Multispectral live-cell imaging with uncompromised spatiotemporal resolution

Multispectral imaging is an established method to extend the number of colours usable in fluorescence imaging beyond the typical limit of three or four. However, standard approaches are poorly suited to live-cell imaging owing to the need to separate light into many spectral channels, and unmixing a...

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Detalles Bibliográficos
Autores: Kumar, Akaash, McNally, Kerrie, Zhang, Yuexuan, Haslett-Saunders, Alex, Wang, Xinru, Guillem Martí, Jordi|||0000-0003-0307-2221, Lee, David, Huang, Buwei, Stallinga, Sjoerd, Kay, Robert, Baker, David, Derivery, Emmanuel, Manton, James
Tipo de recurso: artículo
Fecha de publicación:2025
País:España
Institución:Universitat Politècnica de Catalunya (UPC)
Repositorio:UPCommons. Portal del coneixement obert de la UPC
Idioma:inglés
OAI Identifier:oai:upcommons.upc.edu:2117/443226
Acceso en línea:https://hdl.handle.net/2117/443226
https://dx.doi.org/10.1038/s41566-025-01745-7
Access Level:acceso abierto
Palabra clave:Multispectral imaging
Fluorescence imaging
Imatge multispectral
Imatge de fluorescència
Àrees temàtiques de la UPC::Enginyeria dels materials
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oai_identifier_str oai:upcommons.upc.edu:2117/443226
network_acronym_str ES
network_name_str España
repository_id_str
spelling Multispectral live-cell imaging with uncompromised spatiotemporal resolutionKumar, AkaashMcNally, KerrieZhang, YuexuanHaslett-Saunders, AlexWang, XinruGuillem Martí, Jordi|||0000-0003-0307-2221Lee, DavidHuang, BuweiStallinga, SjoerdKay, RobertBaker, DavidDerivery, EmmanuelManton, JamesMultispectral imagingFluorescence imagingImatge multispectralImatge de fluorescènciaÀrees temàtiques de la UPC::Enginyeria dels materialsMultispectral imaging is an established method to extend the number of colours usable in fluorescence imaging beyond the typical limit of three or four. However, standard approaches are poorly suited to live-cell imaging owing to the need to separate light into many spectral channels, and unmixing algorithms struggle with low signal-to-noise ratio data. Here we introduce an approach for multispectral imaging in live cells that comprises an iterative spectral unmixing algorithm and eight-channel camera-based image-acquisition hardware. This enables the accurate unmixing of low signal-to-noise ratio datasets captured at video rates, while maintaining diffraction-limited spatial resolution. We use this approach on a commercial spinning-disk confocal microscope and a home-built oblique-plane light-sheet microscope to image one to seven spectrally distinct fluorophore species simultaneously, using both fluorescent protein fusions and small-molecule dyes. We further develop protein-binding proteins (minibinders), labelled with organic fluorophores, and use these in combination with our multispectral imaging approach to study the endosomal trafficking of cell-surface receptors at endogenous levels.Peer ReviewedSpringer20252025-09-0820252025-10-07journal articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/2117/443226https://dx.doi.org/10.1038/s41566-025-01745-7reponame:UPCommons. Portal del coneixement obert de la UPCinstname:Universitat Politècnica de Catalunya (UPC)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:upcommons.upc.edu:2117/4432262026-05-27T15:37:01Z
dc.title.none.fl_str_mv Multispectral live-cell imaging with uncompromised spatiotemporal resolution
title Multispectral live-cell imaging with uncompromised spatiotemporal resolution
spellingShingle Multispectral live-cell imaging with uncompromised spatiotemporal resolution
Kumar, Akaash
Multispectral imaging
Fluorescence imaging
Imatge multispectral
Imatge de fluorescència
Àrees temàtiques de la UPC::Enginyeria dels materials
title_short Multispectral live-cell imaging with uncompromised spatiotemporal resolution
title_full Multispectral live-cell imaging with uncompromised spatiotemporal resolution
title_fullStr Multispectral live-cell imaging with uncompromised spatiotemporal resolution
title_full_unstemmed Multispectral live-cell imaging with uncompromised spatiotemporal resolution
title_sort Multispectral live-cell imaging with uncompromised spatiotemporal resolution
dc.creator.none.fl_str_mv Kumar, Akaash
McNally, Kerrie
Zhang, Yuexuan
Haslett-Saunders, Alex
Wang, Xinru
Guillem Martí, Jordi|||0000-0003-0307-2221
Lee, David
Huang, Buwei
Stallinga, Sjoerd
Kay, Robert
Baker, David
Derivery, Emmanuel
Manton, James
author Kumar, Akaash
author_facet Kumar, Akaash
McNally, Kerrie
Zhang, Yuexuan
Haslett-Saunders, Alex
Wang, Xinru
Guillem Martí, Jordi|||0000-0003-0307-2221
Lee, David
Huang, Buwei
Stallinga, Sjoerd
Kay, Robert
Baker, David
Derivery, Emmanuel
Manton, James
author_role author
author2 McNally, Kerrie
Zhang, Yuexuan
Haslett-Saunders, Alex
Wang, Xinru
Guillem Martí, Jordi|||0000-0003-0307-2221
Lee, David
Huang, Buwei
Stallinga, Sjoerd
Kay, Robert
Baker, David
Derivery, Emmanuel
Manton, James
author2_role author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Multispectral imaging
Fluorescence imaging
Imatge multispectral
Imatge de fluorescència
Àrees temàtiques de la UPC::Enginyeria dels materials
topic Multispectral imaging
Fluorescence imaging
Imatge multispectral
Imatge de fluorescència
Àrees temàtiques de la UPC::Enginyeria dels materials
description Multispectral imaging is an established method to extend the number of colours usable in fluorescence imaging beyond the typical limit of three or four. However, standard approaches are poorly suited to live-cell imaging owing to the need to separate light into many spectral channels, and unmixing algorithms struggle with low signal-to-noise ratio data. Here we introduce an approach for multispectral imaging in live cells that comprises an iterative spectral unmixing algorithm and eight-channel camera-based image-acquisition hardware. This enables the accurate unmixing of low signal-to-noise ratio datasets captured at video rates, while maintaining diffraction-limited spatial resolution. We use this approach on a commercial spinning-disk confocal microscope and a home-built oblique-plane light-sheet microscope to image one to seven spectrally distinct fluorophore species simultaneously, using both fluorescent protein fusions and small-molecule dyes. We further develop protein-binding proteins (minibinders), labelled with organic fluorophores, and use these in combination with our multispectral imaging approach to study the endosomal trafficking of cell-surface receptors at endogenous levels.
publishDate 2025
dc.date.none.fl_str_mv 2025
2025-09-08
2025
2025-10-07
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/2117/443226
https://dx.doi.org/10.1038/s41566-025-01745-7
url https://hdl.handle.net/2117/443226
https://dx.doi.org/10.1038/s41566-025-01745-7
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Springer
publisher.none.fl_str_mv Springer
dc.source.none.fl_str_mv reponame:UPCommons. Portal del coneixement obert de la UPC
instname:Universitat Politècnica de Catalunya (UPC)
instname_str Universitat Politècnica de Catalunya (UPC)
reponame_str UPCommons. Portal del coneixement obert de la UPC
collection UPCommons. Portal del coneixement obert de la UPC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,811543

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