Spatiotemporal focusing enables all-optical in situ histology of heterogeneous tissue.

Abstract

Living systems embody heterogeneous tissues with complex opto-mechanical properties. Achieving organ-scale, diffraction-limited volumetric imaging that faithfully captures in vivo architecture requires minimizing sample deformation and preserving vascular and neuronal continuity across delicate tissue interfaces. As a solution to this problem, we developed a robotic nonlinear optical system for iterative multiphoton microscopy and opto-micromachining. Adaptive control enabled days-long autonomous operation, while spatiotemporal line-focused ablation increased the machining efficiency by 100-fold over prior configurations. Using the intact murine craniocerebral system as a test bed, our approach demonstrates the potential for whole-body submicrometer resolution imaging and anatomical reconstruction.