Rapid depth-integrated super-resolution imaging for digital pathology

Image Scanning Microscopy (ISM) is a powerful super-resolution technique that has significantly advanced biological imaging by enabling resolution beyond the diffraction limit. However, as with many high-resolution imaging systems, this resolution often comes at the expense of a shallow depth of focus, necessitating sequential axial scanning to visualize thick biological specimens. In addition, the complexity of instrumentation and alignment can constrain ISM's deployment in clinical or high-throughput environments, where both resolution and volumetric efficiency are critical. Here, we present resonant multifocal scanning microscopy (RSM), an accessible super-resolution imaging platform designed to overcome these challenges through an extended depth of focus (EDoF) architecture. RSM employs stationary multi-foci excitation and continuous sample scanning to achieve super-resolution, alongside tunable optics for rapid axial refocusing to produce a depth-integrated projection, all while being incorporated into a standard epifluorescence platform. Our approach achieves a two-fold resolution enhancement relative to diffraction-limited imaging, while supporting large-area scanning and imaging across extended axial ranges. This capability facilitates efficient visualization of thick tissue sections in a single projected plane, enhancing throughput without compromising spatial detail. By addressing key limitations in depth and scalability, this method offers a practical solution for translating super-resolution imaging into biological and clinical workflows. Its ability to deliver high-resolution projection images across broad fields of view, along with the flexibility to integrate local 3D volumetric imaging, positions it as a promising tool for next-generation digital pathology, where speed, resolution, and volumetric coverage are essential.