Abstract
Purpose: :
SEM characterizes SCE surface topography. However, SEM involves separation of SC inner and outer walls associated with disruption of any structures in SC, processing that often induces up to 20 % shrinkage artifact and is destructive preventing further imaging. Such problems are avoided by the non-destructive technique described in this report, a technique that not only defines surface topography but also outlines surface boundaries of individual cells of SC inner wall (IW), outer wall (OW) and collector channels (CC).
Methods: :
Macaca nemestrina monkey eyes (10), Dissection into quadrants with Healon© injection to dilate SC. The entire limbus was then dissected into 500 u radial segments. Dissecting microscope images were used to identify structures spanning SC, then further characterized by phase and differential interference contrast (Nomarski). Labeling of two selected SC regions with PECAM-1 (CD31) and propidium iodide. Confocal Fluoview imaging of 0.5 u slices (185 images in specimen I (SI), (145 images in specimen II (SII)). Independent two-channel 12 bit image stacks were imaged sequentially to reduce crosstalk and optimize each channel intensity; each channel then was normalized & down sampled to 8 bit/channel to permit Image J native projections tools to generate 3D projections. Images then were combined to produce RGB 3D stack projections rotating 180° around both the X and Y-axis to provide 3 ° increment images and movies.
Results: :
Propidium iodide, selective for DNA in both the nucleus and mitochondria of cytoplasm, stained the entire interior of the cells, while CD31 stained cell membranes. Together the labels outlined cellular topography of the walls of SC, cylindrical tubes spanning SC and the interior of CC. Surfaces completely obscured in one orientation were at times well visualized by rotating the 3D images. SCE cells were generally elongated as were CC cells, but SCE in the region of funnel entrances had a markedly different orientation and configuration with a very narrow cellular profile. Cell margins clearly demarcated in SI: SCE {IW (23), OW (33)}, tube wall (26), CC (18) and in SII: SCE {IW (105), OW (0)}, tube wall (13), CC (63).
Conclusions: :
A new technique involves viscoelastic dilation of SC, then labeling of SCE with propidium iodide and CD31. Subsequent 3D image generation permits examination of SCE surface topography and effectively defines cell wall boundaries while avoiding the need for SC wall separation, the shrinkage artifact and the complex destructive processing required in SEM.
Keywords: trabecular meshwork • outflow: trabecular meshwork • microscopy: light/fluorescence/immunohistochemistry