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

Abstract

Counting molecules in living cells is important for understanding biological phenomena. We used single–step bleaching to estimate the single–fluorophore intensity in a spinning–disk confocal microscope and quantify the amount of EB1–eGFPs in the neurons of fly larvae, allowing us to estimate their binding constant and the GTP–cap size of microtubules.