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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).
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In situ measurement of the isoplanatic patch for imaging through intact bone
Abstract Wavefront‐shaping (WS) enables imaging through scattering tissues like bone, which is important for neuroscience and bone‐regeneration research. WS corrects for the optical aberrations at a given depth and field‐of‐view (FOV) within the sample; the extent of the validity of which is limited to a region known as the isoplanatic patch (IP). Knowing this parameter helps to estimate the number of corrections needed for WS imaging over a given FOV. In this paper, we first present direct transmissive measurement of murine skull IP using digital optical phase conjugation based focusing. Second, we extend our previously reported phase accumulation ray tracing (PART) method to provide in‐situin‐silicoestimation of IP, called correlative PART (cPART). Our results show an IP range of 1 to 3 μm for mice within an age range of 8 to 14 days old and 1.00 ± 0.25 μm in a 12‐week old adult skull. Consistency between the two measurement approaches indicates that cPART can be used to approximate the IP before a WS experiment, which can be used to calculate the number of corrections required within a given field of view.
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- Award ID(s):
- 1706916
- PAR ID:
- 10238346
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Journal of Biophotonics
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 1864-063X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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