARVO (1962-2015); The Authors (2016-present)
To measure the envelope volume of individual epithelial cells in intact sheets of mouse retinal pigment epithelium (RPE) using multiphoton excitation microscopy of fluorescent dyes, and to analyze the distribution and diffusion of a small (623 Da) polar molecule.
Fresh sheets of retinal pigment epithelium (RPE) were harvested by dissection from the eyes of wildtype BALB/c mice; the use of these albino mice obviated interference by melanin during imaging. Isolated tissue fragments were incubated with the non–fluorescent cell–permeant calcein AM, which is cleaved in viable cells to a highly anionic, well–retained green fluorescent dye. Fluorescence of calcein in the cells was produced with multiphoton excitation by a Ti:Sapphire laser tuned to 920 nm. RPE cells were scanned in a 3D volume of 100 x 100 x 35 µm, and the volumes of individual cells were determined using published methods (1). Diffusion of calcein in the RPE cells was measured with methods based on fluorescence recovery after photobleaching (FRAP), and analyzed with customized Matlab software.
The RPE was readily identified in bright field and multiphoton imaging by its regular hexagonal packing and its position among the unstained lamellae of dissected eyes. Vigorous fluorescence of calcein confirmed the viability of the RPE cells. Qualitative analysis of transverse optical sections through en face oriented tissue revealed an even distribution of fluorescence throughout the available cell volume, suggesting no intracellular binding or other sequestration of the dye. Analysis of the calcein images of RPE cells yielded an average volume of 5.5 ± 0.3 x 103 µm3; the average diameter was 23 ± 6 µm and thickness 12 ± 3 µm. Examination of the FRAP data yielded an apparent diffusion coefficient for calcein of 270 µm2/s (T = 22.6°C); this value represents an approximately twofold decrease in the diffusivity as compared to the diffusion coefficient of 550 µm2/s (T = 22.2°C) estimated for calcein in solution.
Viable sheets of mouse RPE cells can be isolated from the eye and characterized with vital dyes. The cells in such sheets exhibit normal morphology, and their morphometric properties can be measured. The diffusion coefficient of soluble fluorescent molecules in the cells can be determined. This preparation offers considerable potential for characterization of RPE cell structure and its molecular and physiological properties. 1. Peet J, Bragin A, Calvert P et al. J Cell Sci. 2004; 117:3049–59
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