Quantitative tools in the Multi-image Phasor Analysis (MiPA)

Yuansheng Sun; Ulas C. Coskun; Shih-Chu Jeff Liao; Beniamino Barbieri

doi:10.1117/12.2626879

3 March 2022 Quantitative tools in the Multi-image Phasor Analysis (MiPA)

Yuansheng Sun, Ulas C. Coskun, Shih-Chu Jeff Liao, Beniamino Barbieri

Proceedings Volume 11965, Multiphoton Microscopy in the Biomedical Sciences XXII; 119650A (2022) https://doi.org/10.1117/12.2626879
Event: SPIE BiOS, 2022, San Francisco, California, United States

Conference Poster

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique that provides a totally new dimension to quantitate the fluorescent probe, in addition to its steady-state intensity and spectral profiles. As the fluorescence lifetime is a parameter specific of each fluorophore, its measurement is instrumental in the identification of the fluorophore in a mixture: this opens a new opportunity for multiplex imaging and improves imaging sensitivity (signal-to-noise ratio) and resolution in many applications. More importantly, with peculiar selectivity of probes, FLIM can provide quantitative information of the probe microenvironment (ions, pH, oxygen content, local electrical fields, index of refractions, etc.); FLIM is one of the most robust ways of quantifying FRET for studying protein-protein interactions in live specimens. Today, turn-key FLIM instruments are a mature technology available from several companies, making the technique easily accessible. However, a major challenge for the user is still the analysis and interpretation of FLIM data. In combination with the digital frequency domain FLIM (known as FastFLIM), the phasor plots approach has been demonstrated to be an effective solution to tackle many challenges in FLIM data analysis. In this report, we describe several tools using the phasor plots method available in the ISS VistaVision software multiimage phasor analysis module, and demonstrate their utilities with examples for various applications including time-resolved multiplexing, NADH imaging, STED and FRET.

Conference Presentation

Citation Download Citation

Yuansheng Sun, Ulas C. Coskun, Shih-Chu Jeff Liao, and Beniamino Barbieri "Quantitative tools in the Multi-image Phasor Analysis (MiPA)", Proc. SPIE 11965, Multiphoton Microscopy in the Biomedical Sciences XXII, 119650A (3 March 2022); https://doi.org/10.1117/12.2626879

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