Counting fluorescently labeled proteins in tissues in the spinning-disk microscope using single-molecule calibrations.

ABSTRACT: Quantification of molecular numbers and concentrations in living cells is critical for testing models of complex biological phenomena. Counting molecules in cells requires estimation of the fluorescence intensity of single molecules, which is generally limited to imaging near cell surfaces, in isolated cells, or where motions are diffusive. To circumvent this difficulty, we have devised a calibration technique for spinning-disk confocal microscopy, commonly used for imaging in tissues, that uses single-step bleaching kinetics to estimate the single-fluorophore intensity. To cross-check our calibrations, we compared the brightness of fluorophores in the SDC microscope to those in the total internal reflection and epifluorescence microscopes. We applied this calibration method to quantify th

SUBMITTER: Liao M 

PROVIDER: S-EPMC9265152 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature