Fig. 1. Exemplary raw PSHG dataset obtained using a laser-scanning PSHG microscope on rat tail tendon and typical outputs from CollagenFitJ. (a) A typical PSHG dataset acquired at polarization angles in steps of 20 deg from 0 to 180 deg. (b) Collagen structure-related images generated using the single-axis molecular model for collagen: FI, CHI3115, CHI3315, CHI3331, and THETA. (c) Fitting assessment images: ERR, SNR, and R2. (d) Dispersion and randomness maps (SD, MAD, and ENT images) displayed only for the FI image, computed using a circular mask with a radius of 5 and a window size of 11.

Fig. 2. Examples of binary collagen masks created for the same PSHG image set as in Fig. 1(a) using the fitting assessment images (ERR, SNR, and R2) by applying different threshold values: 0.1 for ERR, 7 for SNR, and 0.8 for R2 (step 11A in the Supplementary Material). In addition, a custom mask was generated by segmentation of collagen in the average image derived from the entire PSHG stack (step 11B in the Supplementary Material).

Fig. 3. Validation of CollagenFitJ on PSHG image sets from various formalin-fixed paraffin-embedded tissue sections using different techniques and configurations. For each dataset, a PSHG image obtained as the average of the raw PSHG image stack is provided. Also included are the outputs of the CollagenFitJ plugin: collagen structure-related images (FI, CHI3115, CHI3315, CHI3331, and THETA), fitting assessment images (ERR, SNR, and R2), and dispersion and randomness maps (SD, MAD, and ENT) computed for different CollagenFitJ outputs under various settings [Fig. S1(g) in the Supplementary Material] for exemplification. (a) Loose collagen fibrils in rat tail tendon, captured with a custom-modified Leica TCS SP confocal microscope adapted for SHG imaging³⁸ in forward detection. Local statistics images are generated with a radius of 2.5, size 7 circular mask on CHI3331.tif. (b) Follicular thyroid carcinoma nodule capsule, imaged with the same system as in panel (a). Local statistics images are created using a radius of 7.5, size 15 circular mask on THETA.tif. (c) Mouse colon tumor, acquired with a scanning SHG microscope¹⁹ using backward detection. Local statistics images are produced with a radius of 5, size 11 circular mask on CHI3115.tif. (d) Rat tail tendon, imaged with a widefield SHG microscope.¹⁵ Local statistics images are generated with a radius of 10, size 21 circular mask on CHI3315.tif.

Fig. 4. Validation of CollagenFitJ using in vivo PSHG image sets of B16 melanoma. The PSHG image obtained as the average of the raw PSHG image stack provides an overview of the SHG intensity. Also included are the outputs of the CollagenFitJ plugin: collagen structure-related images (FI, CHI3115, CHI3315, CHI3331, and THETA), fitting assessment images (ERR, SNR, and R2), and dispersion and randomness maps (SD, MAD, and ENT) generated with a radius of 5, size 11 circular mask [Fig. S1(g) in the Supplementary Material] applied to CHI3115.tif.