Data for: Multiplexed miniaturized two-photon microscopy (M-MINI2Ps)

Head-mounted miniaturized two-photon microscopes are powerful tools to record neural activity with cellular resolution deep in the mouse brain during unrestrained, free-moving behavior. Two-photon microscopy, however, is traditionally limited in imaging frame rate due to the necessity of raster scanning the laser excitation spot over a large field-of-view (FOV). Here, we present two multiplexed miniature two-photon microscopes (M-MINI2Ps) to increase the imaging frame rate while preserving the spatial resolution. Two different FOVs are imaged simultaneously and then demixed temporally or computationally. We demonstrate large-scale (500×500 µm2 FOV) multiplane calcium imaging in visual cortex and prefrontal cortex in freely moving mice during spontaneous exploration, social behavior, and auditory stimulus. Furthermore, the increased speed of M-MINI2Ps also enables two-photon voltage imaging at 400 Hz over a 380×150 Âµm2 FOV in freely moving mice. M-MINI2Ps have compact footprints and..., , # Data for: Multiplexed miniaturized two-photon microscopy (M-MINI2Ps)

Dataset DOI: [10.5061/dryad.kd51c5bkp](10.5061/dryad.kd51c5bkp)

## Description of the data and file structure

Calcium and Voltage imaging datasets from Multiplexed Miniaturized Two-Photon Microscopy (M-MINI2P)

### Files and variables

#### File: TM_MINI2P_Voltage_Cranial_VisualCortex.zip

**Description:** Voltage imaging dataset acquired in mouse primary visual cortex (V1) using the TM-MINI2P system through a cranial window preparation. This .zip file contains two Tif files, corresponding to the top field of view (FOV) and the bottom field of view (FOV) of the demultiplexed recordings.

#### File: TM_MINI2P_Calcium_GRIN_PFC_Auditory_Free_vs_Headfix.zip

**Description:** Volumetric calcium imaging dataset from mouse prefrontal cortex (PFC) using the TM-MINI2P system with a GRIN lens implant, comparing neural responses during sound stimulation versus quiet periods, under both freely moving and head-fixed conditions...,