Recent advances in nonlinear microscopy: Deep insights and polarized revelations

Nonlinear microscopy is a technique that utilizes nonlinear interactions between light and matter to image fluorescence and scattering phenomena in biological tissues. Very high peak intensities from focused short pulsed lasers are required for nonlinear excitation due to the extremely low probability of the simultaneous arrival of multiple photons of lower energy to excite fluorophores or interact with selective structures for harmonic generation. Combined with reduced scattering from the utilization of longer wavelengths, the inherent spatial confinement associated with achieving simultaneous arrival of photons within the focal volume enables deep imaging with low out-of-focus background for nonlinear imaging. This review provides an introduction to the different contrast mechanisms available with nonlinear imaging and instrumentation commonly used in nonlinear microscopy. Furthermore, we discuss some recent advances in nonlinear microscopy to extend the imaging penetration depth, conduct histopathological investigations on fresh tissues and examine the molecular order and orientation of molecules using polarization nonlinear microscopy.