Strategies for Optimizing the Determination of Second-Order Nonlinear Susceptibility Tensor Coefficients for Collagen in Histological Samples

Quantitative second harmonic generation microscopy methods that provide an estimate for the molecular structure of collagen in tissues by fitting theoretical models with experimental data at pixel-level are gaining increasing momentum. Such methods offer reliable diagnostic-relevant information that complement traditional histology procedures. We provide guidelines on selecting the optimal experimental data acquisition parameters (frame or pixel averaging, pixel dwell time, pixel size, zoom factor) and the most suited pre-processing steps (thresholding, averaging of pixels) for ensuring experimental data fitting with very high performance. Under controlled image acquisition conditions, we also propose pixel fitting efficiency calculation which varies across distinct tissue types and has potential to be used to differentiate tissues exhibiting distinct collagen architectures.