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