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      Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

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          Abstract.

          Significance: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy.

          Aim: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications.

          Approach: This review covers FLIM principles and theory, including advantages over intensity-based fluorescence measurements. Fundamentals of FLIM instrumentation in time- and frequency-domains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell- and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed.

          Conclusions: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.

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            Feature Pyramid Networks for Object Detection

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              Fluorescence lifetime measurements and biological imaging.

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                Author and article information

                Contributors
                Journal
                J Biomed Opt
                J Biomed Opt
                JBOPFO
                JBO
                Journal of Biomedical Optics
                Society of Photo-Optical Instrumentation Engineers
                1083-3668
                1560-2281
                13 May 2020
                July 2020
                13 May 2020
                : 25
                : 7
                : 071203
                Affiliations
                [a ]Morgridge Institute for Research , Madison, Wisconsin, United States
                [b ]University of Wisconsin , Department of Biomedical Engineering, Madison, Wisconsin, United States
                Author notes
                [* ]Address all correspondence to Melissa C. Skala, E-mail: mcskala@ 123456wisc.edu
                Author information
                https://orcid.org/0000-0002-6432-2389
                https://orcid.org/0000-0002-1470-3300
                https://orcid.org/0000-0002-6320-7637
                Article
                JBO-200002SSR 200002SSR
                10.1117/1.JBO.25.7.071203
                7219965
                32406215
                60052769-46fa-4fb8-8794-e886d437da2c
                © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.
                History
                : 06 January 2020
                : 24 April 2020
                Page count
                Figures: 10, Tables: 4, References: 313, Pages: 43
                Funding
                Funded by: National Institutes of Health (NIH)
                Award ID: R01 CA185747
                Award ID: R01 CA205101
                Award ID: R01 CA211082
                Award ID: R21 CA224280
                Award ID: U01 TR002383
                Award ID: R37 CA226526
                Funded by: National Science Foundation (NSF)
                Award ID: CBET-1642287
                Funded by: Stand Up To Cancer
                Award ID: SU2C-AACR-IG-08-16
                Award ID: SU2C-AACR-PS-18
                Funded by: University of Wisconsin Carbone Cancer Center
                Award ID: P30 CA014520
                Categories
                Special Section on Selected Topics in Biophotonics: Fluorescence Lifetime Imaging and Optical Micromechanics
                Paper
                Custom metadata
                Datta et al.: Fluorescence lifetime imaging microscopy: fundamentals and advances…

                Biomedical engineering
                fluorescence lifetime,microscopy,image analysis,cell heterogeneity,review
                Biomedical engineering
                fluorescence lifetime, microscopy, image analysis, cell heterogeneity, review

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