Diesel2p mesoscope with dual independent scan engines for flexible capture of dynamics in distributed neural circuitry

Abstract

Imaging the activity of neurons that are widely distributed across brain regions deep in scattering tissue at high speed remains challenging. Here, we introduce an open-source system with Dual Independent Enhanced Scan Engines for Large Field-of-view Two-Photon imaging (Diesel2p). Combining novel optical design, adaptive optics, and temporal multiplexing, the system offers subcellular resolution over a large field-of-view (∼ 25 mm2) with independent scan engines. We demonstrate the flexibility and various use cases of this system for calcium imaging of neurons in the living brain.

High-Speed, Cortex-Wide Volumetric Recording of Neuroactivity at Cellular Resolution using Light Beads Microscopy

“…Many platforms have demonstrated the requisite mesoscopic optical performance. [17][18][19][20][21][22][23] However, in all of these systems spatiotemporal sampling remains suboptimal: the high repetition rate lasers employed lead to inevitable over-sampling in the lateral planes due to finite resonant scanner speed, and multiple-pulse-per-pixel binning to improve SNR at low pulse energies. Accordingly, performance is limited to at most multi-plane rather than volumetric performance, slow frame rates, and brain-heating-limited SNR, particularly when imaging at depth.…”

“…Temporal multiplexing 30,31,3219,23,25 can be used to increase information throughput by scanning N copies of a single laser pulse which are delayed in time, and directed toward separate regions of the sample. As long as the relative delay between beams exceeds the fluorescence lifetime-limited decay time of the signal from the previous beam (~6-7 ns or ~140-160 MHz for GCaMP 31 ), fluorescence at the detector can be re-assigned in order to reconstitute the FOV scanned by each beam, resulting in an N-fold increase in data rate beyond the inertial limits of the scanning system.…”

“…As long as the relative delay between beams exceeds the fluorescence lifetime-limited decay time of the signal from the previous beam (~6-7 ns or ~140-160 MHz for GCaMP 31 ), fluorescence at the detector can be re-assigned in order to reconstitute the FOV scanned by each beam, resulting in an N-fold increase in data rate beyond the inertial limits of the scanning system. However, multiplexed systems employing conventional high-repetition-rate lasers (typically ~80 MHz) are limited to at most a ~2-fold increase in data rate, 19,23,[30][31][32] in addition to suffering from oversampling inefficiencies as mentioned above. Lowering laser repetition rates to the few MHz regime can simultaneously increase the maximum possible degree of multiplexing N while improving sampling efficiency.…”

“…Lowering laser repetition rates to the few MHz regime can simultaneously increase the maximum possible degree of multiplexing N while improving sampling efficiency. 25 However, many multiplexing platforms use feedforward approaches employing chains of beam splitters and dedicated optical paths for delaying and steering each beam, resulting in unfavorable scaling of system complexity with increasing N, 10,19,23,25,30,31 and as a result can leave the majority of the inter pulse timing interval unexploited for maximizing information throughput. 25 Recently, multi-pass temporal multiplexing schemes have been demonstrated using systems in which the beam is sent through a single set of components which tag the light with a specific delay and focal position in the sample corresponding to each pass through the system 33,34 .…”

Flexible Simultaneous Mesoscale Two-Photon Imaging Of Neural Activity at High Speeds

“…The polarizing beam splitter also rejects any unwanted excess power from any of the four beams controlling the amount of excitation power reaching the sample. The custom objective 22 Point spread function measurements. Fluorescent beads with a diameter of 0.5 µm (T7281, Invitrogen) were used to measure the point spread function of the microscope.…”