Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning
Autor(a) principal: | |
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Data de Publicação: | 2018 |
Outros Autores: | , |
Tipo de documento: | Artigo |
Idioma: | por |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10316/107716 https://doi.org/10.1155/2018/1371386 |
Resumo: | Time-gated fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to assess the biochemistry of cells and tissues. When applied to living thick samples, it is hampered by the lack of optical sectioning and the need of acquiring many images for an accurate measurement of fluorescence lifetimes. Here, we report on the use of processing techniques to overcome these limitations, minimizing the acquisition time, while providing optical sectioning. We evaluated the application of the HiLo and the rapid lifetime determination (RLD) techniques for accurate measurement of fluorescence lifetimes with optical sectioning. HiLo provides optical sectioning by combining the high-frequency content from a standard image, obtained with uniform illumination, with the low-frequency content of a second image, acquired using structured illumination. Our results show that HiLo produces optical sectioning on thick samples without degrading the accuracy of the measured lifetimes. We also show that instrument response function (IRF) deconvolution can be applied with the RLD technique on HiLo images, improving greatly the accuracy of the measured lifetimes. These results open the possibility of using the RLD technique with pulsed diode laser sources to determine accurately fluorescence lifetimes in the subnanosecond range on thick multilayer samples, providing that offline processing is allowed. |
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Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical SectioningAlgorithmsHeLa CellsHumansImage Processing, Computer-AssistedMicroscopy, FluorescenceTime-gated fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to assess the biochemistry of cells and tissues. When applied to living thick samples, it is hampered by the lack of optical sectioning and the need of acquiring many images for an accurate measurement of fluorescence lifetimes. Here, we report on the use of processing techniques to overcome these limitations, minimizing the acquisition time, while providing optical sectioning. We evaluated the application of the HiLo and the rapid lifetime determination (RLD) techniques for accurate measurement of fluorescence lifetimes with optical sectioning. HiLo provides optical sectioning by combining the high-frequency content from a standard image, obtained with uniform illumination, with the low-frequency content of a second image, acquired using structured illumination. Our results show that HiLo produces optical sectioning on thick samples without degrading the accuracy of the measured lifetimes. We also show that instrument response function (IRF) deconvolution can be applied with the RLD technique on HiLo images, improving greatly the accuracy of the measured lifetimes. These results open the possibility of using the RLD technique with pulsed diode laser sources to determine accurately fluorescence lifetimes in the subnanosecond range on thick multilayer samples, providing that offline processing is allowed.Hindawi2018info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://hdl.handle.net/10316/107716http://hdl.handle.net/10316/107716https://doi.org/10.1155/2018/1371386por2040-2295Silva, Susana FigueiredoDomingues, José PauloMorgado, António Miguelinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-28T10:11:38Zoai:estudogeral.uc.pt:10316/107716Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T21:24:02.178298Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning |
title |
Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning |
spellingShingle |
Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning Silva, Susana Figueiredo Algorithms HeLa Cells Humans Image Processing, Computer-Assisted Microscopy, Fluorescence |
title_short |
Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning |
title_full |
Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning |
title_fullStr |
Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning |
title_full_unstemmed |
Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning |
title_sort |
Accurate Rapid Lifetime Determination on Time-Gated FLIM Microscopy with Optical Sectioning |
author |
Silva, Susana Figueiredo |
author_facet |
Silva, Susana Figueiredo Domingues, José Paulo Morgado, António Miguel |
author_role |
author |
author2 |
Domingues, José Paulo Morgado, António Miguel |
author2_role |
author author |
dc.contributor.author.fl_str_mv |
Silva, Susana Figueiredo Domingues, José Paulo Morgado, António Miguel |
dc.subject.por.fl_str_mv |
Algorithms HeLa Cells Humans Image Processing, Computer-Assisted Microscopy, Fluorescence |
topic |
Algorithms HeLa Cells Humans Image Processing, Computer-Assisted Microscopy, Fluorescence |
description |
Time-gated fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to assess the biochemistry of cells and tissues. When applied to living thick samples, it is hampered by the lack of optical sectioning and the need of acquiring many images for an accurate measurement of fluorescence lifetimes. Here, we report on the use of processing techniques to overcome these limitations, minimizing the acquisition time, while providing optical sectioning. We evaluated the application of the HiLo and the rapid lifetime determination (RLD) techniques for accurate measurement of fluorescence lifetimes with optical sectioning. HiLo provides optical sectioning by combining the high-frequency content from a standard image, obtained with uniform illumination, with the low-frequency content of a second image, acquired using structured illumination. Our results show that HiLo produces optical sectioning on thick samples without degrading the accuracy of the measured lifetimes. We also show that instrument response function (IRF) deconvolution can be applied with the RLD technique on HiLo images, improving greatly the accuracy of the measured lifetimes. These results open the possibility of using the RLD technique with pulsed diode laser sources to determine accurately fluorescence lifetimes in the subnanosecond range on thick multilayer samples, providing that offline processing is allowed. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10316/107716 http://hdl.handle.net/10316/107716 https://doi.org/10.1155/2018/1371386 |
url |
http://hdl.handle.net/10316/107716 https://doi.org/10.1155/2018/1371386 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.none.fl_str_mv |
2040-2295 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
Hindawi |
publisher.none.fl_str_mv |
Hindawi |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
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1799134125934772224 |