5/8/2023 0 Comments Uneven contrast cellprofiler![]() ![]() We seek to analyze large numbers of RPE cells in whole mounts to understand direct cell-to-cell and noncontact interactions RPE cell death affects the organization and shape of neighboring cells as they remodel to rescue the barrier function in The etiology of several diseases includes breakdown of the BRB accompanied by RPE cell loss and mosaic remodeling. The RPE is integral to the function of the mammalian outer blood-retinal barrier (BRB). The RPE cells have numerous other functions thatĪre tightly integrated with photoreceptor cells. The RPEĬells function as a barrier between the neuroepithelium and the choroid. The RPE is a continuous monolayer of interconnected, relatively uniform cuboidal cells on a thin spheroidal shell. This method and software can be readily applied to other aspects of vision science, neuroscience, andĮpithelial biology where patterns may exist in a sheet or surface of cells. To deviate from normal near large bumps in the RPE sheet caused by druse or when large frank holes in the RPE sheet are observed In the future, we mayīe able to observe interactions of second, third, or higher ring neighbors and analyze tension in sheets, which might be expected Image analysis of the RPE sheet, we can now analyze cell-to-cell interactions of immediate neighbors. This methodĪllows tens to hundreds of thousands of cells to be analyzed, each with 23 metrics. High-sampling-size approach performed as well as trained human scorers, but was considerably faster and easier. RPE sheet that had not been previously noticed in rd8.Ĭonclusions: An optimized dissection method and a series of programs were used to establish a rapid and hands-off analysis. Rd8 mice exhibited several measurable changes in patterns of RPE cells compared to WT, suggesting a slow degeneration of the Support a role in the maintenance of PhR cells. The health of the RPE cell, while the slow loss of photoreceptor (PhR) cells previously established in this knockout does IRBP −/− mouse RPE sheet did not differ from C57BL/6J (wild type, WT), suggesting that IRBP does not play a direct role in maintaining Subtle differences between the RPE sheet characteristics of young and old mice were identified. We compared normal with diseased RPE cells during aging with quantitative cell Whether tallied manually or automatically with software, the resultingĬell measurements were in close agreement. Human- and computer-analyzed results were compared. Of the shape of each cell, its neighbors, and interactions beyond direct cell–cell contact in the sheet. Record an image suitable for large-scale identification of cell-to-cell boundaries, and then obtained quantitative descriptors ![]() The software-based analysis merged 25 to 36 images into one and adjusted settings to Substantial fractions of the RPE sheet (usually 20–50% of the sheet) could be analyzed. Almost all of the sheet could be routinely imaged, and The dissectionĪnd flatmounting techniques were illustrated in a video recording. Results: A simplified dissection procedure afforded a consistent source of images that could be processed by computer. Photoshop, Java, Perl, and Matlab scripts, as well as CellProfiler, were used to quantify selected parameters.ĭata were exported into Excel spreadsheets for further analysis. The RPE sheet was imaged under fluorescence confocal microscopy after staining for ZO-1 to identify RPEĬell boundaries. The dissection and outcomes were monitored and evaluatedīy video recording. Iris, and the neural retina were removed, leaving the RPE sheet exposed. Four radial cuts were made with iridectomy scissors from the puncture to near the optic nerve head. Eyes were fixed for 10 min, and dissected by puncturing the cornea with a sharp needle or a Mouse eyes were harvested, and extra-orbitalįat and muscles were removed. Of open-access software to rapidly analyze large numbers of flatmount images. Methods: The two principal methodological advances of this study were optimization of a mouse RPE flatmount preparation and refinement (RPE) cells and contact- and noncontact-mediated cell-to-cell interactions across a large series of flatmount RPE images. Purpose: Our goal was to optimize procedures for assessing shapes, sizes, and other quantitative metrics of retinal pigment epithelium
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