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

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 transillumination 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.