New implementation of second harmonic generation microscopy for three-dimensional resolution

We present a fast scanning transmission-mode confocal scanning laser microscope system based on the use of a second harmonic generation (SHG) crystal for signal detection. The quadratic intensity dependence of SHG is exploited to preferentially reveal unscattered signal light and reject out-of-focus scattered background. The SHG crystal plays the role of a virtual pinhole that remains self-aligned without a need for de-scanning. We demonstrate that this new microscope method produces images with higher contrast and less speckle than transmission scanning microscopy with linear detection.