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 60×/1.2NA water immersion primary objective and measure the spatial resolution and fluorescence collection efficiency for illumination angles of 35° and 45° with respect to the coverslip. Illumination at 35° 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°, 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 × 100 × 100 μm<sup>3</sup> to be 0.29, 0.31, 0.83, and 2.45-3.00 μm, 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.

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Publication Details

Journal
PNAS Nexus
Published
2025-11-26
DOI
https://doi.org/10.1093/pnasnexus/pgaf370
Citations
1
Primary Topic
Advanced Fluorescence Microscopy Techniques
Type
article
Field-Weighted Citation Impact
1.63
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article

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

Thomas Phillips, Julien Colombelli, Montserrat Llanses Martinez, Carme Cortina et al.
1 citations
PNAS Nexus
Advanced Fluorescence Microscopy Techniques
1.63
article

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

Thomas Phillips, Julien Colombelli, Montserrat Llanses Martinez, Carme Cortina, Leo Rowe-Brown, Theresa Suckert, Nathan Curry, Hugh Sparks, Wenzhi Hong, Alix Le Marois, Chris Bakal, Jayne Culley, Maddy Parsons, Colin D.H. Ratcliffe, Liuba Dvinskikh, Zhizhen Xu, Edwin García, Neil O. Carragher, Giorgio Stassi, Mar Arias-García, Erik Sahai, Eduard Batlle, Joffrey Pelletier, Nils Gustafsson, Shengjie Zhang, Martin Lee, Claudia Owczarek, Chris Dunsby, Yuriy Alexandrov
article en
1 citations

Abstract

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 60×/1.2NA water immersion primary objective and measure the spatial resolution and fluorescence collection efficiency for illumination angles of 35° and 45° with respect to the coverslip. Illumination at 35° 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°, 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 × 100 × 100 μm<sup>3</sup> to be 0.29, 0.31, 0.83, and 2.45-3.00 μm, 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.

PNAS NexusVol. 4(12)
Institució Catalana de Recerca i Estudis Avançats (ES), Synlab Czech (Czechia) (CZ), Institute of Cancer Research (GB), King's College London (GB), The Francis Crick Institute (GB), Bellvitge University Hospital (ES), Edinburgh Cancer Research (GB), Institut d'Investigació Biomédica de Bellvitge (ES), Ajuntament de L’Hospitalet (ES), Institute for Research in Biomedicine (ES), Centro de Investigación Biomédica en Red de Cáncer (ES), Imperial College London (GB), Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione (IT), University of Palermo (IT)
Openalex Percentile: Top 11%
Advanced Fluorescence Microscopy Techniques
1.63
DOIOpen Access PDFOpenAlex
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