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bioRxiv · 10.64898/2026.03.12.711239

Back-illumination Phase Imaging Enables Nanoscale Drift Stabilization in Non-transparent Biological Tissues

Manko, H.·Tondusson, M.·Boyreau, A.·Meras, M.·Bancelin, S.·Groc, L.·Cognet, L.
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Abstract

High-resolution optical microscopy enables nanoscale investigation of molecular structures but is challenged by sample drift during long acquisitions, particularly in thick biological tissues where trans-illumination is unpractical. Precise stabilization at the nanoscale is critical for high-resolution imaging techniques like localization microscopy and single particle tracking. Here we introduce a method combining homogenized differential phase contrast imaging with cross-correlation-based analysis to achieve automated, precise 3D drift correction applicable under oblique back-illumination. We demonstrate its effectiveness in fixed and live organotypic brain slices, maintaining focus within tens of nanometers and enabling high-quality nanoscale mapping of extracellular structures based on single particle tracking. Furthermore, we illustrate its application to opaque liver tissues combined with near-infrared single particle tracking. Our label-free approach provides a versatile solution for stabilizing optical microscopes in thick non-transparent tissues, facilitating extended high-resolution imaging across increasingly complex biological samples.

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Manko, H., Tondusson, M., Boyreau, A., Meras, M., Bancelin, S., Groc, L., Cognet, L.. 2026-03-14. Back-illumination Phase Imaging Enables Nanoscale Drift Stabilization in Non-transparent Biological Tissues. https://doi.org/10.64898/2026.03.12.711239

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