Nonlinear microscopy

We describe the basic principles of nonlinear optical microscopies based on two-photon excited fluorescence and on second-harmonic generation. Particular attention is given to the physical mechanisms underlying molecular second-harmonic generation, and the features unique to its signal contrast. We provide an overview of some applications of nonlinear microscopy including the visualization of molecular ‘flip–flop’ dynamics in membranes, high-resolution measurements of inter-membrane separations, and high-sensitivity membrane potential imaging. Future developments of nonlinear microscopy are briefly considered.