Bo Li

Synaptic inputs onto the dendrites and cell body integrate and trigger action potentials (APs) at the axon initial segment (AIS). The AIS receives GABAergic synaptic inputs; however, the structure and function of glutamatergic synapses at the AIS remain poorly understood. Here, we show that, in adult mice, the AIS exhibits axonic spines in about half of the neurons examined in three brain regions: the dorsal lateral septum (dLS), bed nucleus of the stria terminalis and striatum. In the dLS, axonic spines express ionotropic glutamate receptors and undergo structural plasticity. Voltage-gated Na⁺ channels at the AIS boost the synaptic responses of axonic spines and thus AP generation. Although hippocampal dorsal CA3 neurons synapse onto both axonic spine neurons (ASNs) and non-ASNs, they preferentially activate ASNs and subsequently inhibit non-ASNs through feedforward inhibition. Together, these results indicate that axonic spines jump-start APs in dLS neurons and route information flow from the hippocampus to downstream brain regions.

Multiphoton microscopy is increasingly pushed toward deep, large-scale and fast functional imaging, where acquisition speed has become a bottleneck. Here we present SPARC, a structure-guided, physics-aware reconstruction framework for scan-limited multiphoton imaging. Rather than treating undersampled recovery as generic super-resolution, SPARC formulates it as a constrained reconstruction problem tailored to point-scanning microscopy by incorporating a sample-matched structural reference from the same field of view and the anisotropic acquisition physics of scan-limited imaging. SPARC jointly integrates denoising and deep upsampling, enabling stable recovery from noisy sparse measurements. In simulated calcium imaging with ground truth, SPARC improved spatial reconstruction fidelity and temporal signal recovery relative to existing methods. In vivo, SPARC improved temporal readout in three-photon and mesoscale two-photon calcium imaging, and enabled a more temporally informative 400-Hz voltage readout on a standard resonant-galvo two-photon microscope. These results suggest SPARC expands the functional operating range of existing multiphoton microscopes under scan-limited conditions.

Three-photon microscopy (3PM) has extended optical imaging beyond the reach of two-photon microscopy (2PM), but its practical depth remains far below theoretical predictions because of low photon efficiency and severe noise contamination. Here we present an integrated hardware and software solution that addresses these limitations. We developed a plug-and-play region-of-interest (ROI) imaging module that selectively excites neuron-occupied regions to improve power efficiency and reduce photothermal stress. The module incorporates frame-partitioned accumulative (FPA) imaging for high signal-to-noise ratio (SNR) structural acquisition, automated neuron segmentation, and motion-robust registration for stable recordings. Complementing this, OptiCal is a cascaded deep-learning framework that removes mixed noise including periodic, motion-induced, and random components. Together, these innovations enable high-fidelity imaging to 1.7 mm depth, extending the practical limit of 3PM by about 400 μm while maintaining low excitation power. Our results reveal reliable calcium activity and behavior-correlated dynamics in deep medial prefrontal cortex of awake mice.

Understanding how neural circuits compute requires capturing voltage dynamics across large neuronal populations with millisecond resolution in vivo . However, two-photon voltage imaging remains fundamentally limited by a trade-off among imaging speed, field of view, and excitation efficiency. We introduce HS2PM, a hybrid scanning two-photon microscope that overcomes this bottleneck, enabling kilohertz-rate imaging over a 650 × 524 μm ² field of view at single-cell resolution while preserving the photon efficiency of single-point excitation. HS2PM stably records deep-layer membrane potentials from over 160 neurons simultaneously, with minimal photobleaching and phototoxicity. It resolves both spikes and subthreshold voltage dynamics in vivo , revealing how these jointly shape sensory adaptation and population coding. Beyond voltage imaging, HS2PM supports high-speed fluorescence lifetime and vascular flow imaging, establishing a multimodal platform for dissecting fast circuit dynamics with precision previously inaccessible to optical methods.

Understanding how distributed brain regions interact across cortical depths is crucial for uncovering the neural basis of behavior and cognition. Two‐photon microscopy (2PM) excels at imaging the superficial layer, while three‐photon microscopy (3PM) penetrates deep tissues but lacks sufficient speed for capturing fast superficial dynamics. Here, we present a compact two‐photon imaging module (2PIM) that seamlessly integrates with existing 3PM to achieve simultaneous, high‐speed imaging of superficial and deep brain regions, while preserving the deep tissue penetration of 3PM. This dual‐modality system allows for remote and independent control of imaging planes in both axial and lateral dimensions. Leveraging this technology, we discovered context‐dependent modulation of hippocampal CA1 and visual cortex responses to identical tactile stimuli, layer‐specific temporal dynamics in sensory cortex during air puff stimulation, and depth‐divergent neuronal activity patterns in motor cortex across acute‐to‐chronic pain. These results highlight the system's potential for dissecting the spatiotemporal coordination of Multi‐Regions Imaging, three‐photon microscopy, and two‐photon microscopy of distributed brain circuits.

Mapping cortex-wide neuronal activity at single-cell resolution has been limited by the physical tradeoff between numerical aperture and field-of-view (FOV) in two-photon microscopes. We present Meso2P, a modular two-photon platform that decouples excitation and detection by introducing a lateral paraboloid fluorescence collector. The design sustains an effective NA 0.87 over a contiguous 6 × 6 mm² FOV at high speed (2,048 × 2,048 pixels at 7.67 Hz). The modular platform can be upgraded with optional modules for simultaneous multi-plane imaging (1-4 planes at full resolution and speed), volumetric imaging (6 × 6 × 0.5 mm³, 2,048 × 2,048 × 28 voxels at 1 Hz capturing > 210,000 neurons), and holographic two-photon optogenetic stimulation for targeted perturbations. To handle the resulting large-scale data, we provide an open-source deep-learning pipeline that automates motion correction, segmentation, and spike inference. We demonstrate cortex-wide sensory responses, layer-specific network synchrony during anaesthesia, and in-vivo tracking of micro- and nanoplastic distribution. Meso2P therefore provides a reproducible route to high-throughput volumetric imaging across almost the entire cortex with high detection efficiency.

Understanding how distributed brain regions interact across cortical depths is crucial for uncovering the neural basis of behavior and cognition. Two-photon microscopy (2PM) excels at imaging the superficial layer, while three-photon microscopy (3PM) penetrates deep tissues but lacks sufficient speed for capturing fast superficial dynamics. Here, we present a compact two-photon imaging module (2PIM) that seamlessly integrates with existing 3PM to achieve simultaneous, high-speed imaging of superficial and deep brain regions, while preserving the deep tissue penetration of 3PM. This dual-modality system allows for remote and independent control of imaging planes in both axial and lateral dimensions. Leveraging this technology, we discovered context-dependent modulation of hippocampal CA1 and visual cortex responses to identical tactile stimuli, layer-specific temporal dynamics in sensory cortex during air puff stimulation, and depth-divergent neuronal activity patterns in motor cortex across acute-to-chronic pain. These results highlight the system’s potential for dissecting the spatiotemporal coordination of distributed brain circuits.

Mapping cortex-wide neuronal activity at single-cell resolution has been limited by the physical trade-off between numerical aperture and field-of-view (FOV) in two-photon microscopes. We present Meso2P, a modular two-photon platform that decouples excitation and detection by introducing a lateral paraboloid fluorescence collector. The design sustains an effective NA 0.87 over a contiguous 6 * 6 mm^2 FOV at high speed (2,048 * 2,048 pixels at 7.67 Hz). The modular platform can be upgraded with optional modules for simultaneous multi-plane imaging (1-4 planes at full resolution and speed), volumetric imaging (6 * 6 * 0.5mm^3, 2,048 * 2,048 * 28 voxels at 1Hz capturing > 210,000 neurons), and holographic two-photon optogenetic stimulation for targeted perturbations. To handle the resulting large-scale data, we provide an open-source deep-learning pipeline that automates motion correction, segmentation, and spike inference. We demonstrate cortex-wide sensory responses, layer-specific network synchrony during anaesthesia, and in-vivo tracking of micro- and nanoplastic distribution. Meso2P therefore provides a reproducible route to high-throughput volumetric imaging across almost the entire cortex with high detection efficiency.

Optical imaging is important for understanding brain function. However, established methods with high spatiotemporal resolution are limited by the potential for laser damage to living tissues. We describe an adaptive femtosecond excitation source that only illuminates the region of interest, which leads to a 30-fold reduction in the power requirement for two- or three-photon imaging of brain activity in awake mice for improved high-speed longitudinal neuroimaging.

Stomatal closure defense and apoplastic defense are two major immunity mechanisms restricting the entry and propagation of microbe pathogens in plants. Surprisingly, activation of plant intracellular immune receptor NLR genes, while enhancing whole plant disease resistance, were sometimes linked to a defective stomatal defense in autoimmune mutants. Here we report the use of high temperature and genetic chimera to investigate the inter-dependence of stomatal and apoplastic defenses in autoimmunity. High temperature inhibits both stomatal and apoplastic defenses in the wild type, and it suppresses constitutive apoplastic defense responses and rescues the deficiency of stomatal closure response in autoimmune mutants. Chimeric plants have been generated to activate NLR only in guard cells or the non-guard cells. NLR activation in guard cells inhibits stomatal closure defense response in a cell autonomous manner likely through repressing ABA responses. At the same time, it leads to increased whole plant resistance accompanied by a slight increase in apoplastic defense. In addition, NLR activation in both guard and non-guard cells affects stomatal aperture and water potential. This study thus reveals that NLR activation has a differential effect on immunity in a cell type specific matter, which adds another layer of immune regulation with spatial information. Plant immunity against foliar pathogens consists of stomatal defense to restrict the entry of pathogens and apoplastic defense to restrict the propagation of pathogens. Plant intracellular immune receptor NLR genes are known to trigger apoplastic defense upon perception of pathogen effectors. In this study, we uncovered a previously unidentified inhibitory role of NLR activation in stomatal defense by using autoimmune mutants where NLR genes are activated without pathogen trigger. We further showed by chimera plants that NLR function is cell autonomous in guard cells for stomatal closure defense and non-cell autonomous for apoplastic defense. In addition, we revealed that high temperature slows down stomatal closure defense in addition to its known effect of inhibiting apoplastic defense. This study therefore reveals an unexpected regulation of stomatal defense by NLR genes and provides the spatial information of these regulations in stomatal defense and apoplastic defense.