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bioRxiv · 10.1101/2025.03.24.645046

Imaging throughput of compact handheld microscopes for quantitative single cell studies

BULLOCH, S.·XU, T.·HERRMANN, D.·TIMPSON, P.·PHAN, T. G.·Lim, Y. J.·Lee, W. M.
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Abstract

High-throughput live-cell imaging within incubator environments often necessitates a compromise between optical resolution and instrument/computational complexity. In this work, we demonstrate that controlled defocusing, a default function in all optical microscopes, can be utilized as a primary contrast mechanism for large-scale cell analysis (7000 cells per field of view). Our results suggest that even at a low numerical aperture (NA [~] 0.01) and using just epi-illumination, defocused images can generate a uniform negative contrast across the cell body. This negative contrast improves automated cell segmentation efficiency over a wide field of view compared to in-focus imaging. We further evaluated the accessibility of this defocus imaging approach for both 2D and 3D cultures by implementing an automated cell segmentation protocol on a commercial off-the-shelf digital Universal Serial Bus (USB) microscope. The compact form factor of the digital USB microscope facilitates minimal pixel sampling, enabling high-throughput single-cell detection and continuous tracking across a large adherent cell population over several days. Our assessments of the utility of defocus imaging for 2D and 3D tissue cultures were further supported by monitoring the negative contrast changes during the migration and dissociation of 3D tissue spheroids. Our results show that negative contrast profiles from defocus images enable the quantification of cell proliferation, division, migration, and cell-to-cluster dissociation within standard culture environments. This defocusing methodology offers a scalable approach to extensive high-content screening through simplified instrumentation controls. Key pointsO_LIDefocused images of adherent cells using low NA optics homogenizes intracellular intensity hotspots, creating uniform negative contrast for whole cell segmentation; similar to shadow imaging in fluorescence. C_LIO_LIInverting look-up-table (LUT) combined with defocus imaging on large population of adherent cells in standard culture flask taken by standard digital USB microscopes allow for rapid, routine tracking of thousands of cells per field of view. C_LIO_LINegative contrast image profiles from defocusing enable the quantification cell proliferation, division, migration for 2D monolayer and 3D tissue spheroid cultures. C_LI

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BULLOCH, S., XU, T., HERRMANN, D., TIMPSON, P., PHAN, T. G., Lim, Y. J., Lee, W. M.. 2025-03-25. Imaging throughput of compact handheld microscopes for quantitative single cell studies. https://doi.org/10.1101/2025.03.24.645046

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