Second Harmonic Generation (SHG) is a high-resolution, nonlinear optical imaging technique used to study intact tissues and cellular structures. SHG arises from media lacking a center of symmetry, such as anisotropic crystals or interfaces like membranes. It can image highly ordered structural proteins without exogenous labels and is particularly useful for biological membrane probes. Collagen, a primary component of connective tissues, has significant nonlinear susceptibility for SHG due to its noncentrosymmetric helix structure. SHG techniques offer advantages such as selective imaging, rejection of surface reflection, and high spatial resolution. The SHG signal intensity is quadratic with peak power and depends on the molecular surface density. SHG has been successfully applied to produce high-resolution images of living cells, 3D imaging of endogenous structural proteins, and biomolecular arrays. It also allows for the study of biochemical and biophysical processes, including collagen nanostructure, spatial orientation of fibrous structures, and microstructural changes in mixed collagen gels. SHG microscopy has potential in clinical studies, such as quantifying disease status, monitoring extracellular matrix restructuring, and visualizing skin lesions. The method is often used in multimodal approaches, combining with techniques like multiphoton fluorescence and coherent anti-Stokes Raman scattering (CARS) to gather more comprehensive information about tissues and cells.Second Harmonic Generation (SHG) is a high-resolution, nonlinear optical imaging technique used to study intact tissues and cellular structures. SHG arises from media lacking a center of symmetry, such as anisotropic crystals or interfaces like membranes. It can image highly ordered structural proteins without exogenous labels and is particularly useful for biological membrane probes. Collagen, a primary component of connective tissues, has significant nonlinear susceptibility for SHG due to its noncentrosymmetric helix structure. SHG techniques offer advantages such as selective imaging, rejection of surface reflection, and high spatial resolution. The SHG signal intensity is quadratic with peak power and depends on the molecular surface density. SHG has been successfully applied to produce high-resolution images of living cells, 3D imaging of endogenous structural proteins, and biomolecular arrays. It also allows for the study of biochemical and biophysical processes, including collagen nanostructure, spatial orientation of fibrous structures, and microstructural changes in mixed collagen gels. SHG microscopy has potential in clinical studies, such as quantifying disease status, monitoring extracellular matrix restructuring, and visualizing skin lesions. The method is often used in multimodal approaches, combining with techniques like multiphoton fluorescence and coherent anti-Stokes Raman scattering (CARS) to gather more comprehensive information about tissues and cells.