Molecular Counting by Photon Statistics in Confocal Fluorescence Imaging

Quantifying molecules in subdiffraction-sized structures is crucial for understanding various fundamental biological processes. Ideally, a non-destructive measurement would result in an image of a cellular structure along with the number of fluorescent molecules in each region of interest. Recently, a considerable amount of work has gone into developing such methods. Counting by Photon Statistics (CoPS) is a promising new technique based on photon antibunching, which exploits the fact that a single molecule can only generate one photon at a time [1]. The number of independent fluorescent emitters can be determined by measuring photon coincidences on a time-resolved confocal microscope with four single- photon detectors (e.g., MicroTime 200, PicoQuant) [2]. Both a molecular brightness and the spatial density of fluorophores per image pixel are estimated. By summing over all corresponding pixels, the number of molecules in a structure can be calculated. The only calibration required is to determine the instrument's Point Spread Function (PSF) size. We investigated imaging with CoPS under a wide range of experimental conditions. The applicability of the method both to artificial samples such as immobilized DNA origami, as well as to biological samples is explored. We also discuss sample preparation requirements and practical guidelines for performing CoPS measurements. [1] Ta, H., Wolfrum, J., Herten, D.-P., An extended scheme for counting fluorescent molecules by photon-antibunching. Laser Phys. 20:119 (2010) [2] Ta, H.et al., Mapping molecules in scanning far-field fluorescence nanoscopy.Nat. Commun. 6:7977 (2015)