Super-resolution live cell microscopy of membrane-proximal fluorophores

Verena Richter, Peter Lanzerstorfer, Julian Weghuber, Herbert Schneckenburger

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Here, we present a simple and robust experimental setup for the super-resolution live cell microscopy of membrane-proximal fluorophores, which is comparably easy to perform and to implement. The method is based on Structured Illumination Microscopy (SIM) with a switchable spatial light modulator (SLM) and exchangeable objective lenses for epi-illumination and total internal reflection fluorescence (TIRF) microscopy. While, in the case of SIM (upon epi-illumination), cell layers of about 1–2 µm in close proximity to the plasma membrane can be selected by software, layers in the 100 nm range are assessed experimentally by TIRF-SIM. To show the applicability of this approach, both methods are used to measure the translocation of the glucose transporter 4 (GLUT4) from intracellular vesicles to the plasma membrane upon stimulation by insulin or insulin-mimetic compounds, with a lateral resolution of around 100 nm and an axial resolution of around 200 nm. While SIM is an appropriate method to visualize the intracellular localization of GLUT4 fused with a green fluorescent protein, TIRF-SIM permits the quantitative evaluation of its fluorescence in the plasma membrane. These imaging methods are discussed in the context of fluorescence lifetime kinetics, providing additional data for the molecular microenvironment.

Original languageEnglish
Article number7099
Pages (from-to)1-12
Number of pages12
JournalInternational Journal of Molecular Sciences
Volume21
Issue number19
DOIs
Publication statusPublished - 26 Sep 2020

Keywords

  • Fluorescence imaging
  • Glucose transporter
  • Insulin
  • Insulin mimetic drugs
  • SIM
  • Super-resolution microscopy
  • TIRF
  • Cricetulus
  • Protein Transport/drug effects
  • Humans
  • Luminescent Proteins/analysis
  • Optical Imaging/methods
  • Madin Darby Canine Kidney Cells
  • Microscopy, Fluorescence/methods
  • Hypoglycemic Agents/pharmacology
  • CHO Cells
  • Cell Survival
  • Imaging, Three-Dimensional/methods
  • Cell Membrane/drug effects
  • Insulin/pharmacology
  • Glucose Transporter Type 4/analysis
  • Animals
  • Dogs
  • Luminescent Agents/analysis
  • Software

Fingerprint Dive into the research topics of 'Super-resolution live cell microscopy of membrane-proximal fluorophores'. Together they form a unique fingerprint.

Cite this