DOI: 10.5061/dryad.kd51c5bkp
title: Data for: Multiplexed miniaturized two-photon microscopy (M-MINI2Ps)

description: 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 µm2
 FOV in freely moving mice. M-MINI2Ps have compact footprints and are
 compatible with the open-source MINI2P. M-MINI2Ps, together with their
 design principles, allow the capture of faster physiological dynamics and
 population recordings over a greater volume than currently possible in
 freely moving mice, and will be a powerful tool in systems neuroscience.
description: # 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. This .zip file
 contains 12 Tif files: top and bottom fields of view (FOVs) of the
 multiplexed recordings at three imaging depths (100 μm, 155 μm, and 240 μm
 from the end of the implanted GRIN lens), with six files from freely
 moving conditions and six files from head-fixed conditions. #### File:
 CM_MINI2P_Calcium_Cranial_VisualCortex_SocialBehavior.zip
 **Description:** Calcium imaging dataset from mouse primary visual cortex
 (V1) using the CM-MINI2P system through a cranial window, recorded during
 social interaction and isolated conditions. This .zip file contains 6 Tif
 files: multiplexed recordings from the top and bottom fields of view
 (FOVs), and single-FOV recordings at two imaging depths (170 µm and 250
 µm). #### File: TM_MINI2P_Calcium_Cranial_VisualCortex.zip
 **Description:** Multi-depth calcium imaging dataset from mouse primary
 visual cortex (V1) using the TM-MINI2P system through a cranial window
 during spontaneous exploration. This .zip file contains 6 Tif files:
 demultiplexed recordings from two fields of view (FOV1 and FOV2) at three
 imaging depths (110 µm, 170 µm, and 230 µm). ## Code/software All datasets
 are in .tiff format and ImageJ can be used for visualization. Analysis of
 calcium imaging data and voltage imaging data were analyzed using CaImAn
 and Volpy, respectively, which are open-source packages available at
 [https://github.com/flatironinstitute/CaImAn](https://github.com/flatironinstitute/CaImAn).