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

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Flow Cytometry Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2779))

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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.

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Abbreviations

N∗(t):

Number of molecules in excited state at time t

k:

Sum of de-excitation rates

knr:

Sum of radiation-less de-excitation pathways

kem:

Rate of fluorescence emission

kr:

Sum of radiative de-excitation pathways

αi:

Pre-exponential factor

E(t):

Emission intensity as a function of time (t)

E0:

Maximum emission intensity

mem:

Depth of modulation of emission signal

Ï•:

Phase of any modulated signal

M:

Total depth of modulation for any modulated signal

Ï•em:

Phase of modulated emission signal

Ï•ex:

Phase of modulated excitation signal

Δϕ:

Phase shift between excitation and emission modulated signals

ω:

Angular modulation frequency

I(t):

Intensity of fluorescence

Ï„:

Fluorescence lifetime of the fluorophore

mex:

Depth of modulation of excitation signal

a:

Laser beam height and to the velocity of a cell crossing the laser beam at t0

G:

Angle of the cosine of the phase shift on a phasor graph

S:

Angle of the sine of the phase shift on a phasor graph

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Authors and Affiliations

  1. Department of Chemical & Materials Engineering, New Mexico State University, Las Cruces, NM, USA

    Jessica P. Houston & Samantha Valentino

  2. Cytek Biosciences, Fremont, CA, USA

    Aric Bitton

Authors
  1. Jessica P. Houston
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  2. Samantha Valentino
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  3. Aric Bitton
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Corresponding author

Correspondence to Jessica P. Houston .

Editor information

Editors and Affiliations

  1. Bethesda, MD, USA

    Teresa S. Hawley

  2. Washington, DC, USA

    Robert G. Hawley

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Houston, J.P., Valentino, S., Bitton, A. (2024). Fluorescence Lifetime Measurements and Analyses: Protocols Using Flow Cytometry and High-Throughput Microscopy. In: Hawley, T.S., Hawley, R.G. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 2779. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3738-8_15

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  • DOI: https://doi.org/10.1007/978-1-0716-3738-8_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3737-1

  • Online ISBN: 978-1-0716-3738-8

  • eBook Packages: Springer Protocols

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