Total internal reflection fluorescence lifetime and anisotropy screening of cell membrane dynamics.

Abstract

A high content screening (HCS) system for fluorescence measurements at surfaces, in particular the plasma membrane of living cells, is described. The method is based on multiple total internal reflections (TIRs) of an incident laser beam within the glass bottom of a microtiter plate such that up to 96 individual samples could be illuminated by an evanescent electromagnetic field. Fluorescence lifetimes and time-resolved fluorescence anisotropies of these samples were assessed. While fluorescence lifetime represents a general measure for the interaction of a marker molecule with its microenvironment, the rotational diffusion time corresponds to the relaxation time of a molecule from a position with a defined orientation into a position with an arbitrary orientation. Thus, time-resolved fluorescence anisotropy reflects the viscosity of the microenvironment, i.e., membrane fluidity in the case of living cells. For all measurements in this study, either human glioblastoma cells incubated with the fluorescent membrane marker NBD-cholesterol or human breast cancer cells expressing a membrane-associating fluorescent protein were used.

• Authors: Thomas Bruns, Wolfgang S L Strauss, Herbert Schneckenburger
• Journal: Journal of biomedical optics (J Biomed Opt) 2008 Jul-Aug Vol. 13 Issue 4 Pg. 041317 ISSN: 1083-3668 [Print] United States
• PMID: 19021325 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Cholesterol

Topics

• Algorithms
• Anisotropy
• Cell Membrane (chemistry, metabolism)
• Cholesterol (chemistry, metabolism)
• Humans
• Image Interpretation, Computer-Assisted (methods)
• Microscopy, Fluorescence (methods)
• Spectrometry, Fluorescence (methods)