Three-dimensional multi-site two-photon excitation for probing neuronal signal integration

Two-photon holographic microscopy (2PHM) offers the advantage of simultaneous multi-site excitation in three dimensions. This is useful for studies of neuronal signal integration, which require multiple controlled synaptic inputs delivered simultaneously onto dendritic trees. In this work, we holographically split a single femtosecond pulse-laser in order to project multiple foci onto the neuron. At each focus, two-photon photolysis of caged neurotransmitter molecules, which bind to receptors and mimic synaptic transmission, is performed, thereby allowing measurement of how neurons integrate multiple synaptic inputs.