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pgaf370.pdf (1.59 MB)

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2025-11-26

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cc-by (c) Sparks, Hugh et al., 2025
Si us plau utilitzeu sempre aquest identificador per citar o enllaçar aquest document: https://hdl.handle.net/2445/226912

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

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Recurs relacionat

https://doi.org/10.1093/pnasnexus/pgaf370

Resum

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.

Matèries

Sistemes d'imatges, Lectors òptics, Microscòpia mèdica

Matèries (anglès)

Imaging systems, Optical scanners, Medical microscopy

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Articles publicats en revistes (Institut d'lnvestigació Biomèdica de Bellvitge (IDIBELL))

Citació

SPARKS, Hugh, et al. High content 3D imaging by dual-view oblique plane microscopy. PNAS Nexus. 2025. Vol. 4, num. 12, pags. pgaf370. ISSN 2752-6542. [consulted: 22 of May of 2026]. Available at: https://hdl.handle.net/2445/226912

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