Super-Resolution Imaging of the Actin Cytoskeleton in Living Cells Using TIRF-SIM

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Super-resolution (SR) imaging techniques have advanced rapidly in recent years, but only a subset of these techniques is gentle enough to be used by cell biologists to study living cells with minimal photodamage. Our research is focused on studies of the dynamic remodeling of the actin cytoskeleton in living pancreatic beta cells during insulin secretion. These studies require super-resolution light microscopic techniques that are gentle enough to record rapid changes of the actin cytoskeleton in real time. In this chapter, we describe an SR technique that breaks the diffraction limit of the conventional light microscope called TIRF-SIM. Using this SR techniques, we have been able to show that (1) microvilli on pancreatic beta cells translocate in the plane of the plasma membrane and (2) the cortical actin network reorganizes when cells are stimulated to secrete insulin. We describe the FIJI plugins that were used to process and analyze the TIRF-SIM images to obtain quantitative data.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages3-24
Number of pages22
DOIs
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2364
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Airyscan
  • CLSM
  • Cortical actin cytoskeleton
  • FIJI
  • Image processing and analysis
  • Live cell imaging
  • Microvilli
  • Pancreatic beta cells
  • Super-resolution microscopy
  • TIRF-SIM

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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