Improved sensitivity for two-photon frequency-domain lifetime measurement

Arnold D Estrada,Andrew K Dunn

doi:10.1364/oe.18.013631

Abstract

We demonstrate a method to improve the measurement sensitivity of two-photon frequency-domain lifetime measurements in poor signal to background conditions. This technique uses sinusoidal modulation of the two-photon excitation source and detection of the second harmonic of the modulation frequency that appears in the emission. Additionally, we present the mathematical model which describes how the observed phase shift and amplitude demodulation factor of two-photon phosphorescence emission are related to the phosphorescence lifetime and modulation frequency. We demonstrate the validity of the model by showing the existence of new frequency terms in the phosphorescence emission generated from the quadratic nature of two-photon absorption and by showing that the phase shift and demodulation match theory for all frequency components.

Highlights

Fluorescence lifetime measurement techniques can be used to probe energy transfer processes between a fluorophore and its local environment
To verify these expressions we first determined the lifetime of sample solutions using a standard time-domain approach with a two-photon excitation source
We performed frequency-domain tests to compare the measured demodulation factor and phase shift of the 1f and 2f components to that predicted by the model given this measured lifetime

Summary

Introduction

Fluorescence lifetime measurement techniques can be used to probe energy transfer processes between a fluorophore and its local environment. Through the use of various fluorescent, phosphorescent and luminescent probe molecules, these techniques have proven to be very effective for quantifying a wide range of phenomena of interest to medical and biological researchers [1,2,3]. Due to their long excited-state lifetimes, phosphorescent probe molecules have been used for measuring local environmental concentrations of quenching agents. The desire to make these measurements depth-resolved has led to the combining of two-photon excitation with time-domain and frequency-domain lifetime determination methods [8,9]

Results

Discussion

Conclusion