Super-resolution re-scan second harmonic generation microscopy

Abstract

Second harmonic generation microscopy (SHG) is generally acknowledged as a powerful tool for the label-free three-dimensional visualization of tissues and advanced materials, with one of its most popular applications being collagen imaging. Despite 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 demonstrate super-resolved re-scan SHG, qualitatively and quantitatively showing on collagenous tissues the available resolution advantage over the diffraction limit. We introduce as well super-resolved re-scan two-photon excited fluorescence microscopy, an imaging modality not explored to date.

Keywords: second harmonic generation; super-resolved optical imaging; two-photon excited fluorescence.

Fig. 1.

Resolution advantage of rSHG vs. SHG on rat tail collagenous tissues (formalin-fixed, unstained) at different levels of detail. Colored masks indicate sample areas where rSHG’s resolving power is well visible. Images collected with a scientific grade Hamamatsu ORCA-Flash 4.0 v3 s-CMOS camera. (Scale bars: 3 µm.)

Fig. 2.

SHG and rSHG images of a tissue fragment (formalin-fixed, H&E-stained) from a negative resection margin adjacent to a breast carcinoma acquired with (A) a Hamamatsu ORCA-Flash 4.0 v3 scientific-grade sCMOS camera and (B) a FLIR Chameleon3 conventional CMOS camera. (Scale bars: 5 μm.)

Fig. 3.

Resolution improvement in rSHG and rTPEF. (A) rSHG and SHG images collected on rat tail tendon collagen fibrils. (B) Examples of profile line pairs transversally drawn across the collagen fibril in the highlighted area. (C) rTPEF and TPEF images collected on an Argo-SIM calibration sample. (D) Examples of profile line pairs transversally drawn across the visible structures. Profile lines: green: rSHG/rTPEF, blue: SHG/TPEF.