We present an optical technique, called AB/FCS-fingerprinting, capable of characterizing fluorophore independence in aqueous solution using two-photon excitation microscopy and time-correlated single photon counting. Fluorescence correlation spectroscopy (FCS) is used to monitor fluctuations in the intensity of emissions. Auto- or cross-correlation analysis of these fluctuations can measure the average number of fluorescent molecules in an aqueous sample. Photon antibunching (AB) is observed from single quantum entities, which can emit only one photon at a time. By recording the number of coincident photons detected as a function of time between photon detections, AB analysis is used to determine the number of independent emitters in a sample. In AB/FCS-fingerprinting, the number of independent emitters in a sample is compared with the average number of fluorescent molecules in the same sample using a microscope that can be rapidly reconfigured to measure either AB or FCS from serial dilutions of a florescent sample. Since the number of fluorescent molecules is not necessarily equal to the number of independent emitters, a comparison of these values can provide insight into the independence of fluorophores in molecular assemblies. We validated this technique by measuring AB/FCS-fingerprinting of serial dilutions of mVenus, mNeonGreen, and an organic fluorophore, Alexa-Fluor-488. Experimental results were in good agreement with Monte-Carlo antibunching simulations for a single quantum emitter and with the predictions of a zero-truncated Poisson distribution model for photon antibunching from monomeric fluorophores in solution.

Date Published: 4 March 2019
PDF
Proc. SPIE 10882, Multiphoton Microscopy in the Biomedical Sciences XIX, 108820P (4 March 2019); doi: 10.1117/12.2513683

Published in SPIE Proceedings Vol. 10882:
Multiphoton Microscopy in the Biomedical Sciences XIX
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)