Spot-Based Global Registration for Subpixel Stitching of Single-Molecule Resolution Images for Tissue-Scale Spatial Transcriptomics.

Single-molecule imaging at the tissue scale has revolutionized our understanding of biology by providing unprecedented insight into the molecular expression of individual cells and their spatial organization within tissues. However, achieving precise image stitching at the single-molecule level remains a challenge, primarily due to heterogeneous background signals and dim labeling signals in single-molecule images. This paper introduces Spot-Based Global Registration (SBGR), a novel strategy that shifts the focus from raw images to identified molecular spots for high-resolution image alignment. The use of spot-based data enables straightforward and robust evaluation of the credibility of estimated translations and stitching performance. The method outperforms existing image-based stitching methods, achieving subpixel accuracy (83 ± 36 nm) with exceptional consistency. Furthermore, SBGR incorporates a mechanism to surgically remove duplicate spots in overlapping regions, maximizing information recovery from duplicate measurements. In conclusion, SBGR emerges as a robust and accurate solution for stitching single-molecule resolution images in tissue-scale spatial transcriptomics, offering versatility and potential for high-resolution spatial analysis.

Multiplexed electrical detection of whole viruses from plasma in a microfluidic platform.

The advancement of point-of-care diagnostics is crucial to improving patient outcomes, especially in areas with low access to hospitals or specialized laboratories. In particular, rapid, sensitive, and multiplexed detection of disease biomarkers has great potential to achieve accurate diagnosis and inform high quality care for patients. Our Coulter counting and immunocapture based detection system has previously shown its broad applicability in the detection of cells, proteins, and nucleic acids. This paper expands the capability of the platform by demonstrating multiplexed detection of whole-virus particles using electrically distinguishable hydrogel beads by demonstrating the capability of our platform to achieve simultaneous detection at clinically relevant concentrations of hepatitis A virus (>2 × 103 IU mL-1) and human parvovirus B19 virus like particles (>106 IU mL-1) from plasma samples. The expanded versatility of the differential electrical counting platform allows for more robust and diverse testing capabilities.

In-drop thermal cycling of microcrystal assembly for senescence control (MASC) with minimal variation in efficacy.

The secretome from mesenchymal stem cells (MSCs) has recently gained attention for new therapeutics. However, clinical application requires in vitro cell manufacturing to attain enough cells. Unfortunately, this process often drives MSCs into a senescent state that drastically changes cellular secretion activities. Antioxidants are used to reverse and prevent the propagation of senescence; however, their activity is short-lived. Polymer-stabilized crystallization of antioxidants has been shown to improve bioactivity, but the broad crystal size distribution (CSD) significantly increases the efficacy variation. Efforts were made to crystalize drugs in microdroplets to narrow the CSD, but the fraction of drops containing at least one crystal can be as low as 20%. To this end, this study demonstrates that in-drop thermal cycling of hyaluronic acid-modified antioxidant crystals, named microcrystal assembly for senescence control (MASC), can drive the fraction of microdrops containing crystals to >86% while achieving significantly narrower CSDs (13±3µm) than in bulk (35±11µm). Therefore, this approach considerably improves the practicality of CSD-control in drops. In addition to exhibiting uniform release, MASC made with antioxidizing N-acetylcysteine extended the release time by 40%. MASC further improves the restoration of reactive oxygen species homeostasis in MSCs, thus minimizing cellular senescence and preserving desired secretion activities. We propose that MASC is broadly useful to controlling senescence of a wide array of therapeutic cells during biomanufacturing.

Spectroscopic Investigation of Phosphorus Mineralization as Affected by the Calcite-Water Interfacial Chemistry.

The mineralization and bioavailability of phytic acid, the predominant organic phosphorus (OP) species in many soils, have generally been rendered limited due to its interaction with soil minerals. In particularly calcareous and neutral to slightly alkaline soils, phytic acid is known to actively react with calcite, although how this interaction affects phytic acid mineralization is still unknown. This study, therefore, investigated the mechanisms regarding how the calcite-water interface influences phytic acid mineralization by phytase, at pHs 6 and 8 using in situ spectroscopic techniques including solution nuclear magnetic resonance and attenuated total reflection Fourier transform infrared spectroscopy. The findings indicated a pH-specific effect of the calcite-water interface. Inhibited phytase activity and thus impaired phytic acid mineralization were induced by calcite at pH 6, while the opposite effect was observed at pH 8. How the interaction between phytic acid and calcite and between phytase and calcite differed between the two pH values contributed to the pH-specific effect. The results demonstrate the importance of soil pH, enzyme-, and OP-clay mineral interactions in controlling the mineralization and transformation of OP and, consequently, the release of phosphate in soils. The findings can also provide implications for the management of calcite-rich and limed soils.

Droplet Microfluidics for High-Resolution Virology.

Microfluidics has enabled a new era of cellular and molecular assays due to the small length scales, parallelization, and the modularity of various analysis and actuation functions. Droplet microfluidics, in particular, has been instrumental in providing new tools for biology with its ability to quickly and reproducibly generate drops that act as individual reactors. A notable beneficiary of this technology has been single-cell RNA sequencing, which has revealed new heterogeneities and interactions for the fundamental unit of life. However, viruses far surpass the diversity of cellular life, affect the dynamics of all ecosystems, and are a chronic source of global health crises. Despite their impact on the world, high-throughput and high-resolution viral profiling has been difficult, with conventional methods being limited to population-level averaging, large sample volumes, and few cultivable hosts. Consequently, most viruses have not been identified and studied. Droplet microfluidics holds the potential to address many of these limitations and offers new levels of sensitivity and throughput for virology. This Feature highlights recent efforts that have applied droplet microfluidics to the detection and study of viruses, including for diagnostics, virus-host interactions, and cell-independent virus assays. In combination with traditional virology methods, droplet microfluidics should prove a potent tool toward achieving a better understanding of the most abundant biological species on Earth.

Inertial Self-Assembly Dynamics of Interacting Droplet Ensembles in Microfluidic Flows.

The multiphase flow of droplets is widespread and used for both biological and nonbiological applications alike. However, the ensemble interactions of such systems are inherently nonlinear and complex, compounded by interfacial effects, making it a difficult many-body problem. In comparison, the self-assembly dynamics of solid particles in flow have long been studied and exploited in the field of inertial microfluidics. Here, we report novel self-assembly dynamics of liquid drops in microfluidic channels that contrast starkly with the established paradigm of inertial microfluidics, which stipulates that higher inertia leads to better spatial ordering. Instead, we find that ordering can be negatively correlated with inertia, while Dean flow can achieve long-range spatial periodicity on length scales at least 3 orders of magnitude greater than the drop diameter. Experimentally, we decouple droplet generation from ordering, enabling independent and systematic variation of key parameters, especially in ranges practical to droplet microfluidics. We find the inertia-dependent emergence of preferred drop separations and show that surfactant effects can influence the longitudinal ordering of multidrop arrays. The dynamics we describe have immediate utility to droplet microfluidics, where the ability to order drops is key to the streamlined integration of on-chip incubation with deterministic drop manipulation downstream─two important functions for biological assays. To this end, we demonstrate the use of passive inertial drop self-assembly to combine a delay line with picoinjection. These results not only present a largely unexplored direction for inertial microfluidics but also show the practical benefit of its unification with the versatile field of droplet microfluidics.

Quantum dot/antibody conjugates for in vivo cytometric imaging in mice.

Multiplexed, phenotypic, intravital cytometric imaging requires novel fluorophore conjugates that have an appropriate size for long circulation and diffusion and show virtually no nonspecific binding to cells/serum while binding to cells of interest with high specificity. In addition, these conjugates must be stable and maintain a high quantum yield in the in vivo environments. Here, we show that this can be achieved using compact (∼15 nm in hydrodynamic diameter) and biocompatible quantum dot (QD) -Ab conjugates. We developed these conjugates by coupling whole mAbs to QDs coated with norbornene-displaying polyimidazole ligands using tetrazine-norbornene cycloaddition. Our QD immunoconstructs were used for in vivo single-cell labeling in bone marrow. The intravital imaging studies using a chronic calvarial bone window showed that our QD-Ab conjugates diffuse into the entire bone marrow and efficiently label single cells belonging to rare populations of hematopoietic stem and progenitor cells (Sca1(+)c-Kit(+) cells). This in vivo cytometric technique may be useful in a wide range of structural and functional imaging to study the interactions between cells and between a cell and its environment in intact and diseased tissues.

Whole-Genome Sequencing of a Single Viral Species from a Highly Heterogeneous Sample.

Metagenomic studies suggest that only a small fraction of the viruses that exist in nature have been identified and studied. Characterization of unknown viral genomes is hindered by the many genomes populating any virus sample. A new method is reported that integrates drop-based microfluidics and computational analysis to enable the purification of any single viral species from a complex mixed virus sample and the retrieval of complete genome sequences. By using this platform, the genome sequence of a 5243 bp dsDNA virus that was spiked into wastewater was retrieved with greater than 96% sequence coverage and more than 99.8% sequence identity. This method holds great potential for virus discovery since it allows enrichment and sequencing of previously undescribed viruses as well as known viruses.

Compact high-quality CdSe-CdS core-shell nanocrystals with narrow emission linewidths and suppressed blinking.

High particle uniformity, high photoluminescence quantum yields, narrow and symmetric emission spectral lineshapes and minimal single-dot emission intermittency (known as blinking) have been recognized as universal requirements for the successful use of colloidal quantum dots in nearly all optical applications. However, synthesizing samples that simultaneously meet all these four criteria has proven challenging. Here, we report the synthesis of such high-quality CdSe-CdS core-shell quantum dots in an optimized process that maintains a slow growth rate of the shell through the use of octanethiol and cadmium oleate as precursors. In contrast with previous observations, single-dot blinking is significantly suppressed with only a relatively thin shell. Furthermore, we demonstrate the elimination of the ensemble luminescence photodarkening that is an intrinsic consequence of quantum dot blinking statistical ageing. Furthermore, the small size and high photoluminescence quantum yields of these novel quantum dots render them superior in vivo imaging agents compared with conventional quantum dots. We anticipate these quantum dots will also result in significant improvement in the performance of quantum dots in other applications such as solid-state lighting and illumination.

Organoborane Se and Te Precursors for Controlled Modulation of Reactivity in Nanomaterial Synthesis.

To exploit the distinctive optoelectrical properties of nanomaterials, precise control over the size, morphology, and interface structure is essential. Achieving a controlled synthesis demands precursors with tailored reactivity and optimal reaction temperatures. Here, we introduce organoborane-based selenium and tellurium precursors borabicyclononane-selenol (BBN-SeH) and tellurol (BBN-TeH). The reactivity of these precursors can be modified by commercially available additives, covering a wide range of intermediate reactivity and filling significant reactivity gaps in existing options. By allowing systematic adjustment of growth conditions, they achieve the controlled growth of quantum dots of various sizes and materials. Operating via a surface-assisted conversion mechanism, these precursors rely on surface coordination for activation and undergo quantitative deposition on coordinating surfaces. These properties allow precise control over the radial distribution and density of different chalcogenide atoms within the nanoparticles. Diborabicyclononanyl selane ((BBN)2Se), an intermediate from the BBN-SeH synthesis, can also serve as a selenium precursor. While BBN-SeH suppresses nucleation, (BBN)2Se exhibits efficient nucleation under specific conditions. By leveraging these distinct activation behaviors, we achieved a controlled synthesis of thermally stable nanoplates with different thicknesses. This study not only bridges critical reactivity gaps but also provides a systematic methodology for precise nanomaterial synthesis.

Unbiased, Cell-free Profiling of Single Influenza Genomes at High-throughput.

The segmented structure of the Influenza A virus (IAV) genome facilitates reassortment, segment exchange during co-infection. When divergent strains mix across human, agricultural, and wildlife reservoirs novel strains are generated, which has been the source of pandemics. Due to the limited throughput and infection-based assays, IAV reassortment studies has been limited to permissive reassortment. We have developed DE-flowSVP to achieve extremely high throughput, direct profiling of as many as 10 5 IAV particles in a single-day experiment and enabled quantitative profiling of reassortment propensity between divergent strains for the first time. By profiling reassortants between two naturally circulating low-pathogenicity avian IAVs, we confirmed that molecular incompatibility yields strong preference toward within-strain mixing. Surprisingly, we revealed that two-to-three particle aggregation contributed primarily to genome mixing (75-99%), suggesting that aggregation mediated by sialic acid binding by viral surface proteins provides a secondary pathway to genome mixing while avoiding the co-packaging fitness cost. We showed that genome mixing is sensitively dependent on co-infection timing, relative segment abundances, and viral surface-protein background. DE-flowSVP enables large-scale survey of reassortment potential among the broad diversity of IAV strains informing pandemic strain emergence.

Versatile Prepolymer Platform for Controlled Tailoring of Quantum Dot Surface Properties.

Quantum dots (QDs) hold immense promise for bioimaging, yet technical challenges in surface engineering limit their wider scientific use. We introduce poly(pentafluorophenyl acrylate) (PPFPA) as a user-friendly prepolymer platform for creating precisely controlled multidentate polymeric ligands for QD surface engineering, accessible to researchers without extensive synthetic expertise. PPFPA combines the benefits of both bottom-up and prepolymer approaches, offering minimal susceptibility to hydrolysis and side reactions for controlled chemical composition, along with simple synthetic procedures using commercially available reagents. Live cell imaging experiments highlighted a significant reduction in nonspecific binding when employing PPFPA, owing to its minimal hydrolysis, in contrast to ligands synthesized by using a conventional prepolymer prone to uncontrolled hydrolysis. This observation underscores the distinct advantage of our prepolymer system. Leveraging PPFPA, we synthesized biomolecule-conjugated QDs and performed QD-based immunofluorescence to detect a cytosolic protein. To effectively label cytosolic targets in such a dense and complex environment, probes must exhibit minimal nonspecific binding and be compact. As a result, QD-immunofluorescence has focused primarily on cell surface targets. By creating compact QD-F(ab')2, we sensitively detected alpha-tubulin with a ∼50-fold higher signal-to-noise ratio compared to organic dye-based labeling. PPFPA represents a versatile and accessible platform for tailoring QD surfaces, offering a pathway to realize the full potential of colloidal QDs in various scientific applications.

Controlled placement of colloidal quantum dots in sub-15 nm clusters.

We demonstrated a technique to control the placement of 6 nm-diameter CdSe and 5 nm-diameter CdSe/CdZnS colloidal quantum dots (QDs) through electron-beam lithography. This QD-placement technique resulted in an average of three QDs in each cluster, and 87% of the templated sites were occupied by at least one QD. These QD clusters could be in close proximity to one another, with a minimum separation of 12 nm. Photoluminescence measurements of the fabricated QD clusters showed intermittent photoluminescence, which indicates that the QDs were optically active after the fabrication process. This optimized top-down lithographic process is a step towards the integration of individual QDs in optoelectronic and nano-optical systems.

Compact zwitterion-coated iron oxide nanoparticles for biological applications.

The potential of superparamagnetic iron oxide nanoparticles (SPIONs) in various biomedical applications, including magnetic resonance imaging (MRI), sensing, and drug delivery, requires that their surface be derivatized to be hydrophilic and biocompatible. We report here the design and synthesis of a compact and water-soluble zwitterionic dopamine sulfonate (ZDS) ligand with strong binding affinity to SPIONs. After ligand exchange, the ZDS-coated SPIONs exhibit small hydrodynamic diameters, and stability with respect to time, pH, and salinity. Furthermore, small ZDS coated SPIONs were found to have a reduced nonspecific affinity (compared to negatively charged SPIONs) toward serum proteins; streptavidin/dye functionalized SPIONs were bioactive and thus specifically targeted biotin receptors.

Double Emulsion Flow Cytometry for Rapid Single Genome Detection.

Established techniques in droplet microfluidics have utilized single emulsion (SE) drops to compartmentalize and analyze single cells achieving high-throughput, low input analysis. Building upon this foundation, double emulsion (DE) droplet microfluidics has emerged with distinct advantages in terms of stable compartmentalization, resistance to merging, and most importantly direct compatibility with flow cytometry. In this chapter, we describe a simple-to-fabricate, single-layer DE drop generation device that achieves spatial control over surface wetting with a plasma treatment step. This easy-to-operate device allows for the robust production of single-core DEs with excellent control over the monodispersity. We further explain the use of these DE drops for single-molecule and single-cell assays. Detailed protocols are described to perform single molecule detection using droplet digital PCR in DE drops and automated detection of DE drops on a fluorescence-activated cell sorter (FACS). Due to the wide availability of FACS instruments, DE methods can facilitate the broader adoption of drop-based screening. As the applications of FACS-compatible DE droplets are immensely varied and extend well beyond what can be explored here, this chapter should be seen as an introduction to DE microfluidics.

Intracellular Spatial Transcriptomic Analysis Toolkit (InSTAnT).

Imaging-based spatial transcriptomics technologies such as MERFISH offer snapshots of cellular processes in unprecedented detail, but new analytic tools are needed to realize their full potential. We present InSTAnT, a computational toolkit for extracting molecular relationships from spatial transcriptomics data at the intra-cellular resolution. InSTAnT detects gene pairs and modules with interesting patterns of mutual co-localization within and across cells, using specialized statistical tests and graph mining. We showcase the toolkit on datasets profiling a human cancer cell line and hypothalamic preoptic region of mouse brain. We performed rigorous statistical assessment of discovered co-localization patterns, found supporting evidence from databases and RNA interactions, and identified subcellular domains associated with RNA-colocalization. We identified several novel cell type-specific gene co-localizations in the brain. Intra-cellular spatial patterns discovered by InSTAnT mirror diverse molecular relationships, including RNA interactions and shared sub-cellular localization or function, providing a rich compendium of testable hypotheses regarding molecular functions.

High-sensitivity whole-genome recovery of single viral species in environmental samples.

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. This work presents a method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A novel gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. Validation experiments using a sewage sample spiked with two known viruses demonstrate the method's efficacy. We achieve 100% recovery of the spiked-in SV40 (Simian virus 40, 5243bp) genome sequence with uniform coverage distribution, and approximately 99.4% for the larger HAd5 genome (Human Adenovirus 5, 35938bp). Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables targeted characterizations of rare viral species and whole-genome amplification of single genomes for accessing the mutational profile in single virus genomes, contributing to an improved understanding of viral ecology.

Electrochemical point-of-care devices for the diagnosis of sepsis.

Sepsis is a life-threatening dysfunction of organ systems caused by a dysregulated immune system because of an infectious process. It remains one of the leading causes of hospital mortality and of hospital readmissions in the United States. Mortality from sepsis increases with each hour of delayed treatment, therefore, diagnostic devices that can reduce the time from the onset of a patient's infection to the delivery of appropriate therapy are urgently needed. Likewise, tools that are capable of high-frequency testing of clinically relevant biomarkers are required to study disease progression. Electrochemical biosensors offer important advantages such as high sensitivity, fast response, miniaturization, and low cost that can be adapted to clinical needs. In this review paper, we discuss the current state, limitations, and future directions of electrochemical-based point-of-care detection platforms that contribute to the diagnosis and monitoring of sepsis.

Simplified, Shear Induced Generation of Double Emulsions for Robust Compartmentalization during Single Genome Analysis.

Drop microfluidics has driven innovations for high throughput, low input analysis techniques such as single-cell RNA-seq. However, the instability of single emulsion (SE) drops occasionally causes significant merging during drop processing, limiting most applications to single-step reactions in drops. Here, we show that double emulsion (DE) drops address this critical limitation and completely prevent drop contents from mixing. DEs show excellent stability during thermal cycling. More importantly, DEs undergo rupture into the continuous phase instead of merging, preventing content mixing and eliminating unstable drops from the downstream analysis. Due to the lack of drop merging, the monodispersity of drops is maintained throughout a workflow, enabling the deterministic manipulation of drops downstream. We also developed a simple, one-layer DE drop maker compatible with simple surface treatment using a plasma cleaner. The device allows for the robust production of single-core DEs at a wide range of flow rates and better control over the shell thickness, both of which have been significant limitations of conventional two-layer devices. This approach makes the fabrication of DE devices much more accessible, facilitating its broader adoption. Finally, we show that DE droplets eliminate content mixing and maintain compartmentalization of single virus genomes during PCR-based amplification and barcoding, while SEs mixed contents due to merging. With their resistance to content mixing, DE drops have key advantages for multistep reactions in drops, which is limited in SEs due to merging and content mixing.

Extending the Bioavailability of Hydrophilic Antioxidants for Metal Ion Detoxification via Crystallization with Polysaccharide Dopamine.

Although metal ions, such as silver and gold, have been shown to have strong antimicrobial properties, their potential to have toxic effects on human and environmental health has gained interest with an improved understanding of their mechanisms to promote oxidative stress. Redox control is a major focus of many drug delivery systems and often incorporates an antioxidant as the active pharmaceutical ingredient (API) to neutralize overproduced reactive oxygen species (ROS). Nevertheless, there are still limitations with bioavailability and extended redox control with regard to antioxidant drug delivery. Herein, this study develops a colloidal antioxidant crystal system that dissolves sustainably through polymer stabilization using sodium hyaluronate conjugated with dopamine (HA-dopa). We explore the role of dopamine incorporation into crystal-stabilizing polymers and quantify the balance between drug-polymer interactions and competing polymer-polymer interactions. We propose that this type of analysis is useful in the engineering of and provides insight into the release behavior of polymer-crystal complexes. In developing our crystal complex, N-acetylcysteine (NAC) was used as the model antioxidant to protect against silver ion toxicity. We found that our optimized HA-dopa-stabilized NAC crystals prolong the release time of NAC 5-fold compared to a polymer-free NAC crystal. Therefore, following sublethal exposure to AgNO3, the extended lifetime of NAC was able to maintain normal intracellular ROS levels, modulate metabolic function, mitigate fluctuations in ATP levels and ATP synthase activity, and preserve contraction frequency in engineered cardiac muscle tissue. Furthermore, the protective effects of the HA-dopa-stabilized NAC crystals were extended to a Daphnia magna model where silver-ion-induced change to both cell-level biochemistry and organ function was alleviated. As such, we propose that the packaging of hydrophilic antioxidants as colloidal crystals drastically extends the lifetime of the API, better maintains ROS homeostasis post metal ion exposure, and therefore preserves both intracellular biochemistry and tissue functionality.