Optimization of second-harmonic generation microscopy

We describe the principles and characteristics of second-harmonic generation imaging (SHGI) and explore various methods for optimization of the technique. Second-harmonic imaging is optimized for ultrashort laser pulses, high numerical aperture microscope objectives, a highly sensitive non-descanned large area detector, pseudo-phase-matching, and specimens with large second-order non linearity or which exhibit surface plasmon enhanced phenomena. We also compare and contrast the techniques of SHGI and two-photon excited fluorescence imaging.