Brightness calibrates particle size in single particle fluorescence imaging.

Abstract

This Letter provides a novel approach to quantify the particle sizes of highly bright semiconductor polymer dots (Pdots) for single-particle imaging and photobleaching studies. A quadratic dependence of single-particle brightness on particle size was determined by single-particle fluorescence imaging and intensity statistics. In terms of the same imaging conditions, the particle diameter can be quantified by comparing the individual brightness intensity with associated calibration curve. Based on this sizing method, photobleaching trajectories and overall photon counts emitted by single particles were analyzed. It is found that photobleaching rate constants of different sized Pdots are not strongly dependent on particle diameter except the sparsely occurring fluorescence blinking in certain dim particles and the rapid photobleaching component in some bright particles. The overall photon counts increase with increasing particle diameter. However, those larger than 30nm deviate away from the increasing tendency. These results reveal the significance of selecting appropriate Pdots (≤30 nm) for single-particle imaging and tracking applications.