Selective visualization of fluorescent sterols in Caenorhabditis elegans by bleach-rate-based image segmentation

Daniel Wüstner, Ane Landt Larsen, Nils J. Færgeman, Jonathan R Brewer, Daniel Sage

Research output: Contribution to journalJournal articleResearchpeer-review

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.
Original languageEnglish
JournalTraffic
Volume11
Issue number4
Pages (from-to)440-54
Number of pages15
ISSN1398-9219
DOIs
Publication statusPublished - 1. Apr 2010

Fingerprint

Caenorhabditis elegans
Sterols
Image segmentation
Visualization
Bleaching
Tissue
Imaging techniques
Nerve Tissue
Kinetics
RNA Interference
Protein C
Fluorescent Dyes
Photons
Oocytes
dehydroergosterol
Microscopy
Rate constants
Microscopic examination
Screening
Microscopes

Keywords

  • Animals
  • Caenorhabditis elegans
  • Caenorhabditis elegans Proteins
  • Chemiluminescent Measurements
  • Ergosterol
  • Fluorescent Dyes
  • Image Enhancement
  • Image Processing, Computer-Assisted
  • Microscopy, Ultraviolet
  • Photobleaching
  • RNA Interference
  • Receptors, LDL
  • Tissue Distribution

Cite this

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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.",
keywords = "Animals, Caenorhabditis elegans, Caenorhabditis elegans Proteins, Chemiluminescent Measurements, Ergosterol, Fluorescent Dyes, Image Enhancement, Image Processing, Computer-Assisted, Microscopy, Ultraviolet, Photobleaching, RNA Interference, Receptors, LDL, Tissue Distribution",
author = "Daniel W{\"u}stner and {Landt Larsen}, Ane and F{\ae}rgeman, {Nils J.} and Brewer, {Jonathan R} and Daniel Sage",
year = "2010",
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language = "English",
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Selective visualization of fluorescent sterols in Caenorhabditis elegans by bleach-rate-based image segmentation. / Wüstner, Daniel; Landt Larsen, Ane; Færgeman, Nils J.; Brewer, Jonathan R; Sage, Daniel.

In: Traffic, Vol. 11, No. 4, 01.04.2010, p. 440-54.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Selective visualization of fluorescent sterols in Caenorhabditis elegans by bleach-rate-based image segmentation

AU - Wüstner, Daniel

AU - Landt Larsen, Ane

AU - Færgeman, Nils J.

AU - Brewer, Jonathan R

AU - Sage, Daniel

PY - 2010/4/1

Y1 - 2010/4/1

N2 - 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.

AB - 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.

KW - Animals

KW - Caenorhabditis elegans

KW - Caenorhabditis elegans Proteins

KW - Chemiluminescent Measurements

KW - Ergosterol

KW - Fluorescent Dyes

KW - Image Enhancement

KW - Image Processing, Computer-Assisted

KW - Microscopy, Ultraviolet

KW - Photobleaching

KW - RNA Interference

KW - Receptors, LDL

KW - Tissue Distribution

U2 - 10.1111/j.1600-0854.2010.01040.x

DO - 10.1111/j.1600-0854.2010.01040.x

M3 - Journal article

C2 - 20070610

VL - 11

SP - 440

EP - 454

JO - Traffic

JF - Traffic

SN - 1398-9219

IS - 4

ER -