The field of optical microscopic imaging has rapidly evolved because of tremendous advances made in laser and detection technology. Various types of linear and nonlinear light-matter interactions have been harnessed for providing contrast in the microscopic images. Simple microscopy techniques such as bright-field and differential-interference-contrast reveal structural information at the cellular level owing to the refractive index contrast of the sample medium. Fluorescence microscopy offers higher chemical specificity and is the most popular contrast mechanism used in biological studies. The contrast is achieved by means of targeted labeling of molecules using exogenous or endogenous fluorophores. However, external fluorophores are often perturbative since they may disrupt the native state of the sample, especially for small molecules whose size may be smaller than the fluorescent label itself. Besides, many molecular species are intrinsically nonfluorescent or only weakly fluorescent. It is also better to avoid external contrast agents for in vivo imaging applications since such contrast agents need concurrent development of appropriate delivery strategies and are often limited by problems of label specificity and induced toxicity. Vibrational microscopy techniques, on the other hand, are inherently label-free. They involve the excitation of molecular vibrations and offer intrinsic chemical specificity. Two such techniques include infrared absorption and Raman microscopy. Out of these, infrared microscopy has low spatial resolution owing to the long infrared wavelengths employed. In addition, water absorption of the infrared light is a major limitation for investigating live biological samples. Raman scattering, on the other hand, is based on the inelastic scattering of light by vibrating molecules and provides a molecular fingerprint of the chemical composition of a living cell or tissue. It offers a powerful label-free contrast mechanism and has been applied in various biological investigations. Linear contrast mechanisms based on fluorescence and Raman scattering typically employ continuous-wave visible light for excitation and sample scanning or laser scanning to generate an image. A confocal pinhole inserted at the detector facilitates a three-dimensionally sectioned image but unfortunately limits the sensitivity of detection.
Department of Physics

Murugkar, S, & Boyd, R.W. (Robert W.). (2017). Overview of second- and third-order nonlinear optical processes for deep imaging. In Deep Imaging in Tissue and Biomedical Materials: Using Linear and Nonlinear Optical Methods (pp. 4–30). doi:10.1201/9781315206554