Journal: Nature Communications

Article Title: High-throughput two-photon volumetric brain imaging in freely moving mice

doi: 10.1038/s41467-025-66922-2

Figure Lengend Snippet: a Photograph of a mouse wearing the microscope headpiece. b Brightfield micrograph of the custom micro-axicon at the LMA-12 fiber tip (top) and ring pattern imaged at ~5-mm distance from the fiber tip (bottom). c , Schematic of miniBB2p showing key elements and excitation beam path (red lines). d Imaging a volume requires a single 2D scan of a Bessel focus but a stack of 2D scans of a Gaussian focus. e Lateral and axial point spread functions measured with 500-nm fluorescent beads. f Image of a ~ 80-μm thick volume of Thy1-GFP-M brain slice by miniBB2p (left) and an image stack at 3-μm step collected by a tabletop Gaussian beam 2PM from the same volume, with structures colored by depth (right). g zoom-in imaging of the boxed areas in f ; white arrowheads denote dendritic spines that were more clearly visualized by Bessel focus. h , Imaging the spontaneous activity of neurons in anterior cingulate cortex (ACC) expressing GCaMP7f in a free-moving mouse with miniBB2p. i Spatial distribution of 1015 neurons extracted by Suite2P in h , randomly colored for visualization. j Example calcium signals from neurons circled in h . k – l Imaging the same volume in awake head-fixed mouse by the tabletop 2PM: Gaussian imaging stack at 3-μm step with structures colored by depth ( k ); neural activity imaging in the top, middle, and bottom layers of the volume, the imaging depth and number of neurons identified by Suite2P are listed at right ( l ). Data in b and e-h are from single representative experiments ( n = 3 fibers in b ; n = 10 beads in e ; n = 5 slices in f , g ; n = 5 animals in h ).

Article Snippet: We recorded neuronal activity in vivo using miniBB2p and later acquired the high-resolution 3D morphological data from the same imaging volume using the tabletop Gaussian-beam 2PM with a 0.8 NA objective (CFI Apo NIR 40×, Nikon) on the same day.

Techniques: Microscopy, Imaging, Slice Preparation, Activity Assay, Expressing