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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. # 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). 

We developed multiplexed miniaturized two-photon microscopes (M-MINI2Ps) that increase imaging speed while preserving high spatial resolution. Using M-MINI2Ps, we performed large-scale volumetric calcium imaging and high-speed voltage imaging in the cortex of freely- behaving mice. 

SUMMARY 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 µm²FOV) multiplane calcium imaging in visual cortex and prefrontal cortex in freely moving mice for spontaneous activity and auditory stimulus evoked responses. Furthermore, the increased speed of M-MINI2Ps also enables two-photon voltage imaging at 400 Hz over a 380×150 µm²FOV 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. 

We demonstrate simultaneous dual-region in-vivo imaging of brain activity in mouse cortex through a miniaturized spatial-multiplexed two-photon microscope platform, which doubles the imaging speed. Neuronal signals from the two regions are computationally demixed and extracted. 

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. 

Noninvasive transabdominal fetal pulse oximetry can provide clinicians critical assessment of fetal health and potentially contribute to improved management of childbirth. Conventional pulse oximetry through continuous wave (CW) light has challenges measuring the signals from deep tissue and separating the weak fetal signal from the strong maternal signal. Here, we propose a new approach for transabdominal fetal pulse oximetry through interferometric near-infrared spectroscopy (iNIRS). This approach provides pathlengths of photons traversing the tissue, which facilitates the extraction of fetal signals by rejecting the very strong maternal signal from superficial layers. We use a multimode fiber combined with a mode-field converter at the detection arm to boost the signal of iNIRS. Together, we can detect signals from deep tissue (>∼1.6 cm in sheep abdomen and in human forearm) at merely 1.1 cm distance from the source. Using a pregnant sheep model, we experimentally measured and extracted the fetal heartbeat signals originating from deep tissue. This validated a key step towards transabdominal fetal pulse oximetry through iNIRS and set a foundation for further development of this method to measure the fetal oxygen saturation.