Multi-Site Reproducibility Study of 3D High-Content Analysis with Dual-View Oblique Plane Microscopy

Sparks, H.; Alexandrov, Y.; Arias-Garcia, M.; Bakal, C.; Batlle, E.; Bousgouni, V.; Carragher, N.; Colombelli, J.; Culley, J.; Curry, N.; Dent, L.; Dunsby, C.; Dvinskikh, L.; Garcia, E.; Giakoumakis, N. N.; Gustafsson, N.; Llanses, M.; Lee, M.; Mandke, K. N.; Marks, D.; McNeish, I.; Ratcliffe, C.; Sahai, E.; Suckert, T.

doi:10.64898/2026.06.29.735376

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bioRxiv · 10.64898/2026.06.29.735376

Multi-Site Reproducibility Study of 3D High-Content Analysis with Dual-View Oblique Plane Microscopy

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Abstract

High content imaging is being applied to achieve quantitative fluorescence readouts in increasingly complex 3-dimensional (3D) cell culture models such as spheroids and organoids. Compared to conventional 2D assays, 3D assays better represent biological heterogeneity but require more complex sample preparation, 3D imaging and 3D image analysis that can affect the accuracy and precision of such assays. We used spheroids formed from the NRAS-activated melanoma cell line 19161 modified to express an ERK kinase translocation reporter (KTR) as an exemplar 3D phenotypic assay carried out in 96-well plates. The spheroids were treated with the ERK activator TPA and a range of concentrations of the MEK inhibitor Binimetinib. 3D live-cell imaging with sub-cellular spatial resolution was performed using a dual-view oblique plane microscope (dOPM) - a form of single-objective light-sheet microscope - and the experiment was performed separately at 4 different institutes. The results were analysed using an identical 3D analysis pipeline and parameters. We assessed the variation in assay readout using a linear mixed effects model. Random variance at the well level was negligible (SD = 0.0048 relative to range of KTR biosensor readout at reference site of 0.17), indicating low technical noise. Treatment effects were dose-dependent and highly statistically significant compared to DMSO control across all sites (Dunnett-corrected p < 0.001). The range in KTR readout between the minimum (3.5 M Binimetinib) and maximum (100 nM TPA) treatments varied between 59 to 96% relative to the reference site. Measured bias in KTR readout between sites was between 6 and 12% of the range of the reference site. This study quantifies the reproducibility of a 3D live spheroid-based assay employing a fluorescence biosensor requiring readout out at the per-cell level using the dOPM platform and discusses areas where experimental protocol could be improved in the future to further improve reproducibility.

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Sparks, H., Alexandrov, Y., Arias-Garcia, M., Bakal, C., Batlle, E., Bousgouni, V., Carragher, N., Colombelli, J., Culley, J., Curry, N., Dent, L., Dunsby, C., Dvinskikh, L., Garcia, E., Giakoumakis, N. N., Gustafsson, N., Llanses, M., Lee, M., Mandke, K. N., Marks, D., McNeish, I., Ratcliffe, C., Sahai, E., Suckert, T.. 2026-07-03. Multi-Site Reproducibility Study of 3D High-Content Analysis with Dual-View Oblique Plane Microscopy. https://doi.org/10.64898/2026.06.29.735376

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