Opto2P-FCM: A MEMS based miniature two-photon microscope with two-photon patterned optogenetic stimulation

Multiphoton microscopy combined with optogenetic photostimulation is a powerful technique in neuroscience enabling precise control of cellular activity to determine the neural basis of behavior in a live animal. Two-photon patterned photostimulation has taken this further by allowing interrogation at the individual neuron level. However, it remains a challenge to implement imaging of neural activity with spatially patterned two-photon photostimulation in a freely moving animal. We developed a miniature microscope for high resolution two-photon fluorescence imaging with patterned two-photon optogenetic stimulation. The design incorporates a MEMS scanner for two-photon imaging and a second beam path for patterned two-photon excitation in a compact and lightweight design that can be head-attached to a freely moving animal. We demonstrate cell-specific optogenetics and high resolution MEMS based two-photon imaging in a freely moving mouse. The new capabilities of this miniature microscope design can enable cell-specific studies of behavior that can only be done in freely moving animals.