  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.
