Bruce E. Cohen

Accessing the rich information carried by infrared light typically relies on bulky, complex optoelectronic systems. Lanthanide-based upconverting nanoparticles (UCNPs) offer a compelling alternative by converting infrared light into visible photons through nonlinear anti-Stokes processes. However, achieving strong upconversion under the low excitation intensities relevant to infrared vision remains challenging, motivating strategies to enhance light–matter interaction. Here, we demonstrate enhanced infrared-to-visible upconversion imaging enabled by integrating alloyed Yb/Er UCNPs with a resonant dielectric metasurface. The metasurface supports an optical resonance aligned with the UCNP excitation band, leading to over three orders of magnitude enhancement in upconversion emission. Crucially, flat-band angular dispersion of this resonance enables uniform enhancement across incident angles relevant to imaging, thereby preserving spatial frequency content and yielding sharp, high-contrast images. In light of ongoing advances in lanthanide-based materials, this metasurface–UCNP hybrid screen provides a promising platform for compact, detector-free, and scalable infrared imaging technologies based on optical upconversion.

Altered extracellular proteolysis has been exploited to selectively activate therapeutics in diseases such as cancer; however, once activated, extracellular drugs can diffuse away, limiting efficacy. We address this challenge by coupling proteolytic activation with membrane tethering to retain drugs within diseased tissue. To accomplish this, we developed “restricted interaction peptides” (RIPs), a delivery platform that leverages elevated proteolytic activity to activate membrane-interacting peptides, localizing cargos near the site of proteolysis. We demonstrate that RIPs can deliver diverse therapeutic cargos, including cytotoxins and radioisotopes. As proof of concept, we engineered “FRIP,” a RIP designed for cleavage by fibroblast activation protein (FAP), an endoprotease upregulated in solid tumors and fibrosis. Efficient P4–P4’ substrate sequences were identified and incorporated into FRIPs. Cell-based studies showed that, upon activation, the peptide adhered to membranes rapidly internalized and successfully delivered therapeutic cargos. Consistent with this, FRIPs delivering MMAE inhibited proliferation in an FAP-dependent manner. Imaging studies confirmed tumor targeting with minimal uptake in normal tissues. Finally, FRIPs delivering MMAE or Cu-67 exhibited potent antitumor effects. These findings establish membrane tethering as a strategy to enhance drug retention.

Electron microscopy (EM) is fundamental to nanocrystal characterization, but some structures degrade quickly under electron beams, limiting advanced structural characterization. Here, we introduce a synthetic strategy that combines layer-by-layer shell growth with in situ annealing to produce NaYb_0.8Er_0.2F₄ alloyed upconverting nanoparticles (UCNPs) with enhanced structural stability and optical properties. Using an automated synthesis platform to control precursor delivery and annealing cycles, high rare-earth ion concentrations are maintained during shell growth and annealing at high temperature, reducing luminescence quenching and degradation under electron beams. This in situ annealing layer-by-layer (ISA-LBL) approach gives rise to alloyed UCNPs (aUCNPs) with exceptional electron-beam stability, reducing beam-induced fractures and voids by >90% compared to conventionally synthesized aUCNPs. ISA-LBL aUCNPs also exhibit enhanced photoluminescence intensity and extended lifetimes, consistent with fewer quenching defects. This demonstrates a synthetic route to nanocrystals with enhanced structural integrity, increasing their compatibility with EM studies and their utility in ionizing environments.

Avalanches within nanoparticles seem like science fiction, but if they are avalanches of photons, they open up real-world innovations in imaging, sensing, optical computing, and other unexplored light-driven technologies. Avalanches are outsized events arising from the integration of many smaller inputs, and photon avalanching (PA) was first reported in bulk crystals in 1979 as an unexpectedly large jump in luminescence as excitation intensity was slowly increased. It would be 41 years before PA would be observed at the nanoscale in photon avalanching nanoparticles (ANPs), Tm³⁺-doped upconverting nanoparticles that show excited-to-ground state absorption inversion greater than 10,000:1 and emission that scales nonlinearly up to the 32nd power of the pump intensity. This extreme nonlinearity enables a real-time 5-fold improvement in the 150-year-old Abbe limit of spatial resolution, achieving 70 nm resolution using only simple scanning confocal microscopy. This extreme nonlinearity also gives rise to a series of highly unusual optical and sensing properties. Tm³⁺ ANPs show NIR-controlled bidirectional photoswitching, lasting over 1000 cycles in ambient or aqueous conditions with no measurable sign of photodegradation. This enables 2- and 3-dimensional optical nanoscale patterning with full erase and rewrite capabilities. Unlimited photoswitching also underlies the super-resolution technique INPALM, which is capable of sub-Ångstrom localization precision and resolving individual ANPs within tightly packed clusters. Nd³⁺-based ANPs show the peculiar property of intrinsic optical bistability (IOB), a form of memory in which emission depends on whether the ANPs have previously undergone PA. This stable, history-dependent contrast makes these ANPs analogous to optical transistors and promising materials for optical computing, neuromorphic circuitry, and related photonic technologies. The steep nonlinearity of PA also makes ANPs exceptional sensors of external perturbations, as tiny environmental changes may be amplified into large changes in optical output. As force sensors, Tm³⁺ ANPs are able to detect forces over a dynamic range of 4 orders of magnitude, from piconewtons to micronewtons, a range that will enable force sensing in complex systems across scales. Application of current ANP designs to imaging and devices, discovery of new PA-associated phenomena, and design of new ANPs with unique properties are all underway as the novelty of this technology cascades toward new fundamental discoveries and applications.

The formation of stable protein complexes enables much of biotechnology, but even high-affinity complexes can dissociate, limiting potential applications in biomaterials, bioimaging, nanomedicine, and other protein-based technologies. Here, we describe lock coupling, a simple and selective one-step reaction between interfacial lysine and glutamate or aspartate side chains to form stable isopeptide bonds and be used for the precise labeling of native antibodies. We identify conditions in which short-lived activated esters formed by the aqueous carbodiimide EDC promote isopeptide bond formation specifically at preassociated amine-acid pairs. Indiscriminate cross-linking is minimized by formation of protein complexes before addition of catalyst, use of acidic pH to suppress exposed Lys reactivity, and limiting the aqueous stability of activated esters. For native antibody (Ab) labeling, we show that the small IgG-binding protein GB1 can be covalently attached to the Ab Fc domain and that introduction of Cys into GB1 loops allows for facile conjugation of fluorophores, micelles, or inorganic nanocrystals for imaging in live cells and animals. By varying Cys substituents and protein stoichiometry, a defined number of probes can be uniformly attached without the need for extensive purification. In live-cell confocal microscopy, labeled GB1 serves as a stable replacement for secondary Abs, enabling simple multicolor immunostaining and imaging. Lock coupling requires just a single reagent in aqueous buffer and leverages both the innate ability of proteins to form high-affinity complexes and the widespread presence of Lys-Glu/Asp pairs at their interfaces, with the potential for precision synthesis of protein-based probes for imaging, biomaterials, biophysics, and medicine.

The formation of stable protein complexes enables much of biotechnology, but even high-affinity complexes can dissociate, limiting potential applications in biomaterials development, bioimaging, nanomedicine, and other protein-based technologies. Here, we describe Lock coupling, a simple and selective one-step reaction between interfacial lysine and glutamate or aspartate sidechains to form stable isopeptide bonds and be used for the precise labeling of native antibodies. We identify conditions in which short-lived activated esters formed by the aqueous carbodiimide EDC promote isopeptide bond formation specifically at pre-associated amine-acid pairs. Indiscriminate crosslinking is minimized by formation of protein complexes before addition of catalyst, use of acidic pH to suppress exposed Lys reactivity, and limiting the aqueous stability of activated esters. For native antibody (Ab) labeling, we show that the small IgG-binding protein GB1 can be covalently attached to the Ab Fc domain, and introduction of Cys into GB1 loops allows for facile conjugation of fluorophores, micelles, or inorganic nanocrystals for imaging in live cells and animals. By varying Cys substituents and protein stoichiometry, a defined number of probes can be uniformly attached without the need for extensive purification. In live-cell confocal microscopy, labeled GB1 serves as a stable replacement for secondary Ab, enabling simple multicolor immunostaining and imaging. Lock coupling requires just a single reagent in aqueous buffer and leverages both the innate ability of proteins to form high-affinity complexes and the widespread presence of Lys-Glu/Asp pairs at their interfaces, with potential for precision synthesis of protein-based probes for imaging, biomaterials, biophysics, and medicine.

Alzheimer's disease has long been associated with the aggregation of amyloid beta peptides (Aβ42) into macroscale plaques, although specific neurodegenerative agents have not been definitively identified. Much evidence has pointed to the soluble nanoscale oligomers that form early in the Aβ42 aggregation pathway, but there is little understanding of these structures, their mechanisms of formation, or how they grow into plaques. Here, we show that a solvatochromic fluorophore with near-infrared (NIR) emission can track synthetic Aβ42 aggregation through environment-sensitive spectral shifts from the earliest time points through plaque formation. This azide-functionalized phosphine oxide azetidine rhodol (Phazr-N₃) shows large polarity-dependent changes in fluorescence emission, with maxima shifting from 630 nm in toluene to 703 nm in aqueous buffer, and a maximum quantum yield of 62%. Upon induction of Aβ42 aggregation, we observe immediate solvatochromic changes in Phazr-N₃ fluorescence, with multiple apparent phases over 12 h, and which culminate before the onset of any major fluorescence changes of conformation-specific aggregation fluorophore thioflavin T. Solution anisotropy measurements show a low micromolar affinity of Phazr-N₃ for disordered, free Aβ42 in solution, and real-time measurements are consistent with rapid liquid-liquid phase separation and slow dehydration of the growing aggregate. Spectral imaging of synthetic plaques stained in the presence of live cells and lipid-binding protein albumin shows over 4000-fold Phazr-N₃ fluorescence intensity above background under no-wash conditions, and over 100-fold intensity above coplated microglial cells or a large excess of albumin. This use of a solvatochromic probe with structure-independent binding to free Aβ42 offers real-time, minimally invasive insight into the full Aβ42 aggregation pathway.

Mechanical force is an essential feature for many physical and biological processes^{1,2,3,4,5,6,7}, and remote measurement of mechanical signals with high sensitivity and spatial resolution is needed for diverse applications, including robotics⁸, biophysics^9,10, energy storage¹¹ and medicine^12,13. Nanoscale luminescent force sensors excel at measuring piconewton forces, whereas larger sensors have proven powerful in probing micronewton forces^14,15,16. However, large gaps remain in the force magnitudes that can be probed remotely from subsurface or interfacial sites, and no individual, non-invasive sensor is capable of measuring over the large dynamic range needed to understand many systems^14,17. Here we demonstrate Tm³⁺-doped avalanching-nanoparticle¹⁸ force sensors that can be addressed remotely by deeply penetrating near-infrared light and can detect piconewton to micronewton forces with a dynamic range spanning more than four orders of magnitude. Using atomic force microscopy coupled with single-nanoparticle optical spectroscopy, we characterize the mechanical sensitivity of the photon-avalanching process and reveal its exceptional force responsiveness. By manipulating the Tm³⁺ concentrations and energy transfer within the nanosensors, we demonstrate different optical force-sensing modalities, including mechanobrightening and mechanochromism. The adaptability of these nanoscale optical force sensors, along with their multiscale-sensing capability, enable operation in the dynamic and versatile environments present in real-world, complex structures spanning biological organisms to nanoelectromechanical systems.

Alzheimer’s disease has long been associated with the aggregation of amyloid beta peptides (Aβ42) into macroscale plaques, although specific neurodegenerative agents have not been definitively identified. Much evidence has pointed to the soluble nanoscale oligomers that form early in the Aβ42 aggregation pathway, but there is little understanding of these structures, their mechanisms of formation, or how they grow into plaques. Here, we show that a solvatochromic fluorophore with near infrared (NIR) emission can track synthetic Aβ42 aggregation through environment-sensitive spectral shifts from the earliest timepoints through plaque formation. This azide-functionalized phosphine oxide azetidine rhodol (Phazr-N3) shows large polarity-dependent changes in fluorescence emission, with maxima shifting from 630 nm in toluene to 703 nm in aqueous buffer, and a maximum quantum yield of 62%. Upon induction of Aβ42 aggregation, we observe immediate solvatochromic changes in Phazr-N3 fluorescence, with multiple apparent phases over 24 h, and which culminate before the onset of any major fluorescence changes of conformation-specific aggregation fluorophore ThT. Solution anisotropy measurements show a low micromolar affinity of Phazr-N3 for non-aggregated Aβ42 in solution, and real-time measurements are consistent with rapid phase separation and slow dehydration of the growing aggregate. Spectral imaging of synthetic plaques stained in the presence of live cells and lipid-binding protein shows over 4000-fold Phazr-N3 fluorescence intensity above background under no-wash conditions, and over 100-fold intensity above co-plated microglial cells. This use of solvatochromic probes and kinetic anisotropy offers real-time, minimally invasive insight into Aβ42 aggregation and the molecular mechanisms of neurodegeneration.