Near-membrane refractometry using supercritical angle fluorescence

TL;DR

This paper introduces a novel method combining TIRF microscopy and supercritical angle fluorescence detection to directly measure cell refractive index with subcellular resolution, enhancing quantitative imaging in live cells.

Contribution

The authors develop a new technique for measuring cell refractive index during imaging using supercritical angle fluorescence, enabling more accurate TIRF microscopy analysis.

Findings

Validated method on mouse embryonic fibroblasts

Achieved subcellular resolution RI measurements

Potential applications in cell imaging and disease detection

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, truely 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 mea-sure the average RI in the 'footprint' region of the cell, during imaging. Our RI measurement is based on the determination on a back-focal plane image of the critical angle separating supercritical and undercritial fluorescence emission components. We validate our method by imaging mouse embryonic fibroblasts. By targeting various dyes and fluorescent-protein chimerae to vesicles, the plasma membrane as well as mitochondria and the ER, we demonstrate local RI measurements with subcellular resolution on a standard TIRF microscope with a removable Bertrand lens as the only modification. Our tech-nique has important applications for imaging axial vesicle dynamics, mitochondrial energy state or detecting cancer cells.