rSHG: Re-scan Second Harmonic Generation Microscopy

TL;DR

This paper introduces rSHG, a super-resolution microscopy technique that enhances Second Harmonic Generation imaging resolution by approximately 1.4 times, enabling detailed 3D visualization of tissues like collagen without labels.

Contribution

The work demonstrates the integration of re-scan microscopy with SHG and TPEF, achieving super-resolution imaging while maintaining the original microscope functionality.

Findings

Super-resolved SHG imaging surpasses diffraction limit by ~1.4x.

Multimodal rSHG/rTPEF/RCM system retains initial confocal functions.

Enhanced imaging capabilities applicable to tissue and material analysis.

Abstract

Second Harmonic Generation Microscopy (SHG) is generally acknowledged as a powerful tool for the label-free 3D visualization of tissues and advanced materials, with one of its most popular applications being collagen imaging. Although the great need, progress in super-resolved SHG imaging lags behind the developments reported over the past years in fluorescence-based optical nanoscopy. In this work, we quantitatively show on collagenous tissues that by combining SHG imaging with re-scan microscopy resolutions that surpass the diffraction limit with ~1.4x become available. Besides Re-scan Second Harmonic Generation Microscopy (rSHG), we demonstrate as well super-resolved Re-scan Two-Photon Excited Fluorescence Microscopy (rTPEF). These two techniques are implemented by modifying a Re-scan Confocal Microscope (RCM), retaining its initial function, resulting thus in a multimodal rSHG/rTPEF/RCM system. Given the simplicity and flexibility of re-scan microscopy, we consider that the reported results are likely to augment the number and nature of applications relying on super-resolved non-linear optical imaging.