Use of nanosecond excitation pulses in fluorescence lifetime measurement via phasor analysis

Dongeun Kim, Sucbei Moon, Wonsang Hwang, Dug Young Kim

Research output: Contribution to journalArticlepeer-review


We investigated the possibility of using long excitation pulses in fluorescence lifetime imaging microscopy (FLIM) using phasor analysis. It has long been believed that the pulse width of an excitation laser must be shorter than the lifetime of a fluorophore in a time-domain FLIM system. Even though phasor analysis can effectively minimize the pulse effect by using deconvolution, the precision of a measured lifetime can be degraded seriously. Here, we provide a fundamental theory on pulse-width-dependent measurement precisions in lifetime measurement in the phasor plane. Our theory predicts that high-precision lifetimes can be obtained even with a laser whose pulse width is four times larger than the lifetime of a fluorophore. We have experimentally demonstrated this by measuring the lifetimes of fluorescence probes with 2.57 ns and 3.75 ns lifetimes by using various pulse widths (0.52–38 ns) and modulation frequencies (10–200 MHz). We believe our results open a new possibility of using long pulse-width lasers for high-precision FLIM.

Original languageEnglish
Pages (from-to)14677-14685
Number of pages9
JournalOptics Express
Issue number9
Publication statusPublished - 2022 Apr 25

Bibliographical note

Funding Information:
National Research Foundation of Korea (NRF-2021R1A2C2009090); Korea Institute for Advancement of Technology (P0011925); Innosuisse - Schweizerische Agentur f?r Innovationsf?rderung (P0011925).

Publisher Copyright:
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement

All Science Journal Classification (ASJC) codes

  • Atomic and Molecular Physics, and Optics


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