Paper

Ultrashort laser based two-photon phase-resolved fluorescence lifetime measurement method

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Published 19 February 2020 © 2020 IOP Publishing Ltd
, , Citation Hafizah Halip et al 2020 Methods Appl. Fluoresc. 8 025003 DOI 10.1088/2050-6120/ab71c2

2050-6120/8/2/025003

Abstract

This paper presents a two-photon phase-resolved fluorescence-lifetime measurement method based on the use of an ultrashort pulse laser. The proposed method also involves the use of a lock-in amplifier to control the phase difference between the reference and fluorescence signals, thereby facilitating the use of an alternative method for determining fluorescence lifetimes. Verification of the fluorescence lifetimes as measured in this study was performed using rhodamine B and a cellular thermoprobe as samples. In this study, we assume that the fluorescence decay was monoexponential in all cases. Rhodamine B was observed to exhibit an average fluorescence lifetime of 2.15 ns, whereas a temperature sensitivity of 1.39 ns C−1 over a temperature range of 33.79–37.2 °C was demonstrated for the cellular thermoprobe. These results validate the feasibility of the proposed method for accurate measurement of fluorescence lifetimes using a simple laser configuration.

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10.1088/2050-6120/ab71c2