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Facilitating trypanosome imaging

Author:
Glogger, Marius, Subota, Ines, Pezzarossa, Anna, Denecke, Anna-Lena, Carrington, Mark, Fenz, Susanne F., Engstler, Markus
Source:
Experimental parasitology 2017
ISSN:
0014-4894
Subject:
cell viability, crosslinking, cytoskeleton, ethylene glycol, fluorescence, hydrogels, image analysis, microscopy, models, plasma membrane, polyethylene glycol, thiols
Abstract:
Research on trypanosomes as a model organism has provided a substantial contribution to a detailed understanding of basic cellular processes within the last few years. At the same time, major advances in super-resolution microscopy have been achieved, facilitating the resolution of biological structures in living cells at a scale of a few nm. However, the motility of trypanosomes has prevented access to high resolution microscopy of live cells. Here, we present a hydrogel based on poly(ethylene glycol) functionalized with either norbornene or thiol moieties for UV induced thiol-ene crosslinking for the embedding and imaging of live trypanosomes. The resulting gel exhibits low autofluorescence properties, immobilizes the cells efficiently on the nanometer scale and is compatible with cell viability for up to one hour at 24 °C. We applied super-resolution imaging to the inner plasma membrane leaflet using lipid-anchored eYFP as a probe. We find specific domains within the membrane where the fluorescence either accumulates or appears diluted rather than being homogenously distributed. Based on a Ripley's analysis, the size of the domains was determined to be raccumulated=170±5 nm and rdilute>115±15 nm. We hypothesize that this structuring of the membrane is associated with the underlying cytoskeleton.
Agid:
5660108