Dual-View Microscope Records Neuronal Activity in 3D at 100 Hz

A new microscopy system can record two-photon images of rapidly changing calcium signals across three-dimensional neuronal structures at a volumetric rate of 100 Hz. The dual-view Bessel two-photon projection microscopy method, or dv-B2PM, was developed by a team led by Leilei Peng of the University of Arizona and Jun B. Ding of Stanford University and reported in Light: Science & Applications. In tests with live motor-cortex brain slices, the instrument captured both the shape of labeled neurons and localized responses to simulated excitatory inputs while reconstructing activity through dendritic branches and the cell body.

The method addresses a central limitation of conventional two-photon microscopy: sequentially scanning individual planes restricts the rate at which a three-dimensional volume can be refreshed. Dendrites can integrate signals over intricate geometries on millisecond and submicrometer scales, so faster volumetric imaging is needed to distinguish events that occur in different parts of a neuron. dv-B2PM uses two objectives positioned at 90 degrees to collect simultaneous, perpendicular projections of the same volume. One objective delivers the Bessel-shaped two-photon excitation beam and detects fluorescence with a photomultiplier tube, while the second objective captures the orthogonal projection and supplies the beam used for two-photon glutamate uncaging. A camera and photomultiplier tube acquire synchronized projection pairs during each volume scan. Overlaps in one view can be resolved using the complementary view, which permits three-dimensional reconstruction of neuronal geometry while reducing the readout burden associated with collecting a full stack of sequential optical sections.

The reported imaging volume was approximately 120 × 80 × 42 µm³, with optical voxel dimensions of 0.5 × 0.5 × 0.75 µm³ and digital sampling of 0.3 µm along all three axes. The authors estimated that matching the system’s 100-Hz volume throughput through sequential scanning would require a hypothetical setup operating at 14,000 frames per second and a pixel rate of 1.7 GHz, illustrating how the dual-projection design avoids a major data-transfer burden. Its performance was evaluated in live brain slices containing motor-cortex neurons labeled with GCaMP8m. A separate 720-nm laser enabled two-photon glutamate uncaging at selected dendritic spines, allowing the researchers to stimulate a localized site while dv-B2PM measured the resulting calcium dynamics.

The stimulation experiments showed that responses depended on intensity. Uncaging near a single dendrite at lower intensity produced subthreshold calcium transients that remained near the stimulation site and decayed before reaching the soma. Stronger stimulation elicited suprathreshold propagation into the cell body and neighboring dendritic branches. Analysis over longer timescales associated dendritic-to-soma propagation with back-propagating action potentials originating in the soma, while short-timescale two-dimensional wavelet analysis along continuous dendrites identified spatially heterogeneous transients with nominal frequencies of 5 to 40 Hz. Some high-frequency events traveled along dendritic pathways at velocities greater than 1 millimeter per second. These findings demonstrate the method’s ability to resolve distinct modes of local and cell-wide activity in a three-dimensional preparation. The authors suggest that such rapid localized events may help investigate dendritic plateau potentials and signal summation, but those broader circuit-level functions remain prospective applications rather than outcomes established by the reported experiments.

Subject of Research: Three-dimensional two-photon imaging of stimulated neuronal activity at 100 Hz

Article Title: Seeing activities in 3D neural structures at 100 Hz

Article References: Seeing activities in 3D neural structures at 100 Hz. Original publication (Bibliographic details unavailable.)

Image Credits: AI Generated

DOI: Not provided

Keywords: neuroscience, two-photon microscopy, 3D imaging, dendrites, calcium imaging, glutamate uncaging, neuronal dynamics, live brain slices, Bessel beam, Seeing, activities, neural