Multiphoton super-resolution imaging via virtual structured illumination

TL;DR

This paper introduces a deep-tissue super-resolution imaging method that uses a virtual structured illumination approach with minimal hardware changes, enabling high-resolution imaging in thick biological tissues despite complex aberrations.

Contribution

The authors develop a matrix-based computational framework that corrects aberrations and achieves super-resolution in thick tissues using a standard laser-scanning microscope with a camera.

Findings

Achieved 130 nm lateral resolution at 180 μm depth in mouse brain tissue.

Validated the method with two-photon super-resolution imaging.

Enhanced image reconstruction for high spatial frequency components.

Abstract

Imaging in thick biological tissues is often degraded by sample-induced aberrations, which reduce image quality and resolution, particularly in super-resolution techniques. While hardware-based adaptive optics, which correct aberrations using wavefront shaping devices, provide an effective solution, their complexity and cost limit accessibility. Computational methods offer simpler alternatives but struggle with complex aberrations due to the incoherent nature of fluorescence. Here, we present a deep-tissue super-resolution imaging framework that addresses these challenges with minimal hardware modification. By replacing the photodetector in a standard laser-scanning microscope with a camera, we measure an incoherent response matrix (IRM). A dual deconvolution algorithm is developed to decompose the IRM into excitation and emission optical transfer functions and the object spectrum. The proposed method simultaneously corrects excitation and emission point-spread functions (PSFs), achieving a resolution of {\lambda}/4, comparable to structured illumination microscopy. Unlike existing computational methods that rely on vector decomposition of a single convoluted PSF, our matrix-based approach enhances image reconstruction, particularly for high spatial frequency components, enabling super-resolution even in the presence of complex aberrations. We validated this framework with two-photon super-resolution imaging, achieving a lateral resolution of 130 nanometers at a depth of 180 micrometers in thick mouse brain tissue.