Super-Resolved Fluorescence Lifetime Imaging of Single Cy3 Molecules and Quantum Dots Using Time-Correlated Single Photon Counting with a Four-Pixel Fiber Optic Array Camera.

Abstract

Time-resolved single molecule localization microscopy (TR-SMLM) with a 2 × 2 pixel fiber optic array camera was combined with time-correlated single photon counting (TCSPC) to obtain super-resolved fluorescence lifetime images of individual Cy3 dye molecules and individual colloidal CdSe/CdS/ZnS core/shell/shell semiconductor quantum dots (QDs). The characteristic blinking and bleaching behavior of the Cy3 and the blinking behavior of the QD emitters were used as distinguishing optical characteristics to isolate them and determine their centroid locations with spatial resolution below the optical diffraction limit. TCSPC was used to characterize the fluorescence lifetime and intensity corresponding to each emitter location. The mean centroid locations of the QDs could be determined with a precision of ∼1-4 nm, and the mean centroid locations of the Cy3 molecules could be determined with a precision of ∼2-9 nm, depending on the number of photons collected during the observation time. In a super-resolved fluorescence lifetime image with a single Cy3 dye molecule and a single QD separated by ∼34 nm, the two emitters were distinguished based on the average photon arrival times with respect to the excitation laser pulse observed during time intervals when only one emitter was in the on state, ∼6 ns for Cy3 and ∼17 ns for the QD. The mean distance between the two emitters was determined with a precision of ∼8 nm. The feasibility of using this super-resolved fluorescence lifetime imaging technique to investigate QD-dye complexes that use Förster resonance energy transfer (FRET) and/or electron transfer to form optical biosensors is discussed.