The field of optical microscopic imaging has rapidly evolved because of tremendous advances made in laser and detection technology. In this chapter, the authors examine the basics of second-order and third-order nonlinear optical processes that are involved in biological imaging, with the emphasis on second harmonic generation (SHG), coherent Raman scattering and self-phase modulation (SPM). In SHG, two photons with the same frequency interacting with a nonlinear optical material are effectively “combined” to generate new photons at twice the frequency of the incident light. SHG imaging is being developed as a powerful tool for investigating the changes to the tumor microenvironment during cancer invasion and metastasis. Polarization-resolved SHG has been applied to yield information related to the molecular organization of collagen, while SHG imaging combined with various image analysis techniques has revealed changes in tissue architecture associated with connective tissue disorders, musculoskeletal diseases, organ fibrosis, cardiovascular pathologies, and cancers.