High-throughput spatial light modulation two-photon microscopy for fast functional imaging.

The optical monitoring of multiple single neuron activities requires high-throughput parallel acquisition of signals at millisecond temporal resolution. To this aim, holographic two-photon microscopy (2PM) based on spatial light modulators (SLMs) has been developed in combination with standard laser scanning microscopes. This requires complex coordinate transformations for the generation of holographic patterns illuminating the points of interest. We present a simpler and fully digital setup (SLM-2PM) which collects three-dimensional two-photon images by only exploiting the SLM. This configuration leads to an accurate placement of laser beamlets over small focal volumes, eliminating mechanically moving parts and making the system stable over long acquisition times. Fluorescence signals are diffraction limited and are acquired through a pixelated detector, setting the actual limit to the acquisition rate. High-resolution structural images were acquired by raster-scanning the sample with a regular grid of excitation focal volumes. These images allowed the selection of the structures to be further investigated through an interactive operator-guided selection process. Functional signals were collected by illuminating all the preselected points with a single hologram. This process is exemplified for high-speed (up to 1 kHz) two-photon calcium imaging on acute cerebellar slices.