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 (SDC) 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 (TIRF) and epifluorescence microscopes. We applied this calibration method to quantify the number of 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 wells as the GTP-tubulin cap size. Our results show the unexplored potential of single-molecule imaging using spinning disk confocal microscopy and provide a straight-forward method to count the absolute number of fluorophores in tissues which can be applied to a wide range of biological systems and imaging techniques.

Chromatin behavior in living cells: Lessons from single‐nucleosome imaging and tracking

“…SDC employs a parallel array of pinholes on a rotating disk to reduce background fluorescence. [144][145][146][147] Although SDC can allow single-nucleosome imaging in rather thicker samples than the oblique illumination microscopy, the disk in an SDC microscope partially blocks some of the emission light. This reduces the number of photons detected and likely results in a lower detection efficiency, which may create critical issues for MSD analysis when a reduced amount of single nucleosomes are tracked.…”

Quantitative Determination of Protein Concentrations in Living Cells

“…Importantly, techniques based on detec?on of single molecules like singlemolecule tracking, and fluorescence correla?on spectroscopy (FCS) are experimentally limited to be conducted at low target-molecule concentra?ons which precludes many biological systems. Recently, fluorescence intensity calibra?on of spinning-disk confocal microscopy images has been performed to quan?fy the number of molecules per area by single-step bleaching [21] in the same manner as single-molecule data is processed [22]. However, this approach is similarly limited to the use of low-concentra?on samples in the range of a few hundreds of receptors per cell.…”