Single-Molecule Fluorescence Imaging in Living Saccharomyces cerevisiae Cells

STAR Protoc. 2020 Oct 17;1(3):100142. doi: 10.1016/j.xpro.2020.100142. eCollection 2020 Dec 18.

Abstract

This protocol describes how to image fluorescently tagged proteins, RNA, or DNA inside living Saccharomyces cerevisiae cells at the single-molecule level. Imaging inside living cells, as opposed to fixed materials, gives access to real-time kinetic information. Although various single-molecule imaging applications are discussed, we focus on imaging of gene transcription at the single-RNA level. To obtain the best possible results, it is important that both imaging parameters and yeast culture conditions are optimized. Here, both aspects are described. For complete details on the use and execution of this protocol, please refer to Lenstra et al. (2015) and Donovan et al. (2019).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescence
  • In Situ Hybridization, Fluorescence / methods
  • Microscopy, Fluorescence / methods
  • Optical Imaging / methods*
  • RNA, Messenger / genetics
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae Proteins / genetics
  • Single Molecule Imaging / methods*
  • Transcription, Genetic / physiology

Substances

  • RNA, Messenger
  • Saccharomyces cerevisiae Proteins