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. Laser ablation of imaged surface layers under adaptive control enables volumetric imaging of samples consisting of heterogeneous tissues, such as the skull and brain.