Near-Membrane Refractometry and Fluorescence Imaging Using Supercritical Angle Fluorescence

Total internal reflection fluorescence (TIRF) microscopy and its variants are standard 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 sample refractive index (RI). Here, we use a combination of TIRF excitation and supercritical angle fluorescence (SAF) emission detection to directly measure the average RI in the "footprint" region of a living 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 in culture. 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, for determining the mitochondrial energy state or detecting metabolically more active cancer cells.

In my talk, I will present data combining near-membrane imaging and refractometry with organelle precision in live cells, I will discuss the interest of combining TIRF excitation and SAF on a single microscope in a type of "co-planar" geometry, and I will swiftly introduce a new geometry allowing simultaneous confocal TIRF and RI imaging with sub-diffraction spatial resolution that we are currently implementing. (martin.oheim@parisdescartes.fr)