Second-harmonic generation (SHG) is proved to be a high spatial resolution, large penetration depth and
non-photobleaching method. In our study, SHG method was used to investigate the normal and cancerous thyroid tissue.
For SHG imaging performance, system parameters were adjusted for high-contrast images acquisition. Each x-y image
was recorded in pseudo-color, which matches the wavelength range in the visible spectrum. The acquisition time for a
512×512-pixels image was 1.57 sec; each acquired image was averaged four frames to improve the signal-to-noise ratio.
Our results indicated that collagen presence as determined by counting the ratio of the SHG pixels over the whole pixels
for normal and cancerous thyroid tissues were 0.48±0.05, 0.33±0.06 respectively. In addition, to quantitatively assess
collagen-related changes, we employed GLCM texture analysis to the SHG images. Corresponding results showed that
the correlation both fell off with distance in normal and cancerous group. Calculated value of Corr50 (the distance where
the correlation crossed 50% of the initial correlation) indicated significant difference. This study demonstrates that SHG
method can be used as a complementary tool in thyroid histopathology.