Related Experiment Video
Updated: Apr 8, 2026
09:03
Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina
Published on: February 13, 2021
5.3K
Multi-view second-harmonic generation imaging of mouse tail tendon via reflective micro-prisms
Optics Letters
|July 1, 2015
Summary
Second-harmonic generation (SHG) imaging misses collagen fibers parallel to laser light. Multi-view SHG microscopy using micro-prisms improves 3D collagen structure imaging, overcoming this limitation.
Area of Science:
- Biomedical Optics
- Microscopy
- Materials Science
Background:
- Second-harmonic generation (SHG) imaging is a label-free technique used to visualize collagen structure.
- Collagen fibers oriented parallel to the laser propagation direction are often missed in standard SHG imaging, limiting structural analysis.
- Understanding collagen organization is crucial in various biological and pathological processes.
Purpose of the Study:
- To investigate the limitations of standard SHG imaging in capturing collagen fiber orientation.
- To develop and demonstrate an alternative multi-view SHG imaging approach for improved 3D collagen visualization.
- To compare SHG imaging with two-photon excited fluorescence (TPEF) for collagen imaging.
Main Methods:
- Experimental demonstration of multi-view SHG imaging using reflective micro-prisms to redirect excitation and emission.
- Imaging of mouse tail tendon to assess collagen fiber structure.
- Utilizing Pearson correlation analysis to quantify image similarities between SHG and TPEF.
- Comparing SHG and TPEF signals for collagen fibers oriented parallel and perpendicular to the laser propagation.
Main Results:
- Standard SHG imaging is insensitive to collagen fibers aligned parallel to the laser propagation direction.
- Multi-view SHG imaging with micro-prisms successfully captured collagen structure regardless of fiber orientation.
- Two-photon excited fluorescence (TPEF) of dye-labeled collagen showed isotropic signal, unaffected by fiber orientation.
- High statistical correlation (0.6-0.8) between SHG and TPEF was observed for perpendicular excitation, but not for parallel excitation.
Conclusions:
- Standard SHG microscopy has inherent limitations in visualizing the complete 3D collagen architecture.
- Multi-view SHG microscopy offers a promising solution to overcome orientation-dependent signal loss.
- The findings suggest that combining multi-view SHG with TPEF or employing multi-view SHG alone can provide more comprehensive structural information of collagenous tissues.
