title: Facilitating trypanosome imaging.
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.
authors.affiliation: Department of Cell and Developmental Biology, Biocenter, University of Würzburg, Würzburg, Germany.
authors.forename: Marius
authors.initials: M
authors.lastname: Glogger
authors.affiliation: Department of Cell and Developmental Biology, Biocenter, University of Würzburg, Würzburg, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Subota
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.
authors.forename: Anna
authors.initials: A
authors.lastname: Pezzarossa
authors.affiliation: Department of Cell and Developmental Biology, Biocenter, University of Würzburg, Würzburg, Germany.
authors.forename: Anna-Lena
authors.initials: AL
authors.lastname: Denecke
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB21QW, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Carrington
authors.affiliation: Department of Cell and Developmental Biology, Biocenter, University of Würzburg, Würzburg, Germany. Electronic address: susanne.fenz@uni-wuerzburg.de.
authors.forename: Susanne F
authors.initials: SF
authors.lastname: Fenz
authors.affiliation: Department of Cell and Developmental Biology, Biocenter, University of Würzburg, Würzburg, Germany. Electronic address: markus.engstler@biozentrum.uni-wuerzburg.de.
authors.forename: Markus
authors.initials: M
authors.lastname: Engstler
mesh_terms: D000953:Antigens, Protozoan / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D015415:Biomarkers / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D020100:Hydrogels; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000235:genetics / Q000648:ultrastructure*
chemical_list: D000953:Antigens, Protozoan; D001426:Bacterial Proteins; D015415:Biomarkers; C401884:HASPB protein, Leishmania; D020100:Hydrogels; D008164:Luminescent Proteins; D015800:Protozoan Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins
keywords: Hydrogel; Membrane; Single-molecule fluorescence microscopy; Trypanosomes