Super-resolved multimodal multiphoton microscopy with spatial frequency-modulated imaging

TL;DR

This paper introduces a novel super-resolution microscopy technique called MP-SPIFI that enables super-resolved imaging of contrast mechanisms via virtual energy states, using single-pixel detection and applicable to biological and inorganic samples.

Contribution

It presents a new super-resolution method based on spatial frequency modulation that works with any contrast mechanism and surpasses the diffraction limit in multiphoton microscopy.

Findings

Achieves spatial resolution up to 2η below diffraction limit

Demonstrates multimodal super-resolved images with TPEF and SHG

Potential for in vivo super-resolved imaging in scattering media

Abstract

Super-resolved far-field microscopy has emerged as a powerful tool for investigating the structure of objects with resolution well below the diffraction limit of light. Nearly all super-resolution imaging techniques reported to date rely on real energy states of probe molecules to circumvent the diffraction limit, preventing super-resolved imaging of contrast mechanisms that occur via virtual energy states such as harmonic generation (HG). Here we report a super-resolution technique based on SPatIal Frequency modulated Imaging (SPIFI) that permits super-resolved nonlinear microscopy with any contrast mechanism, and with single-pixel detection. We show multimodal super-resolved images with two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) from biological and inorganic media. Multiphoton SPIFI (MP-SPIFI) provides spatial resolution up to 2$\eta$ below the diffraction limit, where $\eta$ is the highest power of the nonlinear intensity response. MP-SPIFI has the potential to not only provide enhanced resolution in optically thin media, but shows promise for providing super-resolved imaging at depth in scattering media - opening the possibility of $\textit{in vivo}$ super-resolved imaging.