The photocatalytic performance of two-dimensional (2D) semiconductors is governed by the intricate interplay between nanoscale surface structures and charge-carrier dynamics. However, ensemble-averaged and ex situ measurements obscure the spatial heterogeneity and temporal fluctuations, limiting mechanistic understanding. Here, we employed single-molecule fluorescence imaging to quantitatively visualize the photocatalytic activity of 2D layered InSe at the nanoscale, differentiating the carrier extraction positions and efficiencies of photogenerated holes and electrons for light-driven semiconductor-catalyzed oxidation reactions. We observed that hole-driven activity occurs on both the basal planes and edges, whereas electron-driven reactivity is confined primarily to the edges. Temporal analysis of single-turnover events uncovered catalysis-induced dynamic fluctuations, governed by surface-adsorbate interactions, with thicker direct-band gap flakes exhibiting enhanced temporal stability. Spatially, activity fluctuations correlate positively along individual edges over ∼150 nm, suggesting communication via charge diffusion along the edge. Furthermore, reactivity decays exponentially from the edges into the basal plane over hundreds of nanometers, attributed to carrier redistribution from edge defects to pristine regions. These discoveries offer nanoscale insight into the spatiotemporal heterogeneity of photocatalysis.

Simultaneous determination of spatial location and spectral color of single molecules at large molecular density with high throughput was achieved by combining single-molecule photoswitching and optical in-hardware Fourier transformation of single-molecule emission spectra into the phasor space. The method, named as spectral phasor enabled stochastic optical reconstruction microscopy (SP-STORM), achieved simultaneous super-resolution imaging of five subcellular structures in parallel with minimum crosstalk for the first time. The high-throughput feature of SP-STORM enables these subcellular structures to be readily resolved in about one min, which is more than an order of magnitude faster than other multiplexing single-molecule localization microscopy techniques. The concept of SP-STORM is also compatible with and can be readily applicable to other super-resolution microscopy.

Cell membranes display nanoscale heterogeneity in lipid composition and organization that regulates vital biological processes yet remain challenging to resolve with conventional imaging. We introduce spectral phasor single-molecule localization microscopy (SP-SMLM), a hyperspectral and super-resolution method that combines wavefront-like optical filtering with single-molecule imaging for simultaneous spatial and spectral analysis. A lab-built three-channel imager with sine/cosine filters encodes emission spectra of single molecules into the phasor space, enabling high-throughput, high-SNR mapping of membrane polarity at sub-50 nm spatial and 15 s temporal resolutions. Through simulation, we validate that the phasor angle correlates with the spectral mean for single dye molecules. When applied to Nile red-stained COS-7 cells, SP-SMLM revealed organelle-specific polarity differences and dynamic remodeling of the lipid composition within live cells. The method's hyperspectral capability, rapid acquisition, and compatibility with 2D/3D imaging platforms position SP-SMLM as a powerful tool for studying membrane heterogeneity and dynamics in live cells.

As a major family of red-shifted fluorophores that operate beyond the visible light, polymethine dyes are pivotal in light-based biological techniques. However, the methods for tuning this kind of fluorophores by structural modification remain restricted to bottom-up synthesis and modification using coupling or nucleophilic substitutions. In this study, we introduce a two-step, late-stage functionalization process for heptamethine dyes. This process enables the substitution of the central chloride atom in the commonly used 4'-chloro heptamethine scaffold with various aryl groups using aryllithium reagents. This method borrows the building block and designs from the xanthene dye community, and offers a mild and convenient way for the diversification of heptamethine fluorophores. Notably, this efficient conversion allows for the synthesis of heptamethine-X, the heptamethine scaffold with two ortho-substituents on the 4’-aryl modification, which brings enhanced stability and reduced aggregation to the fluorophore. We showcase the utility of this synthesis by a facile synthesis of a fluorogenic, membrane-localizing fluorophore that outperforms the commercial counterparts with higher brightness and contrast. Overall, this method establishes the synthetic similarities between polymethine and xanthene fluorophores, and provides reference for future optimizing heptamethine fluorophores for their biological applications.

Understanding the host-guest interactions in porous materials is of great importance in the field of separation science. Probing it at the single-molecule level uncovers the inter- and intraparticle inhomogeneity and establishes structure-property relationships for guiding the design of porous materials for better separation performance. In this work, we investigated the dynamics of host-guest interactions in core-shell mesoporous silica particles under in situ conditions by using a fluorogenic reaction-initiated single-molecule tracking (riSMT) approach. Taking advantage of the low fluorescence background, three-dimensional (3D) tracking of the dynamics of the molecules inside the mesoporous silica pore was achieved with high spatial precision. Compared to the commonly used two-dimensional (2D) tracking method, the 3D tracking results show that the diffusion coefficients of the molecules are three times larger on average. Using riSMT, we quantitatively analyzed the mass transfer of probe molecules in the mesoporous silica pore, including the fraction of adsorption versus diffusion, diffusion coefficients, and residence time. Large interparticle inhomogeneity was revealed and is expected to contribute to the peak broadening for separation application at the ensemble level. We further investigated the impact of electrostatic interaction on the mass transfer of molecules in the mesoporous silica pore and discovered that the primary effect is on the fraction rather than their diffusion rates of resorufin molecules undergoing diffusion.

As a major family of red-shifted fluorophores that operate beyond visible light, polymethine dyes are pivotal in light-based biological techniques. However, methods for tuning this kind of fluorophores by structural modification remain restricted to bottom-up synthesis and modification using coupling or nucleophilic substitutions. In this study, we introduce a two-step, late-stage functionalization process for heptamethine dyes. This process enables the substitution of the central chlorine atom in the commonly used 4'-chloro heptamethine scaffold with various aryl groups using aryllithium reagents. This method borrows the building block and designs from the xanthene dye community and offers a mild and convenient way for the diversification of heptamethine fluorophores. Notably, this efficient conversion allows for the synthesis of heptamethine-X, the heptamethine scaffold with two ortho-substituents on the 4'-aryl modification, which brings enhanced stability and reduced aggregation to the fluorophore. We showcase the utility of this method by a facile synthesis of a fluorogenic, membrane-localizing fluorophore that outperforms its commercial counterparts with a significantly higher brightness and contrast. Overall, this method establishes the synthetic similarities between polymethine and xanthene fluorophores and provides a versatile and feasible toolbox for future optimizing heptamethine fluorophores for their biological applications.

Single-molecule localization microscopy (SMLM) has become a strong technique in the toolbox of chemists, biologists, physicists, and engineers in recent years for its unique ability to resolve characteristic features quickly and accurately in complex environments at the nanoscopic level. Multicolor super-resolution imaging has seen the greatest advancement among SMLM techniques, drastically improving the differentiation ability of nanostructures beyond the diffraction limit and increasing the resolution with which previously unresolvable structures are studied. However, current multicolor SMLM methodologies present low spatial resolution and throughput and require complex optical systems. Here, we overcome these drawbacks by developing an ultrahigh-throughput SMLM methodology that allows for ultrahigh throughput multicolor imaging at the nanoscopic level using a color glass filter. Our methodology can readily distinguish fluorophores of close spectral emission and achieves sub-10 nm localization and sub-5nm spectral precisions.### Competing Interest StatementThe authors have declared no competing interest.

Single-molecule localization microscopy (SMLM), a type of super-resolution fluorescence microscopy, has become a strong technique in the toolbox of chemists, biologists, physicists, and engineers in recent years for its unique ability to resolve characteristic features at the nanoscopic level. It drastically improves the resolution of optical microscopes beyond the diffraction limit, with which previously unresolvable structures can now be studied. Spectrally resolved super-resolution fluorescence microscopy via multiplexing of different fluorophores is one of the greatest advancements among SMLM techniques. However, current spectrally resolved SMLM (SR-SMLM) methodologies present low spatial resolution due to loss of photons, low throughput due to spectral interferences, or require complex optical systems. Here, we overcome these drawbacks by developing a SR-SMLM methodology using a color glass filter. It enables high throughput and improved photon usage for hyperspectral imaging at the nanoscopic level. Our methodology can readily distinguish fluorophores of close spectral emission and achieves sub-10 nm localization and sub-5 nm spectral precisions. A SR-SMLM was developed for hyperspectral imaging with sub-10 nm localization and sub-5 nm spectral precisions. It enables high throughput and improved photon usage for hyperspectral imaging at the nanoscopic level.

We present a novel method for the precise tracking of plasmonic gold nanorods (AuNRs) in live cells, enabling a comprehensive understanding of the nanocargo’s cellular dynamics. Traditional single particle tracking (SPT) struggles with accurately determining all five spatial parameters (x, y, z, ϕ, and θ) in live cells due to various challenges. Our innovation combines electronic tunable lens (ETL) technology with bifocal parallax dark-field (DF) microscopy, allowing continuous adjustment of the imaging focal plane for automatic tracking of both translational and rotational movements of AuNRs. This 5D single-particle orientation and rotational tracking (5D SPORT) method achieves remarkable precision, with 3D localization precisions of 9 (x), 10 (y), and 15 nm (z) and angular resolutions below 2°. To showcase its applicability, we investigated intracellular transport of nanocargos using transferrin-modified AuNRs as the imaging probe. Differentiated transport stages, such as active transport and pause period, were clearly unveiled from the observed dynamics in 5D. This advancement in single particle tracking holds promise for a wide range of applications in biomedical research, particularly when combined with other imaging modalities, such as light sheet fluorescence microscopy.

Chronic lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), are major leading causes of death worldwide and are generally associated with poor prognoses. The heterogeneous distribution of collagen, mainly type I collagen associated with excessive collagen deposition, plays a pivotal role in the progressive remodeling of the lung parenchyma to chronic exertional dyspnea for both IPF and COPD. To address the pressing need for noninvasive early diagnosis and drug treatment monitoring of pulmonary fibrosis, we report the development of human collagen-targeted protein MRI contrast agent (hProCA32.collagen) to specifically bind to collagen I overexpressed in multiple lung diseases. When compared to clinically approved Gd3+ contrast agents, hProCA32.collagen exhibits significantly better r1 and r2 relaxivity values, strong metal binding affinity and selectivity, and transmetalation resistance. Here, we report the robust detection of early and late-stage lung fibrosis with stage-dependent MRI signal-to-noise ratio (SNR) increase, with good sensitivity and specificity, using a progressive bleomycin-induced IPF mouse model. Spatial heterogeneous mapping of usual interstitial pneumonia (UIP) patterns with key features closely mimicking human IPF, including cystic clustering, honeycombing, and traction bronchiectasis, were noninvasively detected by multiple MR imaging techniques and verified by histological correlation. We further report the detection of fibrosis in the lung airway of an electronic cigarette-induced COPD mouse model, using hProCA32.collagen-enabled precision MRI (pMRI), and validated by histological analysis. The developed hProCA32.collagen is expected to have strong translational potential for the noninvasive detection and staging of lung diseases, and facilitating effective treatment to halt further chronic lung disease progression.

Gold nanoparticles (AuNPs) have become increasingly useful in recent years for their roles in nanomedicine, cellular biology, energy storage and conversion, photocatalysis, and more. At the single-particle level, AuNPs have heterogeneous physical and chemical properties which are not resolvable in ensemble measurements. In the present study, we developed an ultrahigh-throughput spectroscopy and microscopy imaging system for characterization of AuNPs at the single-particle level using phasor analysis. The developed method enables quantification of spectra and spatial information on large numbers of AuNPs with a single snapshot of an image (1024 × 1024 pixels) at high temporal resolution (26 fps) and localization precision (sub-5 nm). We characterized the localized surface plasmonic resonance (SPR) scattering spectra of gold nanospheres (AuNSs) of four different sizes (40-100 nm). Comparing to the conventional optical grating method which suffers low efficiency in characterization due to spectral interference caused by nearby nanoparticles, the phasor approach enables high-throughput analysis of single-particle SPR properties in high particle density. Up to 10-fold greater efficiency of single-particle spectro-microscopy analysis using the spectra phasor approach when compared to a conventional optical grating method was demonstrated.

Synaptic plasticity relies on rapid, yet spatially precise signaling to alter synaptic strength. Arc is a brain enriched protein that is rapidly expressed during learning-related behaviors and is essential for regulating metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD). We previously showed that disrupting the ubiquitination capacity of Arc enhances mGluR-LTD; however, the consequences of Arc ubiquitination on other mGluR-mediated signaling events is poorly characterized. Here we find that pharmacological activation of Group I mGluRs with S-3,5-dihydroxyphenylglycine (DHPG) increases Ca 2+ release from the endoplasmic reticulum (ER). Disrupting Arc ubiquitination on key amino acid residues enhances DHPG-induced ER-mediated Ca 2+ release. These alterations were observed in all neuronal subregions except secondary branchpoints. Deficits in Arc ubiquitination altered Arc self-assembly and enhanced its interaction with calcium/calmodulin-dependent protein kinase IIb (CaMKIIb) and constitutively active forms of CaMKII in HEK293 cells. Colocalization of Arc and CaMKII was altered in cultured hippocampal neurons, with the notable exception of secondary branchpoints. Finally, disruptions in Arc ubiquitination were found to increase Arc interaction with the integral ER protein Calnexin. These results suggest a previously unknown role for Arc ubiquitination in the fine tuning of ER-mediated Ca 2+ signaling that may support mGluR-LTD, which in turn, may regulate CaMKII and its interactions with Arc.

Optical microscopy has become an invaluable tool for investigating complex samples. Over the years, many advances to optical microscopes have been made that have allowed us to uncover new insights into the samples studied. Dynamic changes in biological and chemical systems are of utmost importance to study. To probe these samples, multidimensional approaches have been developed to acquire a fuller understanding of the system of interest. These dimensions include the spatial information, such as the three-dimensional coordinates and orientation of the optical probes, and additional chemical and physical properties through combining microscopy with various spectroscopic techniques. In this review, we survey the field of multidimensional microscopy and provide an outlook on the field and challenges that may arise.

The development of nanomaterials such as two-dimensional (2D) layered materials advanced applications in many fields, including biosensors format based on field-effect transistors. The unique physical and chemical properties of 2D layered materials enable the detection limit of biomolecules as low as ∼1 pg/mL. The majority of 2D layered materials contain different structural features and defects introduced in chemical synthesis and fabrication processing. These structural features have different physicochemical properties, causing heterogeneous adsorption of bioreceptors like antibodies, enzymes, etc. Understanding the correlation between the adsorption of bioreceptors and properties of structural features is essential for building highly efficient, sensitive biosensors based on 2D layered materials. Here, we utilize a single-molecule localization-based super-resolved fluorescence imaging method to unveil the inhomogeneous adsorption of antibody fragments on 2D layered molybdenum disulfide (MoS2). The surface coverage of antibody fragments on MoS2 thin flakes is quantitatively measured and compared at different structural features and different layer thicknesses. The methodology in the current work can be extended to study bioreceptor adsorption on other types of 2D layered materials and pave a way to improve biosensors' sensitivity based on defect engineering 2D layered materials.

Optical microscopy has become an invaluable tool for investigating complex samples. Over the years, many advances to optical microscopes have been made that have allowed us to uncover new insights into the samples studied. Dynamic changes in biological and chemical systems are of utmost importance to study. To probe these samples, multidimensional approaches have been developed to acquire a fuller understanding of the system of interest. These dimensions include the spatial information, such as the three-dimensional coordinates and orientation of the optical probes, and additional chemical and physical properties through combining microscopy with various spectroscopic techniques. In this review, we survey the field of multidimensional microscopy and provide an outlook on the field and challenges that may arise. Expected final online publication date for the Annual Review of Physical Chemistry, Volume 73 is April 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

A novel single particle tracking (SPT) technique has been developed to visualize the intracellular transport of cargos in five dimensions (5D): the three spatial coordinates (x, y, z) and two orientation angles (azimuthal angle and polar angle) of a probe's transition dipole over a series of time steps. Tracking rotational probes, such as gold nanorods, in the z-axis is challenging because translational motions in the z-axis can be easily confused with rotational motions as both types of motion result in similar changes in signal intensity. The 5D-SPT method overcomes this long-standing challenge by utilizing the concept of parallax to sense the axial movement of the target object and employs an automatic feedback loop algorithm to control the objective's focal plane and bring the target object back to focus repetitively. In such an implementation, the target object is kept in focus to provide the highest possible S/N for 2D localization and orientation determination, while the z positions are recorded from the vertical movement of a high-precision objective scanner. Through the live-cell SPT experiments, we demonstrate that the 5D-SPT technique has potential to significantly improve the capacity to study the intracellular transport of cargos in a cellular environment.

Recessive mutations in RNF216/TRIAD3 cause Gordon Holmes syndrome (GHS), in which dysfunction of the hypothalamic-pituitary-gonadal (HPG) axis and neurodegeneration are thought to be core phenotypes. We knocked out Rnf216/Triad3 in a gonadotropin-releasing hormone (GnRH) hypothalamic cell line. Rnf216/Triad3 knockout (KO) cells had decreased steady-state GnRH and calcium transients. Rnf216/Triad3 KO adult mice had reductions in GnRH neuron soma size and GnRH production without changes in neuron densities. In addition, KO male mice had smaller testicular volumes that were accompanied by an abnormal release of inhibin B and follicle-stimulating hormone, whereas KO females exhibited irregular estrous cycling. KO males, but not females, had reactive microglia in the hypothalamus. Conditional deletion of Rnf216/Triad3 in neural stem cells caused abnormal microglia expression in males, but reproductive function remained unaffected. Our findings show that dysfunction of RNF216/TRIAD3 affects the HPG axis and microglia in a region- and sex-dependent manner, implicating sex-specific therapeutic interventions for GHS.

Noninvasive detection of early-stage liver metastases from different primary cancers is a pressing unmet medical need. The lack of both molecular biomarkers and the sensitive imaging methodology makes the detection challenging. In this study, we observed the elevated expression of chemokine receptor 4 (CXCR4) in uveal melanoma (UM) patient liver tissues, and high CXCR4 expression in liver metastases of UM murine models, regardless of the expression levels in the primary tumors. Based on these findings, we identified CXCR4 as an imaging biomarker and exploited a CXCR4-targeted MRI contrast agent ProCA32.CXCR4 for molecular MRI imaging. ProCA32.CXCR4 has strong CXCR4 binding affinity, high metal selectivity, and r1 and r2 relaxivities, which enables the sensitive detection of liver micrometastases. The MRI imaging capacity for detecting liver metastases was demonstrated in three UM models and one ovarian cancer model. The imaging results were validated by histological and immunohistochemical analysis. ProCA32.CXCR4 has strong potential clinical application for non-invasive diagnosis of liver metastases.

Dynamin has an important role in clathrin-mediated endocytosis by cutting the neck of nascent vesicles from the cell membrane. Here, using gold nanorods as cargos to image dynamin action during live clathrin-mediated endocytosis, we show that, near the peak of dynamin accumulation, the cargo-containing vesicles always exhibit abrupt, right-handed rotations that finish in a short time (~0.28 s). The large and quick twist, herein named the super twist, is the result of the coordinated dynamin helix action upon GTP hydrolysis. After the super twist, the rotational freedom of the vesicle increases substantially, accompanied by simultaneous or delayed translational movement, indicating that it detaches from the cell membrane. These observations suggest that dynamin-mediated scission involves a large torque generated by the coordinated actions of multiple dynamins in the helix, which is the main driving force for vesicle scission.

Single-molecule photocatalysis at defects in 2D layered materials display distinct dynamic features.