Patterned Two-Photon Illumination for High-Speed Functional Imaging of Brain Networks In Vivo

Two-photon functional imaging combined with optogenetic manipulation represents a powerful tool to investigate neural network function and how the activity of specific circuits drives behavior. To fulfill these goals, imaging methods should ideally have a temporal resolution close to the temporal scale of neural activity (i.e., in the millisecond range). Classical laser-scanning two-photon microscopy, however, relies on beam deflection by galvanometric mirrors whose inertia, as well as the time the beam dwells on each image pixel, severely limits its temporal resolution. In this chapter, we focus on one approach to high-speed fluorescence recording, namely scanless imaging using patterned two-photon illumination. We will discuss recent evidence showing that this technique can be used to perform high-speed (up to 1 kHz) recording of calcium signals hundreds of micrometers deep in the rodent brain. Moreover, we describe the combination of scanless imaging with single-photon optogenetic manipulation for all-optical readout and perturbation of brain networks and with microendoscopic imaging for high-speed monitoring of circuit dynamics in deeper brain areas. Finally, we discuss the advantages and limitations of scanless imaging as well as possible future technical improvements that will likely contribute to significantly increasing our understanding of the role of neural networks in driving brain function and behavior.