Selective imaging in second-harmonic-generation microscopy with anisotropic radiation.

Area of Science:

• Biomedical Optics
• Microscopy
• Biophysics

Background:

• Second-harmonic generation (SHG) microscopy offers intrinsic optical sectioning, noninvasiveness, specificity, and high penetrability for biomedical imaging.
• Epicollection of backward propagating SHG is crucial for biomedical applications, but thick tissues preferentially exhibit forward propagation due to phase-matching constraints.
• Myosin and collagen, abundant vertebrate fibrous proteins, possess strong second-harmonic responses.

Purpose of the Study:

• To demonstrate selective imaging of intertwining muscle fibers and type I collagen fibrils.
• To leverage distinct radiation patterns of myosin and collagen for differentiation.
• To utilize forward and backward SHG modalities for selective tissue component visualization.

Main Methods:

• Utilizing the distinct coherence effects and radiation patterns of myosin-based muscle fibers and collagen fibrils.
• Employing forward and backward second-harmonic generation (SHG) microscopy modalities.
• Analyzing the preferential propagation directions of SHG signals from different tissue components.

Main Results:

• Selective imaging of muscle fibers and collagen fibrils was achieved using forward and backward SHG.
• Thick muscle fibers predominantly contributed to the forward SHG signal.
• Collagen fibril distribution was preferentially resolved in the backward SHG channel, with minimal muscle interference.
• Well-formed collagen fibrils were highlighted by forward SHG, while loosely arranged collagen matrices were outlined by backward SHG.

Conclusions:

• Asymmetric radiation patterns of myosin and collagen enable selective SHG imaging.
• Forward and backward SHG channels can differentiate between muscle and collagen in thick tissues.
• This technique provides a novel approach for visualizing the microarchitecture of collagen and muscle.