Observation of living glaucoma cells by video DIC microscopy
Purpose. Trabecular meshwork (TM) tissue excised from patients with primary open angle glaucoma (POAG) rarely survive passage to secondary culture. Our hypothesis is that the poor growth of these cells might indicate a cytoskeletal abnormality. This study focuses on developing a method for observation of dynamic processes within living normal and PO/AG TM cells as they grow from excised tissue. Methods. Human TM tissue from trabeculectomy or cadaver eyes was placed on gelatin-covered glass coverslips in supplemented DMEM medium at 37°. Upon growth of cells from the tissue, the coverslips were placed on a glass slide with warmed media, sealed, and placed on a warmed microscope stage. Cells were observed using enhanced video differential interference contrast (DIC) light microscopy at a final magnification of 10,000X to the video screen. Noise reduction and contrast enhancement was accomplished using a Hammatsu Argus 10 real time image processor, and the final image w is stored on S-VHS videotape. Results. Real time video images of POAG and control cells showed movements of vesicles and organelles within the cells as well as membrane ruffling and filopodial movements. Cells maintained these movements on the stage for more than three hours. Preliminary observations pointed to several apparent differences in POAG cells compared to controls. Vesicle movements appeared less organized and directed, filopodia appeared to be much less stable, often bending and breaking halfway down their length soon after completing full extension, and the membranes of POAG cells near areas of active filopodial growth appeared exceptionally fluid and unstable. Conclusions. Our method does allow observation of dynamic cell processes in living TM cells from POAG eyes. Alteration in vesicle movement (primarily along microtubules), filopodial stability (dependent on actin polymerization) and membrane stability may reflect important abnormalities in these systems. We will now begin to quantitate the differences we observe.
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