Cargando…

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

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Datta, Rupsa, Heaster, Tiffany M., Sharick, Joe T., Gillette, Amani A., Skala, Melissa C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Society of Photo-Optical Instrumentation Engineers 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7219965/
https://www.ncbi.nlm.nih.gov/pubmed/32406215
http://dx.doi.org/10.1117/1.JBO.25.7.071203
_version_ 1783533063465074688
author Datta, Rupsa
Heaster, Tiffany M.
Sharick, Joe T.
Gillette, Amani A.
Skala, Melissa C.
author_facet Datta, Rupsa
Heaster, Tiffany M.
Sharick, Joe T.
Gillette, Amani A.
Skala, Melissa C.
author_sort Datta, Rupsa
collection PubMed
description 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.
format Online
Article
Text
id pubmed-7219965
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Society of Photo-Optical Instrumentation Engineers
record_format MEDLINE/PubMed
spelling pubmed-72199652020-05-20 Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications Datta, Rupsa Heaster, Tiffany M. Sharick, Joe T. Gillette, Amani A. Skala, Melissa C. J Biomed Opt Special Section on Selected Topics in Biophotonics: Fluorescence Lifetime Imaging and Optical Micromechanics 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. Society of Photo-Optical Instrumentation Engineers 2020-05-13 2020-07 /pmc/articles/PMC7219965/ /pubmed/32406215 http://dx.doi.org/10.1117/1.JBO.25.7.071203 Text en © 2020 The Authors https://creativecommons.org/licenses/by/4.0/ 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.
spellingShingle Special Section on Selected Topics in Biophotonics: Fluorescence Lifetime Imaging and Optical Micromechanics
Datta, Rupsa
Heaster, Tiffany M.
Sharick, Joe T.
Gillette, Amani A.
Skala, Melissa C.
Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications
title Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications
title_full Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications
title_fullStr Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications
title_full_unstemmed Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications
title_short Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications
title_sort fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications
topic Special Section on Selected Topics in Biophotonics: Fluorescence Lifetime Imaging and Optical Micromechanics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7219965/
https://www.ncbi.nlm.nih.gov/pubmed/32406215
http://dx.doi.org/10.1117/1.JBO.25.7.071203
work_keys_str_mv AT dattarupsa fluorescencelifetimeimagingmicroscopyfundamentalsandadvancesininstrumentationanalysisandapplications
AT heastertiffanym fluorescencelifetimeimagingmicroscopyfundamentalsandadvancesininstrumentationanalysisandapplications
AT sharickjoet fluorescencelifetimeimagingmicroscopyfundamentalsandadvancesininstrumentationanalysisandapplications
AT gilletteamania fluorescencelifetimeimagingmicroscopyfundamentalsandadvancesininstrumentationanalysisandapplications
AT skalamelissac fluorescencelifetimeimagingmicroscopyfundamentalsandadvancesininstrumentationanalysisandapplications