title: Selective visualization of fluorescent sterols in Caenorhabditis elegans by bleach-rate-based image segmentation.
abstract: The nematode Caenorhabditis elegans is a genetically tractable model organism to investigate sterol transport. In vivo imaging of the fluorescent sterol, dehydroergosterol (DHE), is challenged by C. elegans' high autofluorescence in the same spectral region as emission of DHE. We present a method to detect DHE selectively, based on its rapid bleaching kinetics compared to cellular autofluorescence. Worms were repeatedly imaged on an ultraviolet-sensitive wide field (UV-WF) microscope, and bleaching kinetics of DHE were fitted on a pixel-basis to mathematical models describing the intensity decay. Bleach-rate constants were determined for DHE in vivo and confirmed in model membranes. Using this method, we could detect enrichment of DHE in specific tissues like the nerve ring, the spermateca and oocytes. We confirm these results in C. elegans gut-granule-loss (glo) mutants with reduced autofluorescence and compare our method with three-photon excitation microscopy of sterol in selected tissues. Bleach-rate-based UV-WF imaging is a useful tool for genetic screening experiments on sterol transport, as exemplified by RNA interference against the rme-2 gene coding for the yolk receptor and for worm homologues of Niemann-Pick C disease proteins. Our approach is generally useful for identifying fluorescent probes in the presence of high cellular autofluorescence.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark. wuestner@bmb.sdu.dk
authors.forename: Daniel
authors.initials: D
authors.lastname: Wüstner
authors.forename: Ane
authors.initials: A
authors.lastname: Landt Larsen
authors.forename: Nils J
authors.initials: NJ
authors.lastname: Faergeman
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Brewer
authors.forename: Daniel
authors.initials: D
authors.lastname: Sage
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000032:analysis; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D007089:Image Enhancement / Q000379:methods; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods*; D008860:Microscopy, Ultraviolet; D038761:Photobleaching; D034622:RNA Interference; D011973:Receptors, LDL / Q000032:analysis; D014018:Tissue Distribution
chemical_list: D029742:Caenorhabditis elegans Proteins; D005456:Fluorescent Dyes; C495203:NCR-1 protein, C elegans; C514538:RME-2 protein, C elegans; D011973:Receptors, LDL; C010905:dehydroergosterol; D004875:Ergosterol