Multifocus nonlinear optical microscopy based on SLM

We developed a novel addressable scanless multifocal multiphoton microscope. This microscope works in a fast scanless mode. Subjectively selected sample (or multiple samples located in separated areas) in a large field of view can be imaged by illuminating only the area (or areas) where the target sample (or samples) locate(s). In this way, by precisely designing the multiple foci according to the size and position of the area of interest, we can concentrate all the laser energy and dwell time on that area of the sample, making full use of the available laser power and avoiding photodamage in other areas. Since no scanning is involved, the acquisition time of a multiphoton image is decided only by the sensitivity and readout time of the CCD camera. Moreover, the interfocal distance of the multiple foci matches the lateral resolution of the imaging system, so that the two-photon image was recorded with high lateral resolution. However, crosstalk (spatial interference) on out-of-focus planes occurs between adjacent points when they are too close, degrading the resolution, especially the axial resolution of the imaging system.