Real-time monitoring of changes in plasma membrane potential via imaging of fluorescence resonance energy transfer at individual cell resolution in suspension

Abstract

A method for monitoring heterogeneity in changes of plasma membrane potential (PMP) at an individual cell resolution while in suspension, utilizing a simple and low-cost wide-field illumination arrangement, is presented. The method is modeled via HEK-293 cell line in suspension, double stained with coumarin and oxonol (donor and acceptor), which were loaded into an array of nanoliter wells, each designed to preserve the individuality of the nontethered cell it holds during vigorous biomanipulation. Depolarization of PMP was induced by high K(+) solution, reducing the proximity between the membrane fluorophores and subsequently reducing the efficiency (E%) of resonance energy transfer between them. Spatial plots of E% were produced from both images of fluorescence intensity and polarization. The spatial resolution of E% plots seem to be higher, and their contrast greater, when calculated from the polarization, rather than from the intensity of the fluorescence.

Cellular Redox Profiling Using High-content Microscopy

“…However, these limitations could be overcome using micropatterned cell culture supports that keep individual cells (adherent or non-adherent) trapped in small micro wells while maintaining their viability 30 , or by the use of a thermo-reversible hydrogel to trap cells during imaging 31 . These methods have already been used for high-content screening of plasma membrane potential, or cellular oxygen in individual suspension cells 32,33 or the imaging of intracellular markers in the living, highly motile parasites 31 . Adaptations would also have to be made to the imaging modality, since morphological analysis of the mitochondrial network in these cells would require rapid 3D-acquisition capabilities such as spinning disk confocal or Bessel beam light sheet microscopy 34,35 , as well as to the analysis pipeline, to include the Zdimension while calculating morphological parameters.…”

Cellular Redox Profiling Using High-content Microscopy

“…. These methods have already been used for high-content screening of plasma membrane potential, or cellular oxygen in individual suspension cells 32,33 or the imaging of intracellular markers in the living, highly motile parasites…”