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  • Primer
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Fluorescence lifetime imaging microscopy

Nature Reviews Methods Primers volume 4, Article number: 80 (2024) Cite this article

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Abstract

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique offering profound insights into a broad spectrum of biological processes such as metabolic imaging, protein–protein interactions and live-cell intracellular dynamics. The future of FLIM appears promising, with continuous technological advancements for time-resolved measurements pushing the boundaries for spatiotemporal information. However, the growth of the FLIM community has been slower, owing to the requirement for specialized training and technology. This Primer aims to address this gap by providing a comprehensive overview of FLIM principles, methods and analysis. We discuss various methods for measuring fluorescence lifetimes, including time-tagging and phase-modulation shift methods, along with their implementations and setup variations. Additionally, we explore different avenues for data analysis, with a specific focus on the phasor approach and its crucial considerations. Furthermore, we present a range of applications demonstrating versatility and usability of FLIM. Limitations and optimization strategies are also discussed, covering methodological constraints, equipment limitations and potential errors, along with their solutions. By sharing our expertise, we aim to expand FLIM to broader audiences while reinforcing concepts within the FLIM community. This Primer seeks to inspire bioimaging researchers to fully embrace FLIM, thereby advancing our understanding of complex biological systems.

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Fig. 1: Overview of fluorescence lifetime imaging microscopy basic concepts.
Fig. 2: Fluorescence lifetime imaging microscopy methods and implementations.
Fig. 3: Analysis of fluorescence lifetime with the phasor approach.
Fig. 4: FLIM-NADH in cells and tissues.
Fig. 5: Phasor-FLIM applications.
Fig. 6: Fluorescence lifetime imaging microscopy limitations and workarounds.

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Acknowledgements

The authors thank A. Rossetta (FLIM Labs) for sharing valuable information and engaging in insightful discussions for this Primer. The authors also thank E. Sisamakis (PicoQuant), G. Ossato (Leica Microsystems), B. Barbieri and U. Coskun (ISS) for their helpful discussions on the topics covered in this Primer. L.M. and B.P. were supported by grants 2020-225439, 2021-240122 and 2022-252604 from the Chan Zuckerberg Initiative DAF, an advised fund of the Silicon Valley Community Foundation. L.M. and B.P. were supported by Fondo para la Convergencia Estructural del Mercosur (COF 03/11).

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Author notes

  1. These authors contributed equally: Belen Torrado, Bruno Pannunzio.

Authors and Affiliations

  1. Laboratory for Fluorescence Dynamics, Biomedical Engineering Department, University of California, Irvine, CA, USA

    Belen Torrado & Michelle A. Digman

  2. Beckman Laser Institute and Medical Clinic, University of California, Irvine, CA, USA

    Belen Torrado & Michelle A. Digman

  3. Advanced Bioimaging Unit, Institut Pasteur de Montevideo and Universidad de la República, Montevideo, Uruguay

    Bruno Pannunzio & Leonel Malacrida

  4. Unidad Académica de Histología y Embriología, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

    Bruno Pannunzio

  5. Unidad Académica de Fisiopatología, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay

    Leonel Malacrida

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  2. Bruno Pannunzio
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Contributions

Introduction (B.P., L.M., B.T. and M.A.D.); Experimentation (B.T. and M.A.D.); Results (B.P.); Applications (B.P. and B.T.); Reproducibility and data deposition (L.M. and M.D.); Limitations and optimizations (B.T.); Outlook (B.P. and B.T.); overview of the Primer (L.M., B.T. and M.A.D.).

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Correspondence to Belen Torrado, Leonel Malacrida or Michelle A. Digman.

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Nature Reviews Methods Primers thanks Laura Marcu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

BrightEyes-MCS: https://github.com/VicidominiLab/BrightEyes-MCS

FLIM STUDIO: https://www.flimlabs.com/flim-studio-software/

FLIMfit: https://github.com/flimfit/FLIMfit

flimview: https://github.com/Biophotonics-COMI/flimview

Fluorescence Lifetime Standards: https://iss.com/resources#fluorescence-lifetime-standards

FluorescenceLifetime: https://github.com/CreLox/FluorescenceLifetime

FLUTE: https://github.com/LaboratoryOpticsBiosciences/FLUTE

Leica Application Suite X (LAS X): https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/

LIFA: https://lambertinstruments.com/lifa-software

OpenFLIM: https://github.com/imperial-photonics/openFLIM-GOI

PAM: https://gitlab.com/PAM-PIE/PAM

PhasorPy: https://www.phasorpy.org/stable/index.html

SimFCS: https://www.lfd.uci.edu/globals/

SPCImage: https://www.becker-hickl.com/products/spcimage/

SymPhoTime 64: https://www.picoquant.com/products/category/software/symphotime-64-fluorescence-lifetime-imaging-and-correlation-software

VistaVision: https://iss.com/software/vistavision

Supplementary information

Glossary

Analog frequency domain

A technique in which fluorescence lifetime measurements are obtained by analysing the modulation of the fluorescence signal at a given frequency or different frequencies.

Digital frequency domain

(DFD). A technique used to define data collected with pulsed laser excitation followed by a conversion of the acquired signal into the frequency domain such as the phasor transformation or phasor approach.

Digital heterodyne principle

A method that combines two signals with slightly different frequencies to generate a new signal representing their difference.

Fluorescence lifetime

The average duration a fluorophore remains in its excited state before emitting a photon and returning to its ground state, typically measured in nanoseconds.

Phase

The timing of laser pulses, corresponding to the frequency at which the laser pulses occur.

Phase-modulation

Involves modulating the excitation light at frequencies in the range of several megahertz to tens or hundreds of megahertz to accurately capture fluorescence lifetimes in the nanosecond range and analysing the phase shift between the excitation and emission signals to determine the fluorescence lifetime of the sample.

Phasor approach

A mathematical method used in fluorescence lifetime imaging microscopy for data analysis and visualization represents the fluorescence lifetimes as points on a Cartesian coordinate system. This complex plane is a 2D graph in which the horizontal axis represents a real part of a complex number and the vertical axis represents the imaginary part.

Phasor transformation

Involves the conversion of fluorescence decay data from the time domain at each pixel to the frequency domain using Fourier transformation.

Reciprocity principle

A technique used in the phasor approach that facilitates the identification of pixels within designated regions of interest on the phasor plot by selecting a region of interest in the image. This enables the determination of the distribution of their corresponding phasor points.

Time-correlated single-photon counting

(TCSPC). A method that operates by identifying single photons within a periodic light or laser signal, registering the precise times of photon arrival and subsequently reconstructing the waveform using these recorded data.

Time-domain

A method in which the fluorescence lifetime of fluorophores is determined by analysing the temporal delay between the excitation pulse and the emitted fluorescence signal.

Time-tagging

A method to record the arrival times of individual photons emitted by fluorophores. This technique involves assigning a time stamp to each detected photon relative to the excitation pulse.

Window

The frequency generated by the digital heterodyne principle, which is used to create a low-frequency replica of the fluorescence decay.

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Torrado, B., Pannunzio, B., Malacrida, L. et al. Fluorescence lifetime imaging microscopy. Nat Rev Methods Primers 4, 80 (2024). https://doi.org/10.1038/s43586-024-00358-8

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