This technology enables microlevel neuron and neuron-environment analyses by use of multi-focal holographic 2P optogenetic stimulation in 3D at micron spatial and millisecond temporal resolution.
Advances in optical engineering and development of soma-targeted opsins activated at near infrared wavelengths have made it possible to perform optogenetic manipulation of single or multiple selected brain cells in 3D at the same time as the effects are imaged with 3D multi-photon microscopy at micron spatial and millisecond temporal resolution. Temporal focusing is used to penetrate deeply into tissue while ensuring good signal-to-background ratio for imaging. This all-optical strategy is applicable in awake and sleeping animals, allowing precise photo-activation of selected cells, groups of cells or even subcellular compartments. The system is based on a conventional 2P laser microscope system completed with a blazed grating and lenses for TF and a dedicated holographic unit, the “spatial light modulator” (SLM). With this instrumentation, multi-focal holographic 2P optogenetic stimulation in 3D at micron spatial and millisecond temporal resolution is feasible.