P J Szczesny1, P Walther, M Müller. 1. Laboratory for Electron Microscopy 1, Swiss Federal Institute of Technology, Zurich, Switzerland.
Abstract
PURPOSE: In this electron microscopical study, we compared effects of chemical fixation versus cryofixation on the ultrastructure of acute rod outer segment alterations in the rat retina. METHODS: The alterations were induced by toxic levels of diffuse white light. Albino rats were exposed to 2000 lux for 30 min. Samples from one eye of each animal were fixed by high pressure freezing and samples from the other eye were fixed by standard glutaraldehyde procedures. RESULTS: Light exposed retina showed major differences in their rod outer segments, inner segments and photoreceptor synaptic regions in chemical fixation. In particular gross vesiculations of outer segment membranes were produced in light exposed experiments. In contrast, in cryo-fixed samples such prominent changes were not observed in outer segment membranes. There was, however, occasional formation of small vesicles and a reduction of the cilium diameter in response to light damage. In the dark adapted control group the morphology of chemically fixed and cryo-fixed photoreceptors was similar. CONCLUSIONS: We conclude, that cryo-fixed samples better represent the living state of the retina, because high pressure freezing is a purely physical method and acts much faster than chemical fixation.
PURPOSE: In this electron microscopical study, we compared effects of chemical fixation versus cryofixation on the ultrastructure of acute rod outer segment alterations in the rat retina. METHODS: The alterations were induced by toxic levels of diffuse white light. Albino rats were exposed to 2000 lux for 30 min. Samples from one eye of each animal were fixed by high pressure freezing and samples from the other eye were fixed by standard glutaraldehyde procedures. RESULTS: Light exposed retina showed major differences in their rod outer segments, inner segments and photoreceptor synaptic regions in chemical fixation. In particular gross vesiculations of outer segment membranes were produced in light exposed experiments. In contrast, in cryo-fixed samples such prominent changes were not observed in outer segment membranes. There was, however, occasional formation of small vesicles and a reduction of the cilium diameter in response to light damage. In the dark adapted control group the morphology of chemically fixed and cryo-fixed photoreceptors was similar. CONCLUSIONS: We conclude, that cryo-fixed samples better represent the living state of the retina, because high pressure freezing is a purely physical method and acts much faster than chemical fixation.
Authors: Wiebke Schlörmann; Frank Steiniger; Walter Richter; Roland Kaufmann; Gerd Hause; Cornelius Lemke; Martin Westermann Journal: Histochem Cell Biol Date: 2009-10-23 Impact factor: 4.304