Time-Multiplexed Miniaturized Two-Photon Microscopy

Abstract

We propose a time-multiplexed miniaturized two-photon microscope (TM-MINI2P), enabling a two-fold increase in imaging speed while maintaining a high spatial resolution. Using TM-MINI2P, we conducted high-speed in-vivo calcium imaging in mouse cortex.

Fig. 1.

(a) Layout of the TM-MINI2P. Two laser pulse trains with 2 MHz repetition rate are temporally separated by time τ and directed towards two distinct regions on the sample. (b-d) Fluorescence images of 12-μm fluorescent beads sample with (b) channel 1 excitation (green) only, (c) channel 2 excitation (red) only, and (d) dual channel excitation. (e) Temporal demultiplexed signal from (d). The overlapped imaging region from the two channels is colored as yellow. Scale bar: 50 μm. GRIN lens, graded-index lens.

Fig. 2.

Calcium imaging of mouse cortex in vivo through TM-MINI2P. (a-c) Max-correlation projection of the recording of (a) channel 1 and (b) channel 2 by blocking one excitation path, and (c) dual channel, with the CNMF-extracted neuronal cell body contours overlaid. (d) Demultiplexed dual channel recording [from (c)] with the CNMF-extracted neuronal activity traces of selected cells for each channel (green/red). The spatial footprint and temporal activity trace of the neurons in the overlapped regions of the two channels were labeled in yellow. Galvo-galvo scanning mode were used, resulting in a frame rate of 1.62 fps. (e) Same as (d), but with galvo-resonant-scanner mode, at a frame rate of 17.17 fps. Scale bar: 50 μm.