Near-Membrane Refractometry Using Supercritical Angle Fluorescence

Biophysical Journal
Maia BrunsteinM Oheim

Abstract

Total internal reflection fluorescence (TIRF) microscopy and its variants are key technologies for visualizing the dynamics of single molecules or organelles in live cells. Yet truly quantitative TIRF remains problematic. One unknown hampering the interpretation of evanescent-wave excited fluorescence intensities is the undetermined cell refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence emission detection to directly measure the average RI in the "footprint" region of the cell during image acquisition. Our RI measurement is based on the determination on a back-focal plane image of the critical angle separating evanescent and far-field fluorescence emission components. We validate our method by imaging mouse embryonic fibroblasts and BON cells. By targeting various dyes and fluorescent-protein chimeras to vesicles, the plasma membrane, as well as mitochondria and the endoplasmic reticulum, we demonstrate local RI measurements with subcellular resolution on a standard TIRF microscope, with a removable Bertrand lens as the only modification. Our technique has important applications for imaging axial vesicle dynamics and the mitochondrial energy state or detecting metabolicall...Continue Reading

Citations

Apr 30, 2020·Biophysical Journal·Martin OheimMaia Brunstein
Nov 27, 2018·ACS Nano·Boris FerdmanYoav Shechtman
Apr 19, 2019·ACS Nano·Maia BrunsteinMartin Oheim
Sep 13, 2021·Biophysical Journal·Carine Julien, Martin Oheim

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