Nano Letters

Scanning ion conductance microscopy (SICM) provides gentle, non-contact cell surface imaging, but it has not been used to investigate intracellular structures because the plasma membrane restricts nanopipette access. Here, we combined SICM with microsonication-based cell de-roofing to expose intracellular actin stress fibers (SFs) in U2OS cells for nanotopographical and -mechanical characterization. Importantly, the de-roofing conditions preserved actomyosin contractility, allowing analysis of SF structural and biomechanical changes during ATP-induced contraction. Resting SFs displayed an average height of 203{+/-}38 nm and width of 357{+/-}73 nm, and a complex surface architecture characterized by regularly spaced long-range height modulations (~500 nm periodicity; Wq ~25 nm) and smaller irregular corrugations (Ra ~19.2 nm). ATP stimulation reduced SF height and width by ~39% and ~15%, respectively, while largely preserving surface corrugation patterns. During contraction, some SFs separated into two longitudinal strands. High-resolution SICM imaging also revealed filamentous crosslinks mechanically coupling neighboring SFs, and nanomechanical measurements demonstrated local stiffening during contraction. These findings provide new insight into the structural and mechanical regulation of SF contraction and highlight the potential of SICM combined with cell de-roofing as a powerful platform for studying dynamic intracellular processes at nanometer resolution.

The protein corona influences the in vivo biodistribution of ionizable lipid nanoparticles (LNPs) in nucleic acid delivery, yet its structural architecture remains poorly defined. Using cryo-transmission electron microscopy, we visualized LNP-protein interactions in their native state. We show that, unlike the discrete "fuzzy" shells observed on hard nanoparticles, LNPs displayed no peripheral protein shell. Instead, controlled incubation and competitive "dual-particle" assays, supported by molecular dynamics simulations, indicate that LNP membranes undergo localized thickening and electron-dense remodeling consistent with lipoprotein integration rather than surface adsorption. Similar features were observed in extracellular vesicles, suggesting this behavior is shared among lipid-based carriers, and proteomic analysis identified apolipoproteins as the dominant associated proteins. Together, these findings support a model in which the biological identity of LNPs arises through membrane remodeling rather than shell-like adsorption, and provide a framework for the rational design of targeted nanomedicines. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/695162v2_ufig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@5275d3org.highwire.dtl.DTLVardef@1b59ae4org.highwire.dtl.DTLVardef@1cc290eorg.highwire.dtl.DTLVardef@9b7bfb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Direct validation of the nanoscale structural organization of membrane proteins requires localization precision that matches their molecular dimensions. The sodium-potassium pump, or the Na,K-ATPase is an integral membrane protein responsible for maintaining electrochemical gradients and cellular energy homeostasis. Although its crystal structure is characterized, the organization of the Na,K-ATPase within native plasma membranes, particularly whether it forms functional oligomers, remains an open question. Here, we combined 3D MINFLUX nanoscopy with DNA-PAINT with sub-10 nm localization precision to map the clustering topology of the Na,K-ATPase in mammalian cells. By targeting EGFP-tagged Na,K-ATPase 1 and {beta}1 subunits using anti-GFP nanobodies, we obtained high-density 3D localization maps of the protein in the plasma membrane. To evaluate the point patterns, we developed a computational data-driven spatial point assignment approach that segments apical and basal localizations, mitigating clustering artifacts produced by imaging two membranes in close proximity. Furthermore, we used a spatial statistical approach analyzing sequential nearest-neighbour distances to elucidate supramolecular arrangement information. Our data reveal a preferential nearest-neighbour distance of approximately 7 nm, providing direct visual confirmation of Na,K-ATPase dimerization. Additionally, we identified higher-order nanoclusters composed of up to 21 proteins. These findings provide definitive structural evidence of the dimeric configuration of Na,K-ATPase, establishing a foundation for future research on the functional and regulatory implications of Na,K-ATPase clustering.

Spatial organization and temporal regulation of membrane components are essential for achieving complex functions in artificial cells, such as cell division and signalling. DNA-based molecular tools provide a powerful means to control biomolecular interactions with high precision. Here, we investigate the phase behavior of cholesterol-modified, star-shaped DNA nanomotifs anchored to the lipid bilayers of giant unilamellar vesicles (GUVs), by using fluorescence confocal microscopy and cryo-electron microscopy. These motifs spontaneously anchor to the lipid bilayers via hydrophobic interactions and exhibit distinct spatial organization depending on their sticky end sequences. Motifs with complementary sticky end sequences interact and distribute uniformly, while orthogonal motifs with different sticky end sequences segregate into isolated gel-like domains with limited lateral mobility. Notably, the phase separation of motifs does not require lipid phase separation, indicating that DNA-driven organization can take place independently of lipid phase separation. The behavior of this system is governed by the interplay of three key parameters: (i) hydrophobic anchoring via cholesterol, (ii) electrostatic repulsion between negatively charged DNA nanomotifs, and (iii) sticky end interactions. The observed two-dimensional phase separation of orthogonal DNA nanomotifs at the GUV interface presents a novel strategy for controlling lateral membrane organization in GUV systems. This approach would offer flexibility in membrane composition and enables molecular positioning, thereby achieving a high degree of organization on the surface in artificial cell models.

Single-particle tracking (SPT) over time enables direct observation of molecular transport and interactions in living cells. Fluorescence-based SPT has provided insights into intracellular processes such as endocytosis, receptor signaling, and drug delivery. Extending the observation window to several hours and beyond is critical for capturing slow intracellular dynamics, including the full course of endosomal trafficking, the long-term accumulation of particles within subcellular compartments, and transitions between transport modes that occur on hour-scale timescales. However, long-term intracellular SPT under visible-wavelength excitation remains challenging because fluorescence probes generally suffer from photobleaching and phototoxicity. While near-infrared (NIR) excitation can simultaneously mitigate these issues, generally weak emission of NIR-emitting dyes has hindered its wide application in long-term SPT. Here, we demonstrate long-term NIR SPT using atomically precise gold quantum needles, Au42(PET)32 (PET = 2-phenylethanethiolate). Continuous tracking of intracellular particles in living HEK293 cells was achieved for up to 12 h. Trajectory analysis revealed temporal transitions between directional and diffusive transport, as well as the accumulation of multiple particles within localized intracellular domains over several-hour timescales. The high photostability of Au42, combined with low phototoxicity of NIR excitation, enables visualization of intracellular transport dynamics over timescales difficult to access using conventional visible fluorescent probes. These results establish Au42-based NIR imaging as a platform for long-term, low-phototoxicity intracellular SPT and provide a framework for investigating slow intracellular dynamics in living systems.

Adoptive cell therapy (ACT) remains challenging in solid tumors, where poor tumor infiltration, metabolic heterogeneity, and an immunosuppressive tumor microenvironment (TME) constrain therapeutic efficacy. Here, we developed a magnetically actuated macrophage-based immune microrobot (immunobot) for active solid tumor immunotherapy. Immunobots were constructed by loading bone marrow-derived macrophages (BMDMs) with lipopolysaccharide-modified Janus L1-FePt magnetic microrollers (LMRs), enabling hard-magnetic actuation. Optimized LMR loading supported robust propulsion, retention under flow, and enhanced barrier penetration. LMRs further promoted M1-like polarization through LPS-driven inflammatory activation and FePt-derived labile iron-amplified oxidative stress, with altered iron homeostasis, increased reactive oxygen species (ROS), and enhanced NF-{kappa}B signaling. Immunobots also induced ferroptosis-associated immunogenic cell death in tumor cells. In vivo, magnetically guided immunobots suppressed tumor growth, reprogrammed tumor-associated macrophages (TAMs), promoted dendritic cell maturation, and enhanced CD8 T cell activation. This work establishes a microrobotic immunotherapy platform for active magnetic transport, iron-metabolic regulation, and immune remodeling in solid tumors.

Biohybrid robots combining compliant synthetic support structures with biological actuators could enable future applications ranging from precision microsurgery to unmanned exploration. Machines actuated by living skeletal muscles are capable of adaptive behaviors, such as sensing and responding to environmental stimuli in real-time, offering functional advantages over non-biological actuators. However, typical skeletal muscle-powered biohybrid robots depend on 3D tissues which require large cell volumes and offer limited control of muscle fiber alignment, thus reducing efficiency of force generation and transduction. Here, we present a locomotive biohybrid robot powered by 2D monolayers, or thin films, of precisely aligned skeletal muscle fibers on a micropatterned hydrogel skeleton. We demonstrate how varying skeleton design parameters, ranging from material stiffness to microscale topology, impacts muscle fiber alignment and resultant actuation strains, generating forces 10X higher than previous 2D skeletal muscle actuators, improving untethered actuation longevity by [~]4500X from < 10 minutes to > 30 days, and increasing efficiency of muscle force output (force per unit volume of muscle) by 20X as compared to 3D muscles. Utilizing our optimized design for skeletal muscle thin films, we create a multi-limbed robot composed of independent muscle-powered fins capable of on/off control and frequency-dependent speed control. With these control inputs, we achieve steered multi-directional locomotion at speeds up to 4 body lengths per minute in straight movement and 1200 degrees per minute in rotational movement, highlighting potential for such actuators to be transformed into long-lasting functional soft robots.

Biomolecular condensates are important regulators of cellular compartmentalization and biochemical processes. Understanding their material properties is critical to elucidate how they control molecular organization and dynamics within cells. However, quantitatively probing these properties remains challenging due to the wide range of length scales, concentrations, and timescales over which condensates operate, as well as the limited force ranges accessible to current nanoscale mechanical mapping methods. We explored the use of a non-contact 3D imaging tool Scanning Ion Conductance Microscopy (SICM) for stiffness measurements of liquid-liquid phase-separated biomolecular condensates. We focus on the Dhh1 protein, which is a regulator of cytoplasmic processing bodies (PBs) membrane-less cytoplasmic condensates that control the storage and degradation of untranslated mRNA. In our study, we investigate the properties of mCherry2- or His-mCherry2- tagged full-length Dhh1 and N- or C-terminus tail-deletion constructs, as well as the catalytically inactive mutant DQAD, under different pH and incubation times. We mapped both spatial and temporal changes in the material properties of the condensates, highlighting the capabilities of the instrument. We found that the removal of either of the two tails led to an increase in condensate stiffness upon shifting the pH from a stress-associated cellular environment (pH 6.5) to physiological conditions (pH 7.5). Additionally, the choice of protein tags led to vastly different results depending on the pH where mCherry2-Dhh1 exhibited a stiffening going from pH 6.0 to 6.5 while the double-tagged His-mCherry2 did not. Our measurements are verified and corroborated by established techniques such as optical tweezer-based fusion assays and fluorescence recovery after photobleaching (FRAP). Furthermore, we were able to track the same biomolecular condensate sample for up to 20 days getting insights on the ageing and evolution of the condensates. Overall, our study demonstrates the applicability of SICM for direct measurement of the material properties of biomolecular condensate.

Microtubules are central components of cytoskeletal transport systems and have been widely repurposed as active elements in motor-driven nanodevices. However, site-specific functionalization of stabilized microtubules remains a fundamental challenge, as the tubulin lattice presents chemically indistinguishable binding sites along its length. Here we report a strategy for selective end-functionalization of stabilized microtubules using DNA origami nanostructures. By coupling DNA origami to Fab fragments targeting acetylated -tubulin Lys40 within the microtubule lumen, and exploiting steric exclusion of the origami from the lattice interior, binding is confined to accessible sites at microtubule ends and lattice defects. Using a six-helix bundle origami as a minimal construct, we demonstrate selective tip labelling of gliding microtubules without perturbing kinesin-driven motility. The same structures additionally mark lattice defects, enabling dynamic visualization of defect sites during transport. Furthermore, we show that tip-bound origami can hybridize with complementary DNA strands to capture cargo from surfaces in motion, establishing programmable, end-specific loading. This approach introduces a generalizable route to spatially controlled functionalization of cytoskeletal filaments, enabling new capabilities in molecular transport, nanoscale assembly, and the study of microtubule integrity and repair.

Cerebrovascular inflammation is a critical driver of secondary injury following traumatic brain injury (TBI), yet no noninvasive tool currently exists to map its spatial heterogeneity across the injured brain. Here, we report MoNP-SPION, a monocyte-mimetic nanoprobe that selectively targets activated cerebrovascular endothelium after TBI and enables quantitative MRI of spatiotemporally heterogeneous cerebrovascular inflammation. MoNP-SPION binding to inflamed brain endothelium scaled with activation state, enabling rapid, spatially heterogeneous targeting of injured cerebral vasculature. MoNP-SPION-enhanced MRI revealed dynamic reductions in T2* throughout the brain that extended beyond the injury penumbra and captured both subacute vascular injury and therapy-induced recovery. Importantly, spatiotemporal reductions in T2* strongly correlated with VCAM1 upregulation, supporting MoNP-SPION as a molecularly specific readout of endothelial activation. Together, these findings establish MoNP-SPION as a promising platform for longitudinal, noninvasive monitoring of cerebrovascular inflammation in TBI and other neurological disorders involving vascular dysfunction. TEASERCell-mimicking MRI nanoprobe maps cerebrovascular injury and recovery dynamics following brain trauma.

DNA-programmed assembly of plasmonic nanostructures provides a powerful route to couple molecular recognition with optical signal generation. Here, we report the sequence-specific assembly of DNA-functionalized gold nanorods using a sesame allergen-derived DNA biomarker as a molecular bridge. Target-induced assembly produces concentration-dependent assembly growth, plasmon coupling, and distinct assembly kinetics that are readily monitored by absorption spectroscopy, enabling label-free detection of the target DNA in the nanomolar concentration range. The assembled nanorods further produce strong surface-enhanced Raman scattering (SERS) signals arising from plasmonic coupling within the assemblies, extending detection to the picomolar regime without the use of Raman reporters. Quantitative analysis reveals that both the extent and rate of assembly formation are governed by target DNA concentration. These results establish a direct relationship between molecular recognition, assembly growth, plasmonic coupling, and spectroscopic response, highlighting DNA-programmed gold nanorod assembly as a versatile platform for investigating hybridization-driven plasmonic self-assembly and nucleic acid detection. Table of Content O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/732610v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@6a8f42org.highwire.dtl.DTLVardef@1e36b9corg.highwire.dtl.DTLVardef@1ade546org.highwire.dtl.DTLVardef@1a787bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biological energy conversion relies on highly efficient electron transfer. The chirality induced spin selectivity (CISS) effect, which couples electron spin to momentum in chiral molecules, is hypothesized to promote this efficiency. While observed in isolated biomolecules, the physiological relevance of CISS during active cellular metabolism remains unknown. Here, we demonstrate that CISS influences extracellular electron transfer in living Geobacter sulfurreducens biofilms. Cultivation on ferromagnetic electrodes yields a significant asymmetry in respiratory current between opposite substrate spin states. Furthermore, in situ magnetization reversal induces reversible changes in respiratory flux. These results provide the first in vivo demonstration that spin selectivity directly impacts respiration. By revealing a quantum feature of extracellular respiration, our findings offer a strategy to exploit the spin degree of freedom in bioelectronics.

Transport in complex biological tissues is governed by local rheological heterogeneity at the nanoscale, yet probing such environments deep inside living systems remains challenging. Here, we introduce an orientation-sensitive single-particle tracking (SPoT) approach that simultaneously resolves translational and rotational dynamics of individual carbon nanotubes deep within biological tissue. By exploiting the intrinsic dipole-like emission and shortwave infrared luminescence of carbon nanotubes enhanced through the incorporation of quantum color-centers our method enables long-duration tracking with high signal-to-noise ratio in optically dense environments. Crucially, the length of these nanotubes can be precisely shortened down to a few tens of nanometers to adapt to diffusion environmental dimensions, further optimizing the tracking applicability. SPoT of single carbon nanotubes provides access to relative changes in local viscosity, steric constraints, and environmental anisotropy. When applied to the brain extracellular space, SPoT demonstrates that local variations in the translational and rotational diffusion of tracers are heterogeneous and not systematically correlated. This allows to disentangle the local effects of viscosity and spatial tortuosity within the brain extracellular space, which are distinct features that would otherwise remain undetected through translational diffusion analysis alone. By enabling combined translational and rotational tracking of nano-emitters over unprecedented depths and timescales, this work establishes a new framework for probing nanoscale transport and rheological heterogeneity in intact biological tissues and more generally in complex diffusive environments.

Tracking immune cells deep within living tissue remains a fundamental challenge due to the diffraction-limited resolution of ultrasound imaging and the inability to resolve dense cellular populations. Here, we introduce an intracellular super-resolution ultrasound imaging framework based on stochastic phase-changing nanodroplets (NDs) and ultrasound localization microscopy (ULM). We engineer [~]170 nm perfluorocarbon NDs that undergo reversible, stochastic liquid-gas transitions under acoustic excitation, generating temporally sparse "blinking" signals. Leveraging the intrinsic endocytic activity of macrophages, these NDs are internalized, enabling intracellular contrast generation independent of vascular flow. We validate this approach across imaging scales, from controlled phantoms and in vitro systems to in vivo tumor models, demonstrating robust intracellular blinking, high cell viability, and consistent super-resolution reconstruction in dense cellular environments. The stochastic blinking of internalized NDs provides the temporal separation required to localize individual sources, overcoming a central limitation of conventional ULM. Following systemic administration, ND-labeled macrophages are tracked in vivo after homing to the liver, where super-resolution ULM resolves cellular distributions with a spatial resolution of 26.3 {+/-} 3.2 {micro}m, corresponding to a 6.1-fold improvement over diffraction-limited imaging. This work establishes a previously unexplored paradigm for ultrasound-based intracellular super-resolution imaging, enabling non-invasive visualization of immune cell organization in deep tissue. By introducing spatiotemporally programmable intracellular contrast, this approach expands ultrasound beyond vascular imaging toward functional cellular imaging, with broad implications for immunology, diagnostics, and cell-based therapies.

Surface-enhanced Raman scattering (SERS) nanotags provide highly sensitive platforms for in vitro diagnostics but often require complex, disease-specific customization that limits clinical translation. Biomineralization, in which biomolecules mediate inorganic material synthesis, offers a versatile yet underexplored strategy for generating functional SERS nanotags. Here, we demonstrate that biomolecule-directed biomineralization of gold nanoparticles (AuNPs) using amino acids and exosomes generates distinct nano-bio interfacial architectures that encode biomolecular identity into machine learning-resolvable SERS fingerprints through modulation of plasmonic coupling and Raman reporter organization. As a proof-of-concept system, amino acid-biomineralized AuNPs were synthesized using biomolecules with diverse physicochemical properties, including differences in size, polarity, and charge. The resulting nanotags were characterized using UV-Vis spectroscopy, SERS, fluorescence spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Random forest and support vector machine (SVM) models successfully differentiated amino acid-dependent SERS signatures with near-perfect classification performance. Extending this approach to a biologically complex preclinical cancer model, exosome-biomineralized AuNP nanotags were generated using exosomes derived from clinically relevant pediatric patient-derived osteosarcoma and neuroblastoma tumors. Distinct exosome-dependent spectral fingerprints enabled SVM classification with 93.9% accuracy, while Shapley Additive exPlanations (SHAP) and t-distributed stochastic neighbor embedding (t-SNE) analyses identified diagnostically relevant spectral regions and visualized clustering between tumor classes. Collectively, this work establishes biomineralization as a strategy for transforming complex biomolecular and cellular information into computationally resolvable optical fingerprints, enabling scalable and label-free diagnostic classification of patient-derived biomolecular samples.

Transmembrane signaling mediated by cytokine receptors orchestrates key cellular processes such as proliferation, differentiation, and immune responses. While numerous high-resolution structures of cytokine receptor ectodomains are available, the structural organization of the largely disordered intracellular domain (ICD) has remained unclear. Here, we interrogate the axial organization of cytokine receptor signaling complexes at the plasma membrane by metal-induced energy transfer (MIET). For this purpose, we leveraged biofunctionalized nanodot arrays (bNDAs) to capture cell surface receptors at a defined distance from the substrate. Readout by fluorescence lifetime imaging microscopy enabled quantifying axial distances of proteins in the plasma membrane of cells at both ensemble and single-molecule levels with a resolution of [~]1 nm. Using the prototypic, biomedically relevant class I cytokine receptor GP130 as a model system, we uncover by MIET that the ICD extends randomly into the cytosol in the resting state, but surprisingly undergoes an axial compaction upon signal activation. These results demonstrate the potential of bNDA-supported MIET for resolving the axial architecture of signaling complexes within the cellular context.

The accurate quantification of biological nanoparticles, such as small extracellular vesicles (sEVs), is fundamentally hindered by a resolution-coincidence trade-off in digital assays. While physical confinement can isolate single particles, conventional optical readouts remain diffraction-limited, causing multi-particle occupancy to be miscounted as single events and thereby restricting the analytical dynamic range. Here, we report a nanoplasmonic platform that overcomes this limit by introducing a geometry-defined interface that uniquely unifies nanoscale compartmentalisation and near-field-assisted super-resolution imaging. Utilising a gold plasmonic nanohole array, the strict geometric periodicity of the lattice simultaneously serves as a template for single-vesicle confinement and a deterministic grid that generates an array of localised surface plasmon resonance near-field hotspots. This position-deterministic illumination pattern imposes known geometric priors on the excitation field, shifting high-spatial-frequency information into the detectable bandwidth to achieve sub-100 nm lateral resolution. This dual-purpose geometric determinism enables high-fidelity digital readout of individual vesicles with significantly fewer sub-images than stochastic, speckle-based metasurface structured illumination microscopy approaches. The assay achieves an analytical limit of detection of 143 sEVs/{micro}L, matching the performance of state-of-the-art single-EV counting technologies. It successfully differentiates distinct sEV subpopulations based on surface biomarker expression, establishing a clear pathway for future clinical liquid biopsy applications. By replacing stochastic loading and illumination with geometric design, this work establishes a robust framework for precise vesicle interrogation with broad implications for emerging translational applications and fundamental biology.

Intratumoral (i.t.) delivery of nanoparticles (NPs) is widely used to achieve high local NP concentrations. However, the temporal fate of i.t.-injected NPs remains poorly understood. We present a quantitative approach using whole-body magnetic particle imaging (MPI) to track magnetic NPs (MNPs) following i.t. injection. Using fiducial-calibrated imaging, we quantified MNP mass over time in subcutaneous 4T1 breast tumors. Longitudinal imaging revealed progressive loss of i.t. MNP content and heterogeneous systemic redistribution across animals despite standardized delivery conditions. Ex vivo MPI confirmed off-target accumulation primarily in the liver and spleen, consistent with reticuloendothelial clearance pathways. Histological analysis demonstrated spatially heterogeneous i.t. MNP deposition, potentially associated with local vascular features and tumor microenvironmental heterogeneity that may influence i.t. MNP retention or MNP clearance from the tumor. These findings highlight the importance of quantitative longitudinal whole-body MPI for understanding the fate of MNPs for informing localized nanotherapy.

Many biological macromolecules are activated upon ligand binding at multiple specific binding domains. However, how these domains interact and the transient intermediate conformations that connect binding events to protein activity are typically unknown. Ensemble-averaged measures over stochastic binding events are challenged to resolve the underlying asynchronous dynamics. Single-molecule resolution of these dynamics offers an attractive approach to investigate the ligand-activation process. Optical methods using fluorescently labeled ligands enable observation of individual binding events that report on the energetics of early ligand-bound conformational changes. However, diffraction-limited microscopy limits these methods to low ligand concentrations, often below what is required for physiologically relevant activation. Here, we overcome this limitation using nanophotonic zero-mode waveguides to observe the sequential binding of a fluorescent cyclic nucleotide to each of four subunits in TAX-4 cyclic nucleotide-gated ion channels in cell-derived vesicles. Our observations suggest that binding at one domain positively promotes binding at other domains, and that binding induces an isomerization of the binding domain in individual subunits which we attribute to a sequence of pre-activated intermediate states. This approach provides a broadly applicable tool to dissect the energetic landscape of ligand-binding at macromolecules in native cell membranes.

Light scattering in scintillators is a pervasive problem and a key factor limiting X-ray imaging resolution. Here, we shift scintillator radioluminescence from the traditional visible range into the short-wave infrared (SWIR) or near-infrared II (NIR-II, 1000-3000 nm) window to mitigate light scattering and thereby enhance light penetration and X-ray imaging resolution. We present an NIR II MgGa2O4:Ni2+ scintillator with peak emission at 1340 nm, achieving a threefold improvement in X-ray imaging resolution compared with visible scintillators owing to reduced light scattering. This heavy-metal-free NIR-II scintillator exhibits intense radioluminescence comparable to that of conventional visible-emitting CsI:Tl, achieving a detection limit of 56 nanograys per second, ~100-fold lower than typical doses used in medical imaging. We show that this NIR-II scintillator enables high-resolution X-ray radiography of electronic circuit boards and biological tissues.