Fluorescence Lifetime Measurements and Analyses: Protocols Using Flow Cytometry and High-Throughput Microscopy

Jessica P Houston; Samantha Valentino; Aric Bitton

doi:10.1007/978-1-0716-3738-8_15

Fluorescence Lifetime Measurements and Analyses: Protocols Using Flow Cytometry and High-Throughput Microscopy

Methods Mol Biol. 2024:2779:323-351. doi: 10.1007/978-1-0716-3738-8_15.

Authors

Jessica P Houston¹, Samantha Valentino², Aric Bitton³

Affiliations

¹ Department of Chemical & Materials Engineering, New Mexico State University, Las Cruces, NM, USA. jph@nmsu.edu.
² Department of Chemical & Materials Engineering, New Mexico State University, Las Cruces, NM, USA.
³ Cytek Biosciences, Fremont, CA, USA.

PMID: 38526793
DOI: 10.1007/978-1-0716-3738-8_15

Abstract

This chapter focuses on applications and protocols that involve the measurement of the fluorescence lifetime as an informative cytometric parameter. The timing of fluorescence decay has been well-studied for cell counting, sorting, and imaging. Therefore, provided herein is an overview of the techniques used, how they enhance cytometry protocols, and the modern techniques used for lifetime analysis. The background and theory behind fluorescence decay kinetic measurements in cells is first discussed followed by the history of the development of time-resolved flow cytometry. These sections are followed by a review of applications that benefit from the quantitative nature of fluorescence lifetimes as a photophysical trait. Lastly, perspectives on the modern ways in which the fluorescence lifetime is scanned at high throughputs which include high-speed microscopy and machine learning are provided.

Keywords: FRET; Fluorescence lifetime; Frequency domain; Time domain; Time-resolved flow cytometry.

MeSH terms

Flow Cytometry / methods
Fluorescence
Fluorescent Dyes*
Kinetics
Microscopy, Fluorescence / methods
Review Literature as Topic*

Substances

Fluorescent Dyes