Rigorous investigation and comparison of different fluorescence lifetime imaging microscopy (FLIM) techniques analyzed using the phasor plot

Thomas Kellerer; Janko Janusch; Lukas Moser; Tanja Grawert; Patrick Byers; Tillmann Spellauge; Joachim Rädler; Thomas Hellerer

doi:10.1117/12.3002648

12 March 2024 Rigorous investigation and comparison of different fluorescence lifetime imaging microscopy (FLIM) techniques analyzed using the phasor plot

Thomas Kellerer, Janko Janusch, Lukas Moser, Tanja Grawert, Patrick Byers, Tillmann Spellauge, Joachim Rädler, Thomas Hellerer

Author Affiliations +

Proceedings Volume 12847, Multiphoton Microscopy in the Biomedical Sciences XXIV; 128470O (2024) https://doi.org/10.1117/12.3002648
Event: SPIE BiOS, 2024, San Francisco, California, United States

Conference Poster

Abstract

In microscopy, quantitative information such as fluorescence lifetime allows researchers to provide detailed information about the microenvironment and the physicochemical state of the molecule under study. However, the high number of influencing factors might be an explanation for the strongly deviating values of fluorescent lifetimes for the same fluorophore as reported in literature. This could be the reason for the impression that inconsistent results are obtained depending on which FLIM technique is used. To clarify this controversy, the two most common as well as two newly developed techniques for measuring fluorescence lifetimes in the time-domain (TD) and in the frequency-domain (FD) were implemented in one single microscopy setup. Furthermore, we applied each of them to a variety of fluorophores under different environmental conditions such as pH-value, temperature, solvent polarity, etc., along with distinct state forms that depend, for example, on the concentration. From a vast amount of measurement results, both setup- and sample-dependent parameters were extracted and represented using a single display form, the phasor-plot. The measurements showed consistent results between the techniques and revealed which of the tested parameters has the strongest influence on the fluorescence lifetime. In addition, together with the instant FLIM setup we present a new technique that we coined Speed-Up PhasE-Resolved (SUPER)-FLIM, which we adapted from elsewhere such that it combines the advantages from TD- with FD-FLIM. For the first time, we demonstrate that this method opens the door for very fast (124ns/px) FLIM measurements required for imaging with a resonant galvo scanner.

(2024) Published by SPIE. Downloading of the abstract is permitted for personal use only.

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Thomas Kellerer, Janko Janusch, Lukas Moser, Tanja Grawert, Patrick Byers, Tillmann Spellauge, Joachim Rädler, and Thomas Hellerer "Rigorous investigation and comparison of different fluorescence lifetime imaging microscopy (FLIM) techniques analyzed using the phasor plot", Proc. SPIE 12847, Multiphoton Microscopy in the Biomedical Sciences XXIV, 128470O (12 March 2024); https://doi.org/10.1117/12.3002648

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