simultaneous two - photon calcium imaging at different depths with spatiotemporal multiplexing

nature methods | ADVANCE ONLINE PUBLICATION | Optical probing of neuronal activity with fluorescent calcium indicators is a powerful approach to study information processing in the brain1. In particular, calcium imaging with two-photon laser scanning microscopy (2PLSM)2 is an ideal tool for recording network activity in vivo because it is less invasive than electrophysiology, more cells can be monitored simultaneously, and one can identify the cells being recorded3. Recently, several approaches have been introduced to overcome the traditionally slow raster scanning in two-photon calcium imaging (2PCI), including acoustooptic deflectors (AODs) or targeted path scanning with closedloop scanning mirrors4–7. Thus, as better fluorescent probes and methods for enhancing excitation and detection become available, temporal resolution in 2PCI will improve and reporting of action potential firing will be possible for large groups of neurons in volumes of tissue in vivo. Another way to improve temporal resolution in 2PCI is to scan the sample with multiple beams in parallel8,9. In fact, because time resolution, signal-to-noise ratio and field of view are closely related in 2PCI, multifocal scanning would lead to improvements in all of these areas. For instance, in cases for which nonlinear photodamage is a limitation10, scanning with multiple beams simultaneously and increasing the rate at which laser pulses are delivered to the sample will increase the signal-to-noise ratio11. Another potential simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing Adrian Cheng1–3,5, J Tiago Gonçalves2,5, Peyman Golshani2, Katsushi Arisaka1,4 & Carlos Portera-Cailliau2,3