Measuring photophysical transition rates with fluorescence correlation spectroscopy and antibunching

TL;DR

This paper introduces a combined fluorescence correlation spectroscopy and antibunching technique to accurately measure photophysical transition rates of fluorescent molecules, providing a new tool for detailed molecular analysis.

Contribution

The paper presents a novel method integrating FCS and antibunching measurements to determine absolute photophysical rate constants, including intersystem crossing and triplet state lifetimes.

Findings

Successfully measured rate constants for Rhodamine 110 and ATTO 655.

Bias in measurements estimated to be less than 5%.

Method applicable to common fluorescent dyes.

Abstract

We present a new method that combines fluorescence correlation spectroscopy (FCS) on the microsecond time scale with fluorescence antibunching measurements on the nanosecond time scale for measuring photophysical rate constants of fluorescent molecules. The antibunching measurements allow us to quantify the average excitation rate of fluorescent molecules within the confocal detection volume of the FCS measurement setup. Knowledge of this value allows us then to quantify, in an absolute manner, the intersystem crossing rate and triplet state lifetime from the microsecond temporal decay of the FCS curves. We present a theoretical analysis of the method and estimate the maximum bias caused by the averaging of all quantities (excitation rate, photophysical rates) over the confocal detection volume, and we show that this bias is smaller than 5\% in most cases. We apply the method for measuring the photophysical rate constants of the widely used dyes Rhodamine~110 and ATTO~655.