Unlimited field-of-view focus stacking microscopy based on tilted imaging

Abstract

Focus stacking microscopy achieves high-resolution, all-in-focus imaging through multi-focus image fusion and is widely applied in fields such as biology, materials science, and semiconductor inspection. However, for large-scale samples, conventional methods require pausing at each depth-of-field imaging position to capture a multi-focus image stack, which introduces mechanical backlash errors, reducing inspection efficiency and stitching accuracy, and thus fails to meet the requirements for all-in-focus, large field-of-view (FOV), and high-throughput inspection. Here, we propose an unlimited FOV focus stacking microscopy method based on tilted imaging (TI-FSM). By tilting the microscopy system, we decompose the sample's one-dimensional displacement into motion along and perpendicular to the optical axis, where the former is used to acquire a multi-focus image stack for all-in-focus scanning, while the latter enables unlimited FOV expansion. Additionally, we employ a joint texture–topography reconstruction algorithm to achieve simultaneous reconstruction of texture and 3D topography. Experimental results demonstrate that using a microscopy system with an NA of 0.28, TI-FSM achieves a vertical reconstruction accuracy of 16.2 μm. It enables unlimited FOV expansion and exhibits strong all-in-focus imaging and 3D topography measurement capabilities for large-scale, complex-structured surfaces.