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

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 the number of end–binding protein 1 (EB1)–eGFP in the comets of growing microtubule ends and to measure the cytoplasmic concentration of EB1–eGFP in sensory neurons in fly larvae. These measurements allowed us to estimate the dissociation constant of EB1–eGFP from the microtubules as well as the GTP–tubulin cap size. Our results show the unexplored potential of single–molecule imaging using spinning–disk confocal microscopy and provide a straightforward method to count the absolute number of fluorophores in tissues that can be applied to a wide range of biological systems and imaging techniques.