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Many advantages make optical methods more and more interesting for neuroscientists. Thanks to the development of new voltage-sensitive dyes and of optogenetic probes, photon-based exploration of neuronal electrical activity is rapidly overcoming most of the electron-based techniques for electrophysiological investigations. These innovative labeling strategies have been recently combined with nonlinear imaging techniques, giving new trigger to the investigation of neuronal circuitry. Advances in nonlinear optical imaging of neuron voltage now provide not only a readout of the electrical activity of each neuron through wide microcircuits but also a map of the functionality of dendritic trees and synaptic compartments. Furthermore, improved optogenetic tools can now be easily combined with two-photon excitation, allowing light stimulation of single cells in vivo . This review will describe the main advantages of combining nonlinear laser imaging with specific reporters that allow reconstructing the neuronal circuitry by specifically interrogating or recording from single neurons. We will focus on two outstanding studies of 2012 that took advantage of two-photon microscopy to increase the spatial resolution, opening promising prospective in neuronal activity investigation.

Breakthroughs in Photonics 2012: Nonlinear Laser Imaging for Neuroscience