# Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application
## 3 Second Harmonic Generation Microscopy (SHGM)
As SHG is highly sensitive to collagen fibril/fiber structure [70; 81], SHG imaging can be used to detect collagen-associated changes occurred in various diseases [82], such as cancer, fibrosis, and connective tissue disorders. Dynamic reorganization of the collagen network in ex vivo murine skin dermis was imaged rapidly by polarization-resolved SHGM during mechanical assays with acquisition time of 27 ms per line [83]. Ovarian cancer was studied by SHGM to detect the structure changes of the ovarian extracellular matrix in human normal and malignant ex vivo biopsies (Figure 5a) [84]. The results showed that the SHG emission distribution and bulk optical parameters are related to the tissue structure and significantly different in the tumors. Combining with autofluorescence (AF), SHGM can prove a quantitative analysis to different collagen content in the process of tumor progression and recession post-chemotherapy via calculating  
Figure 4: (**a**) Energy-level diagram of SHG [66] (reprinted with permission from [66], Copyright 2010 PNAS). (**b**) Experimental setup of SHGM with both forward and backward collection geometries [69] (reprinted with permission from [69], © The Optical Society). the ratio of SHG/AF (Figure 5b) [85]. Polarization-dependent SHGM was developed for accurately diagnosing the tumor state of breast cancer by analyzing anisotropy and the \"ratio parameter\" values of SHG signal (Figure 6a) [86]. Through further data analysis, parameters of PIPO (polarization-in, polarization-out) SHGM, i.e., susceptibility ratios and degree of linear polarization, can be used to distinguish tumor from normal tissue (Figure 6b) [87]. Furthermore, some automated or semi-automated solutions have been developed to analyze SHG images for evaluating the healthy and abnormal tissue. Early embryogenesis of zebrafish embryos was automatically reconstructed and analyzed with conformal scanning harmonic microscopy scheme for better imaging quality [88]. Fast Fourier transform analysis and the gray-level co-occurrence matrix have also been applied to analyze biopsy samples of human ovarian epithelial cancer at different stages [89]. It showed the feasibility of identifying collagen fibril morphology based on first and second-order texture statistical parameters from SHG images. In addition, radiomics feature analysis, tree-based pipeline optimization tool [90] and 3D texture analysis based on k-nearest neighbor classification are also proposed for differentiating healthy and pathologic tissues [91]. Figure 5: (**a**) 3D Second Harmonic Generation (SHG) renderings (left panels) and hematoxylin and eosin (H&E) staining (center panels) of normal (top) and malignant ovarian biopsies (bottom). Averaged forward/backward SHG intensities as a function of depth for normal (blue circles) and malignant (red squares) ovaries (right panels) [84] (reprinted with permission from [84], Copyright 2010 Springer Nature). (**b**) SHG images and correcting autofluorescence (AF) images of the collagen in different tissues status (left panels). SHG images of (A) normal stroma, (B) invasive ductal carcinoma, (C) post-chemotherapy. AF images of (D) normal stroma, (E) invasive ductal carcinoma, (F) post-chemotherapy. The ratio of SHG/AF and the collagen density of the three status of breast tissues (right panels) [85] (reprinted with permission from [85], © The Optical Society).