High content 3D imaging by dual-view oblique plane microscopy

Oblique plane microscopy (OPM) is a form of light-sheet fluorescence microscopy (LSFM) employing a single microscope objective at the sample for both fluorescence excitation and detection. Dual-view OPM (dOPM) is an optically folded form of OPM. We present an improved dOPM system employing a 60x/1.2...

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Detalles Bibliográficos
Autores: Sparks, Hugh, Llanses Martínez, Montserrat, Suckert, Theresa, Pelletier, Joffrey, Cortina, Carme, Batlle, Eduard, Colombelli, Julien, Dunsby, Chris
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2025
País:España
Institución:Universidad de Barcelona
Repositorio:Dipòsit Digital de la UB
OAI Identifier:oai:diposit.ub.edu:2445/226912
Acceso en línea:https://hdl.handle.net/2445/226912
Access Level:acceso abierto
Palabra clave:Sistemes d'imatges
Lectors òptics
Microscòpia mèdica
Imaging systems
Optical scanners
Medical microscopy
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spelling High content 3D imaging by dual-view oblique plane microscopySparks, HughLlanses Martínez, Montserrat Suckert, TheresaPelletier, JoffreyCortina, CarmeBatlle, EduardColombelli, JulienDunsby, ChrisSistemes d'imatgesLectors òpticsMicroscòpia mèdicaImaging systemsOptical scannersMedical microscopyOblique plane microscopy (OPM) is a form of light-sheet fluorescence microscopy (LSFM) employing a single microscope objective at the sample for both fluorescence excitation and detection. Dual-view OPM (dOPM) is an optically folded form of OPM. We present an improved dOPM system employing a 60x/1.2NA water immersion primary objective and measure the spatial resolution and fluorescence collection efficiency for illumination angles of 35 degrees and 45 degrees with respect to the coverslip. Illumination at 35 degrees provides slightly better lateral resolution and collection efficiency. Collection efficiency measurements are compared to a full vectorial raytracing simulation of the system. Using a light-sheet angle of 35 degrees, the median bead FWHM for 100 nm diameter fluorescent beads in x, y, and z and the optical sectioning strength were measured over a volume of 100 x 100 x 100 mu m3 to be 0.29, 0.31, 0.83, and 2.45-3.00 mu m, respectively when the two dOPM views are fused. We demonstrate less photobleaching in time-lapse dOPM of live mEmerald-expressing organoids compared to widefield epi-fluorescence z-stack imaging under the condition of equal detected fluorescence signal from a point object in focus. We demonstrate dOPM for multifield-of-view 3D imaging of biological samples in 96-well plates and apply it to imaging cells in collagen gel and quantifying the FUCCI cell-cycle reporter to provide drug dose-response curves in spheroids. We also use it to perform time-lapse multifield-of-view imaging and demonstrate the detection of organoid lumen closure and reopening, organoid migration within a collagen gel and observing dynamic events in arrays of ex vivo tissue slices.Oxford University Press (OUP)2025info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://hdl.handle.net/2445/226912Articles publicats en revistes (Institut d'lnvestigació Biomèdica de Bellvitge (IDIBELL))reponame:Dipòsit Digital de la UBinstname:Universidad de BarcelonaInglésReproducció del document publicat a: https://doi.org/10.1093/pnasnexus/pgaf370PNAS Nexus, 2025, vol. 4, num. 12, pgaf370https://doi.org/10.1093/pnasnexus/pgaf370cc-by (c) Sparks, Hugh et al., 2025https://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:diposit.ub.edu:2445/2269122026-05-27T06:46:51Z
dc.title.none.fl_str_mv High content 3D imaging by dual-view oblique plane microscopy
title High content 3D imaging by dual-view oblique plane microscopy
spellingShingle High content 3D imaging by dual-view oblique plane microscopy
Sparks, Hugh
Sistemes d'imatges
Lectors òptics
Microscòpia mèdica
Imaging systems
Optical scanners
Medical microscopy
title_short High content 3D imaging by dual-view oblique plane microscopy
title_full High content 3D imaging by dual-view oblique plane microscopy
title_fullStr High content 3D imaging by dual-view oblique plane microscopy
title_full_unstemmed High content 3D imaging by dual-view oblique plane microscopy
title_sort High content 3D imaging by dual-view oblique plane microscopy
dc.creator.none.fl_str_mv Sparks, Hugh
Llanses Martínez, Montserrat
Suckert, Theresa
Pelletier, Joffrey
Cortina, Carme
Batlle, Eduard
Colombelli, Julien
Dunsby, Chris
author Sparks, Hugh
author_facet Sparks, Hugh
Llanses Martínez, Montserrat
Suckert, Theresa
Pelletier, Joffrey
Cortina, Carme
Batlle, Eduard
Colombelli, Julien
Dunsby, Chris
author_role author
author2 Llanses Martínez, Montserrat
Suckert, Theresa
Pelletier, Joffrey
Cortina, Carme
Batlle, Eduard
Colombelli, Julien
Dunsby, Chris
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Sistemes d'imatges
Lectors òptics
Microscòpia mèdica
Imaging systems
Optical scanners
Medical microscopy
topic Sistemes d'imatges
Lectors òptics
Microscòpia mèdica
Imaging systems
Optical scanners
Medical microscopy
description Oblique plane microscopy (OPM) is a form of light-sheet fluorescence microscopy (LSFM) employing a single microscope objective at the sample for both fluorescence excitation and detection. Dual-view OPM (dOPM) is an optically folded form of OPM. We present an improved dOPM system employing a 60x/1.2NA water immersion primary objective and measure the spatial resolution and fluorescence collection efficiency for illumination angles of 35 degrees and 45 degrees with respect to the coverslip. Illumination at 35 degrees provides slightly better lateral resolution and collection efficiency. Collection efficiency measurements are compared to a full vectorial raytracing simulation of the system. Using a light-sheet angle of 35 degrees, the median bead FWHM for 100 nm diameter fluorescent beads in x, y, and z and the optical sectioning strength were measured over a volume of 100 x 100 x 100 mu m3 to be 0.29, 0.31, 0.83, and 2.45-3.00 mu m, respectively when the two dOPM views are fused. We demonstrate less photobleaching in time-lapse dOPM of live mEmerald-expressing organoids compared to widefield epi-fluorescence z-stack imaging under the condition of equal detected fluorescence signal from a point object in focus. We demonstrate dOPM for multifield-of-view 3D imaging of biological samples in 96-well plates and apply it to imaging cells in collagen gel and quantifying the FUCCI cell-cycle reporter to provide drug dose-response curves in spheroids. We also use it to perform time-lapse multifield-of-view imaging and demonstrate the detection of organoid lumen closure and reopening, organoid migration within a collagen gel and observing dynamic events in arrays of ex vivo tissue slices.
publishDate 2025
dc.date.none.fl_str_mv 2025
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv https://hdl.handle.net/2445/226912
url https://hdl.handle.net/2445/226912
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Reproducció del document publicat a: https://doi.org/10.1093/pnasnexus/pgaf370
PNAS Nexus, 2025, vol. 4, num. 12, pgaf370
https://doi.org/10.1093/pnasnexus/pgaf370
dc.rights.none.fl_str_mv cc-by (c) Sparks, Hugh et al., 2025
https://creativecommons.org/licenses/by/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv cc-by (c) Sparks, Hugh et al., 2025
https://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 Oxford University Press (OUP)
publisher.none.fl_str_mv Oxford University Press (OUP)
dc.source.none.fl_str_mv Articles publicats en revistes (Institut d'lnvestigació Biomèdica de Bellvitge (IDIBELL))
reponame:Dipòsit Digital de la UB
instname:Universidad de Barcelona
instname_str Universidad de Barcelona
reponame_str Dipòsit Digital de la UB
collection Dipòsit Digital de la UB
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,811543

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