Biology subjects

Ji, N.

Publications and source records attributed to Ji, N..

At least 19 recordsLinked to original sources

bioRxiv2026

High Axial Resolution Is Necessary for Quantitative Two-Photon Calcium Imaging of Neuronal Populations

Two-photon calcium imaging is a standard tool for measuring neuronal population activity in vivo, yet how axial resolution, sensor expression strategy, and analysis pipeline jointly affect data accuracy remains poorly characterized. Here, we imaged the same L2/3 neurons in mouse primary visual cortex at five axial resolutions (3.6-21.0 m), spanning current two-photon systems from benchtop microscopes to large-field-of-view and miniaturized designs. We expressed cytosolic, transgenic, and soma-targeted GCaMP variants and applied five analysis pipelines. Reducing axial resolution systematically attenuated {Delta}F/F0, corrupted visual responsiveness and orientation tuning classifications, and biased pairwise correlations. No pipeline corrected these resolution-dependent artifacts, and pipeline choice alone produced quantitatively divergent results even at the highest resolution. Soma-targeted sensors mitigated but did not eliminate these artifacts. Our findings demonstrate that high axial resolution is necessary for accurate quantitative population imaging, and that robust separation of somatic from neuropil signals remains an unresolved challenge. In BriefImaging the same cortical neurons across five axial resolutions and five analysis pipelines, Ji and colleagues show that lower resolution corrupts neuronal tuning and population correlations. No pipeline corrects these artifacts, and pipeline choice yields divergent results even at high resolution. Soma-targeted sensors mitigate but do not eliminate these failures.

neuroscience↗

bioRxiv2026

A red-emitting, genetically encoded indicator for two-photon voltage recording in vivo

Genetically encoded voltage indicators (GEVIs) enable minimally invasive, cell-type-specific optical measurements of neuronal membrane potential with millisecond temporal resolution. Red-shifted GEVIs are especially advantageous because they permit spectral multiplexing with complementary sensors and enable all-optical circuit interrogation in combination with blue-light-activated opsins. Despite these advantages, existing red GEVIs remain poorly suited for in vivo use due to limited performance under two-photon (2P) excitation, the predominant modality for deep-tissue imaging. Here, we introduce VADER1, a red GEVI that overcomes this limitation and enables reliable spike detection in vivo under 2P illumination. Under 2P excitation, VADER1 supports extended voltage imaging with both random-access and resonant-scanning microscopy, enables recordings from neurons as deep as cortical layer 5, and allows dual-color imaging with calcium indicators. By filling a critical spectral gap, VADER1 enables integrated optical measurements of fast electrical activity alongside other neural signals and establishes a foundation for two-photon all-optical electrophysiology.

neuroscience↗

bioRxiv2026

Cholesteryl Ester as a Prognostic Biomarker In IDH-wildtype Glioblastoma

Current treatment of IDH-wildtype glioblastoma (GBM) relies on the first-line chemotherapy-temozolomide. Although MGMT methylation is routinely conducted to predict chemosensitivity, its efficacy is often compromised. Thus, there is an urgent need to discover more accurate prognostic biomarkers. Cholesteryl ester (CE) has been recently recognized as a key feature of GBM, however, its role in GBM prognosis remains poorly understood. We first employed label-free stimulated Raman scattering (SRS) imaging to quantitatively analyze CE level in intact tumor tissues obtained from IDH-wildtype GBM patients. Our result revealed significantly prolonged 2-year overall survival (OS) in patients with CE level [≥] 40% compared to those with CE level < 40%. CE outperformed MGMT methylation for 2-year OS prognosis (AUC: 0.836 vs. 0.763). Importantly, CE also achieved superior prognostic performance over MGMT methylation on an independent cohort, with higher sensitivity (0.856 vs. 0.667), specificity (0.833 vs. 0.583), NPV (1.00 vs. 0.667), PPV (0.833 vs. 0.583). Given synergistic effects between CE and MGMT methylation, we developed a prognostic model combining these two biomarkers. Specially, machine learning (XGBoost) model exhibited optimal performance in the training cohort (AUC: 0.920), and maintained its superior performance on the independent cohort (sensitivity: 0.946, specificity: 0.873, NPV: 1.00; PPV: 0.917). Mechanistically, integrative analysis of TCGA database linked poor prognosis to the coordinated upregulation of genes involved in cholesterol efflux, hydrolysis, transport, and inhibition of de novo synthesis, unraveling a possible underlying mechanism between poor prognosis and cholesterol metabolism. This work identified CE as a prognostic biomarker for IDH-wildtype GBM.

neuroscience↗

bioRxiv2026

Magnetogenetic control of endogenous calcium signaling via ROS-sensitive ion channels reveals astrocytic regulation of neural circuit activity

Astrocytes exhibit complex calcium signaling proposed to detect, integrate, and modulate neuronal activity; however, their causal contribution to neural circuit function remains unresolved. This is partly due to the lack of tools that recapitulate physiological astrocyte calcium signaling. Here, we show that experimental magnetic control of endogenous calcium signaling in astrocytes is sufficient to modulate neural circuit dynamics. Using pmFeRIC, a magnetogenetic tool for remote, noninvasive, and on-demand activation of endogenous calcium pathways, we demonstrate that selective activation of astrocyte calcium signaling drives neuronal activity. pmFeRIC uses magnetic fields and endogenous ferritin to generate localized reactive oxygen species (ROS) at the plasma membrane, activating native ROS-sensitive ion channels, inducing calcium influx. In hippocampal circuits in vitro, magnetic activation of endogenous astrocytic calcium signaling produced robust, state-dependent changes in neuronal circuit activity, in part by increasing presynaptic release probability, postsynaptic burst firing, and glutamatergic transmission. These effects were mediated by transient receptor potential channels, amplified by intracellular calcium stores, and abolished when astrocyte calcium signaling was disrupted, establishing a causal role in neural circuit dynamics. In vivo, pmFeRIC activates endogenous calcium signaling in astrocytes with kinetics comparable to locomotion-induced activity, enabling control of astrocyte calcium dynamics that resemble physiological behavioral responses. Together, these findings establish that endogenous astrocytic calcium signaling is sufficient to modulate neural circuit function and introduce a broadly applicable platform for cell-specific, wireless, on-demand control of endogenous calcium signaling through magnetic activation of ROS-sensitive ion channels.

neuroscience↗

bioRxiv2026

Task-space dimensions guide human exploration in complex environments

Efficient exploration is essential for rapid learning in high-dimensional environments, where rewards depend on a sparse and unknown subset of task dimensions. Here, we developed a multidimensional value-learning task in which participants explored without explicit knowledge of the underlying structure. Across multiple task designs, participants consistently demonstrated successful learning under structural uncertainty. Behavioral analyses revealed that over half of participants adopted a structured exploration strategy--Full Dimension Scan (FDS)--with three key properties. First, participants systematically isolated and sampled one candidate dimension at a time, effectively reducing search complexity. Second, FDS was temporally persistent within dimensions while maintaining diverse sampling of feature values, enabling efficient local inference. Third, participants sequentially deployed FDS across dimensions, achieving broad coverage of the task space. Importantly, this strategy transferred across tasks: prior experience in lower-dimensional environments promoted stronger FDS use and improved performance in higher-dimensional settings. Consistent with these findings, a dimension-guided exploration model incorporating dimension-level attentional control best captured human behavior. Together, these results identify dimension-guided exploration as a core principle that transforms high-dimensional search into tractable, structured learning, providing new insight into adaptive behavior in complex environments.

animal behavior and cognition↗

bioRxiv2026

Designer indicators for two-photon recording of subthreshold voltage dynamics

Subthreshold voltage dynamics are critical for neuronal information integration, yet they remain understudied in vivo due to the limitations of current tools. While genetically encoded voltage indicators (GEVIs) offer a promising alternative, their application for deep-tissue recording using two-photon (2P) microscopy--a preferred method for deep-tissue recording--has been hindered by insufficient sensitivity for detecting millivolt-scale subthreshold signals. Here, we refined our multiparametric two-photon high-throughput screening platform to develop two novel GEVIs, JEDI3sub and JEDI3hyp, tailored explicitly for subthreshold voltage detection. Through fast 2P optical recording in awake, behaving mice, we demonstrated the superior sensitivity of JEDI3 indicators compared to JEDI-2P. We also showed that JEDI3sub can track population-level subthreshold optical tuning, while JEDI3hyp reliably captured subthreshold dynamics associated with sharp-wave ripple oscillations in hippocampal PV interneurons. Finally, JEDI3hyp facilitated extended imaging of brain-state-dependent, millivolt-scale subthreshold voltage changes across deep-layer somas, fine dendritic structures, and diverse cell types. By addressing the critical gap in 2P optical recording of subthreshold voltage dynamics, JEDI3 indicators open new avenues for studying neural information processing and its alterations in health and disease.

neuroscience↗

bioRxiv2026

A versatile, positive-going voltage indicator that enables accessible two-photon recordings in vivo

Genetically encoded voltage indicators (GEVIs) enable cell-type-specific optical readout of membrane potential, but two-photon (2P) spike detection has been hampered by low signal-to-noise and ultrafast off-kinetics, restricting use to specialized microscopes. We introduce FORCE1s, a green, positive-going GEVI engineered to make robust 2P voltage imaging broadly accessible. FORCE1s brightens from a dark baseline during depolarization, reports spikes with [~]100% {Delta}F/F in awake mice, and displays repolarization kinetics that are tuned for reliable spike detection at sub-kilohertz frame rates. As a result, FORCE1s supports spike-resolved multi-cell recordings on standard resonant-scanning microscopes, and further scales to larger fields of view and neuron counts on advanced modalities. FORCE1s also enables multiplexed voltage-neurotransmitter imaging and extended recordings in freely moving mice using a compact, affordable MEMS-based 2P miniscope. Together, these advances establish FORCE1s as a community-ready tool that democratizes deep-tissue voltage imaging across platforms and experimental contexts.

neuroscience↗

bioRxiv2026

In vivo aberration measurement and correction for ultrafast FACED two-photon fluorescence microscopy of the brain

Ultrafast two-photon fluorescence microscopy (2PFM) based on free-space angular-chirp-enhanced delay (FACED) enables megahertz line scanning and kilohertz frame rates for in vivo brain imaging. However, optical aberrations from the imaging system and brain tissue degrade spatial resolution, signal, and contrast at depth. Here we integrate adaptive optics (AO) with FACED 2PFM to achieve synapse-resolving ultrafast imaging in the living mouse brain. Because FACED generates a one-dimensional array of temporally delayed, spatially separated excitation foci at 1 gigahertz, we developed a focus-averaging, frequency-multiplexed aberration measurement method that simultaneously measures and corrects the average aberration across all FACED foci using a segmented deformable mirror. We validated the accuracy of our method in correcting both system and artificial aberrations. When applied to in vivo morphological imaging of the mouse brain, AO enhances resolution, signal, contrast of dendritic shafts, spines, and boutons. Functionally, AO improves cerebral blood flow imaging by increasing plasma signal and kymograph contrast over large fields of view; when used for glutamate imaging, it amplifies transient amplitudes and reveals visually evoked glutamate release that were undetectable without correction. Together, these results establish AO-FACED 2PFM as a powerful approach that combines ultrafast imaging with high spatial resolution for structural and functional imaging in the living mouse brain.

neuroscience↗

bioRxiv2026

Convergence of Cortical and Thalamic Origins of Free Behavior Modulation of Mouse Primary Visual Cortex

The quality of sensory information flow in the brain may be affected by uninstructed free animal behavior. However, the specific circuit pathways that merge sensory and free behavior remain obscure. In mouse primary visual cortex (V1), we combine two-photon calcium imaging of cortical neurons and thalamo-cortical LGN (lateral geniculate nucleus) boutons with simultaneous measurements of facial movements. When controlling for spurious time-structured nonsense correlations, we observed representations of both eye movements and non-ocular facial movement features in both LGN bouton and V1 cortical neuron activity during grating input stimulation. Further, the correlation between V1 neurons and facial movement is larger for grating stimulation compared to blank stimulation, likely due to the integration of LGN inputs and modulation of V1 from higher brain centers during grating stimulation. Together, our results suggest a convergence of uninstructed non-visual signals from a persistent top-down pathway and a stimulus-gated bottom-up pathway in primary visual cortex.

neuroscience↗

bioRxiv2025

Multimodal Data Fusion Reveals Morpho-Genetic Variations in Human Cortical Neurons Associated with Tumor Infiltration

We introduce LetsACT (Light-Electron-Transcriptome synergistic ACTomography), a multimodal integration platform that overcomes the limitations of single-modality data acquisition and analysis of human brain cells while synergistically leveraging the strengths of each modality. Our approach enables the rapid sample preparation, cell injection, imaging, and multimodal integration of large-scale human neuronal datasets at single-cell resolution. By generating initial laser-scanning-microscopy based optical reconstruction of neuron morphologies followed by refining them using electron-microscopy derived morphological priors, we have assembled one of the largest human cortical morphology datasets to date: 8,398 neurons from 58 donors, with high cortical coverage. This platform is then applied to studying morphological impact of tumor infiltration. Pyramidal neurons in glioma-infiltrated tissues display clear volume shrinkage in somas and branches, tapering from the soma to nearby dendritic compartments. By integrating these morphological variations with spatial and bulk transcriptomic profiles, we find that glioblastoma tissues exhibit dysregulation of 15.29% of genes, including overexpression of TERT, whereas infiltrated tissues show 7.74% gene dysregulation, characterized by overexpression of tumor suppressors such as CDKN2A and TP53. Our analysis implies that pyramidal neurons observed in these infiltrated tissues may involve an active defense instead of undergoing passive apoptosis. Our finding also indicates that LetsACT establishes a valuable resource for the large-scale, comprehensive morpho-genetic analysis of human tissues.

neuroscience↗

bioRxiv2025

Retinal ganglion cell input to superior colliculus encodes salient information

The superior colliculus (SC) is critical in detecting and responding to salient stimuli. However, it is unknown whether salience signals are inherited from external sources such as retina and cortex, or arise from the intrinsic circuitry within SC. We used in vivo 2-photon calcium imaging with adaptive optics to examine the activity of retinal ganglion cell (RGC) axon terminals in superficial SC (sSC). We found that RGC boutons exhibit similar saliency-related activity as sSC neurons. A majority of boutons respond more strongly to salient discontinuities of visual features, and change their orientation preference depending on stimulus properties such as the orientations of edges of drifting gratings. We furthermore demonstrate that these effects arise from the center-surround receptive field structure of RGCs. Our results show that the orientation tuning of RGCs is highly flexible, and that saliency encoding originates in retina rather than sSC.

neuroscience↗

bioRxiv2025

Stimulation with ECoG electrodes modulates cortical activity and sensory processing in the awake mouse brain

Electrical stimulation has been widely used to probe neural network properties and treat dysfunction. Electrocorticography (ECoG) electrodes, long used for activity monitoring, can also stimulate the brain in a minimally invasive and chronic manner. However, how cortical surface electrical stimulation impacts cortical network activity remains poorly understood. Using in vivo calcium imaging in the awake mouse brain with chronically implanted ECoG electrodes, we measured how electrical stimulation modulates the activity of visual cortical neurons, including during concurrent visual stimulation. We found that cortical surface electrical stimulation initially activates L2/3 neurons followed by prolonged inhibition lasting seconds after stimulation. Electrical stimulation suppresses the activity of neurons at their preferred grating orientation but enhances their responses to non-preferred visual stimuli, thereby reducing sensory feature selectivity. By measuring how electrical stimulation modulates the activity of inhibitory neuron subtypes including PV, SST, and NDNF interneurons, we propose a circuit model in which L1 NDNF interneurons are strongly activated by cortical electrical stimulation and, in turn, inhibit L2/3 excitatory neurons and PV interneurons through volume transmission of GABA.

neuroscience↗

bioRxiv2025

Parallel frequency-multiplexed aberration measurement for widefield fluorescence microscopy

Widefield fluorescence microscopy is widely used for imaging at subcellular resolution, but its performance in complex samples is degraded by optical aberrations. Because aberrations can vary spatially across the field of view (FOV), accurate aberration measurement and correction at multiple FOV locations are essential for achieving high-quality imaging over large areas. Here, we introduce parallel frequency-multiplexed aberration measurement (PFAM) to perform massively parallel aberration measurements across an extended FOV. We validated PFAM using fluorescent beads and demonstrated simultaneous measurement and effective correction of spatially varying aberrations at 125 FOV locations. To address the challenges of wavefront sensing in complex samples, we further developed PFAM-SIFT by integrating structured illumination, thereby achieving robust aberration measurement in both brain slices and the mouse brain in vivo. Together, PFAM and PFAM-SIFT provide accurate and scalable wavefront sensing solutions for widefield imaging, enabling simultaneous aberration measurement of spatially varying aberrations in complex biological samples.

biophysics↗

bioRxiv2025

Structure and Function of Lens Suture Examined by 2-photon Fluorescence Microscopic Imaging

We have applied 2-photon fluorescence microscopy (2PFM) to investigate the structures and functions of mouse eye lenses, especially lens sutures, using wildtype (WT) and KLPH-KO suture-cataract lenses in vivo. Dynamic structures of lens sutures have been hypothesized to act as pathways carrying fluid containing ions, nutrients, and other factors as part of microcirculation for maintaining lens homeostasis and transparency. 3D imaging results show both typical "Y" and "double Y" shaped suture structures in WT lenses, diverse suture patterns including "star" shapes with variations appearing at different depths, and misaligned suture planes in KLPH-KO lenses. The variability in suture patterns, quantified using the mean of image stacks similarity indices (SSIM) to the mean projection of the stack, reveals a significant increase (p<0.05) in pattern randomization for KLPH-KO lenses. Both WT and KLPH-KO lenses exhibit voids around sutures and enlarged vacuoles throughout the lens structure in vivo. Pathological features like irregular and larger central voids containing 2~5-{micro}m-diameter amorphous structures that might result from membrane protein remnants/aggregates in the KLPH-KO mouse eye lens were observed. In conclusion, this work provides new morphological markers for characterizing suture cataract and fiber pathological features. Lens sutures are stabilized structures that are impermeable to dyes, and the extracellular spaces of lens peripheral fibers provide efficient diffusion pathways for lens microcirculation.

biophysics↗

bioRxiv2025

Suite3D: Volumetric cell detection for two-photon microscopy

In two-photon imaging of neuronal activity it is increasingly common to acquire 3-dimensional volumes. However, these volumes are typically processed plane by plane, leading to uncorrected axial movement, duplicated cells across planes, reduced signal-to-noise ratio per cell, and missed cells. To overcome these limitations, we introduce Suite3D, a volumetric cell detection pipeline. Suite3D corrects for rigid and non-rigid 3D brain motion, both lateral and axial. It detects neurons using 3D correlation, improving detectability. Finally, it performs 3D segmentation, identifying cells across imaging planes. We validated Suite3D with data from conventional multi-plane microscopes and advanced volumetric microscopes, at multiple resolutions and in multiple brain regions, and with ground truth anatomical labelling. Suite3D successfully detected cells appearing on multiple imaging planes, improving cell detectability and signal quality, avoiding duplications, and running faster than a prior volumetric pipeline. Suite3D offers a powerful solution for analyzing volumetric two-photon data.

neuroscience↗

bioRxiv2025

FACED 2.0 enables large-scale voltage and calcium imaging in vivo

Monitoring neuronal activity at large scale and high spatiotemporal resolution is crucial for understanding information processing within the brain. We optimized a kilohertz-frame-rate two-photon fluorescence microscope with all-optical megahertz line-scan rate to achieve ultrafast imaging across large areas and volumes at subcellular resolution. Applying this technique to voltage and calcium imaging in vivo, we demonstrated simultaneous recording of voltage activity over 200 neurons and calcium activity over 14,000 neurons.

neuroscience↗

bioRxiv2025

Circuit-Based Understanding of Fine Spatial Scale Clustering of Orientation Tuning in Mouse Visual Cortex

In sensory cortex of brain it is often the case that neurons are spatially organized by their functional properties. A hallmark of primary visual cortex (V1) in higher mammals is a columnar functional map, where neurons tuned to different stimuli features are regularly organized in space. However, rodent visual cortex is at odds with this rule and lacks any spatially ordered functional architecture, and rather neuron feature preference is haphazardly organized in patterns termed salt-and-pepper. This sharp contrast in feature organization between the visual cortices of rodents and higher mammals has been a persistent mystery, fueled in part by abundant evidence of conserved cortical physiology between species. In this work, we applied a novel GCaMP indicator that are localized in the nucleus of neurons during two-photon imaging in mouse V1, which enabled us to overcome most spurious spatially correlated activity due to fluorescence contamination, and to ensure a faithful observation of functional organization over space. We found that the orientation tuning properties of distant neuron pairs (> 20 {micro}m) are irregularly and randomly organized, while neuron pairs that are extremely close (< 20 {micro}m) have strongly correlated orientation tuning, indicating a narrow yet strong spatially clustered organization of orientation preference, which we term micro-clustered organization. Exploring a circuit-based model of recurrently coupled mouse V1 we derived two key predictions for the micro-cluster: spatially localized recurrent connections over a comparable narrow spatial scale, and common relative spatial spreads of balanced excitation and inhibition in the network over broad spatial scales. These predictions are validated by both anatomical and optogenetic-based physiological circuit mapping experiments. Altogether, our work takes an important step in building a circuit-based theory of visual processing in mouse V1 over spatial scales that are often ignored, yet contain powerful synaptic interactions.

neuroscience↗

bioRxiv2024

Adaptive optical correction in in vivo two-photon fluorescence microscopy with neural fields

Adaptive optics (AO) restore ideal imaging performance in complex samples by measuring and correcting optical aberrations, but often require custom-built microscopes with carefully aligned wavefront sensing/shaping devices and can be susceptible to sample motion. Here we describe NeAT, a computational framework using neural fields for AO two-photon fluorescence microscopy. NeAT estimates wavefront aberration and recovers sample structure from a 3D image stack without requiring external datasets for training. Incorporating motion correction in learning and correcting conjugation errors commonly found in commercial microscopes, NeAT is designed for deployment in biological laboratories for in vivo imaging. We validate NeATs performance using a custom-built microscope with a wavefront sensor under varying signal-to-noise ratios, aberration, and motion conditions. With a commercial microscope, we demonstrate real-time aberration correction for in vivo morphological and functional imaging in the living mouse brain, with NeAT improving signal and accuracy of glutamate and calcium imaging of synapses and neurons.

neuroscience↗