skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


This content will become publicly available on March 10, 2026

Title: High-Speed Neural Imaging with Multiplexed Miniature Two-Photon Microscopy (M-MINI2P)
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.  more » « less
Award ID(s):
1847141
PAR ID:
10644639
Author(s) / Creator(s):
; ; ; ; ;
Publisher / Repository:
bioRxiv
Date Published:
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; (, Dryad)
    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). 
    more » « less
  2. ; ; ; ; ; (, Optica Publishing Group)
    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. 
    more » « less
  3. ; ; ; (, Biomedical Optics Express)
    Traditional miniaturized fluorescence microscopes are critical tools for modern biology. Invariably, they struggle to simultaneously image with a high spatial resolution and a large field of view (FOV). Lensless microscopes offer a solution to this limitation. However, real-time visualization of samples is not possible with lensless imaging, as image reconstruction can take minutes to complete. This poses a challenge for usability, as real-time visualization is a crucial feature that assists users in identifying and locating the imaging target. The issue is particularly pronounced in lensless microscopes that operate at close imaging distances. Imaging at close distances requires shift-varying deconvolution to account for the variation of the point spread function (PSF) across the FOV. Here, we present a lensless microscope that achieves real-time image reconstruction by eliminating the use of an iterative reconstruction algorithm. The neural network-based reconstruction method we show here, achieves more than 10000 times increase in reconstruction speed compared to iterative reconstruction. The increased reconstruction speed allows us to visualize the results of our lensless microscope at more than 25 frames per second (fps), while achieving better than 7 µm resolution over a FOV of 10 mm2. This ability to reconstruct and visualize samples in real-time empowers a more user-friendly interaction with lensless microscopes. The users are able to use these microscopes much like they currently do with conventional microscopes. 
    more » « less
  4. ; ; (, Science Advances)
    Mask-based integrated fluorescence microscopy is a compact imaging technique for biomedical research. It can perform snapshot 3D imaging through a thin optical mask with a scalable field of view (FOV). Integrated microscopy uses computational algorithms for object reconstruction, but efficient reconstruction algorithms for large-scale data have been lacking. Here, we developed DeepInMiniscope, a miniaturized integrated microscope featuring a custom-designed optical mask and an efficient physics-informed deep learning model that markedly reduces computational demand. Parts of the 3D object can be individually reconstructed and combined. Our deep learning algorithm can reconstruct object volumes over 4 millimeters by 6 millimeters by 0.6 millimeters. We demonstrated substantial improvement in both reconstruction quality and speed compared to traditional methods for large-scale data. Notably, we imaged neuronal activity with near-cellular resolution in awake mouse cortex, representing a substantial leap over existing integrated microscopes. DeepInMiniscope holds great promise for scalable, large-FOV, high-speed, 3D imaging applications with compact device footprint. 
    more » « less
  5. ; ; ; ; ; ; ; ; ; ; et al (, Nature Biomedical Engineering)
    Abstract The simple and compact optics of lensless microscopes and the associated computational algorithms allow for large fields of view and the refocusing of the captured images. However, existing lensless techniques cannot accurately reconstruct the typical low-contrast images of optically dense biological tissue. Here we show that lensless imaging of tissue in vivo can be achieved via an optical phase mask designed to create a point spread function consisting of high-contrast contours with a broad spectrum of spatial frequencies. We built a prototype lensless microscope incorporating the ‘contour’ phase mask and used it to image calcium dynamics in the cortex of live mice (over a field of view of about 16 mm 2 ) and in freely moving Hydra vulgaris , as well as microvasculature in the oral mucosa of volunteers. The low cost, small form factor and computational refocusing capability of in vivo lensless microscopy may open it up to clinical uses, especially for imaging difficult-to-reach areas of the body. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email