Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images

Area of Science:

• Neuroscience
• Biomedical Imaging
• Optical Microscopy

Background:

• Third harmonic generation (THG) microscopy offers label-free imaging for rapid brain tissue pathology.
• Current interpretation of THG brain images lacks quantitative correlation with standard imaging techniques.
• Establishing a quantitative link is crucial for reliable diagnostic applications.

Purpose of the Study:

• To establish a quantitative link between THG and fluorescence images of mouse brain tissue.
• To develop and validate a segmentation workflow for comparative analysis of THG and fluorescence images.
• To provide quantitative evidence for the interpretation of THG image features in relation to cellular structures.

Main Methods:

• Simultaneous acquisition of THG and all-nuclei-highlighted fluorescence images from the same tissue area.
• Development of a novel segmentation workflow applicable to both THG and fluorescence images.
• Quantitative comparison of image features and spatial correlations between the two modalities.

Main Results:

• Achieved segmentation precision of 91.3% for THG and 95.8% for fluorescence images.
• Demonstrated an 88.9% correspondence between main features in THG and fluorescence images.
• Confirmed that 80% of bright objects in THG images correspond to nuclei, with distinct morphological differences observed.

Conclusions:

• The study establishes a robust quantitative link between THG and fluorescence imaging of brain tissue.
• The developed segmentation workflow enables precise comparative analysis for improved image interpretation.
• Findings support the potential of THG microscopy for quantitative pathology and cell morphology recognition in neuroscience.