Back-Illumination Phase Imaging Enables Nanoscale Drift Stabilization in Nontransparent 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 trans-illumination is unpractical. Precise stabilization at the nanoscale is critical for high-resolution imaging techniques, such as localization microscopy and single-particle tracking. Here, we introduce a method combining homogenized differential phase contrast imaging with cross-correlation-based analysis to achieve an 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 nontransparent tissues, facilitating extended high-resolution imaging across increasingly complex biological samples.