Quantifying the backscattering of second harmonic generation in tissues with confocal multiphoton microscopy.

Area of Science:

• Biomedical Optics
• Microscopy Techniques
• Tissue Imaging

Background:

• Second Harmonic Generation (SHG) is a nonlinear optical process used for imaging biological tissues.
• Backward SHG (B-SHG) collection is crucial for certain microscopy applications, but its generation mechanisms require further elucidation.
• Understanding the contributions of directly generated B-SHG versus back-scattered forward SHG (BS-SHG) is essential for accurate interpretation.

Purpose of the Study:

• To investigate and quantify the contributions of different SHG components in mouse tendon tissues.
• To develop and validate a method for differentiating between backward-generated SHG and back-scattered forward SHG.
• To compare these contributions in different types of mouse tendons.

Main Methods:

• Utilized confocal multiphoton microscopy to study SHG in mouse tissues.
• Employed varying confocal pinhole sizes to distinguish between Gaussian (backward-generated) and uniform (BS-SHG) distributions.
• Validated the approach using Monte Carlo simulations before application to tissue samples.

Main Results:

• BS-SHG contributed less to total B-SHG in mouse tail tendon compared to Achilles tendon (~300 μm thickness).
• In thicker Achilles tendon (1000 μm), up to 10% of forward SHG was backscattered and collected.
• BS-SHG appears to be a minor contributor to B-SHG in tail tendon but potentially significant in Achilles tendon.

Conclusions:

• The developed method allows for noninvasive investigation of SHG generation mechanisms in tissues.
• Results provide critical information for optimizing backward SHG microscopy and spectroscopy measurements.
• Tissue-specific differences in SHG generation and scattering are highlighted, impacting imaging interpretation.