Small Methods

Spatial transcriptomics (ST) has emerged as a transformative tool for resolving the molecular heterogeneity of complex tissues within their native anatomical context. However, next-generation sequencing (NGS)-based ST platforms frequently encounter sensitivity bottlenecks arising from sub-optimal probe architectures on solid substrates. Conventional single-stranded DNA coupling methods often lead to disordered interfacial molecular conformations due to non-specific nucleobase-mediated surface tethering, which creates steric hindrance and inhibits the enzymatic efficiency of in situ library preparation. Here, we present Decomap (double-strand protected combinatorial barcoding microarray chip), a high-performance ST platform utilizing a triple-segment (dsZ-X-Y) fabrication strategy to achieve superior transcript capture efficiency. This structural optimization significantly enhances DNA ligation kinetics and subsequent polymerase-mediated extension, overcoming the fundamental limitations of traditional single-stranded coupling strategies. Decomap-seq achieved a median detection of 7,200 genes and 29,097 UMIs per 50 m-spot at a sequencing saturation of 50.1%. These results validate Decomap as a highly sensitive and robust tool for spatial transcriptomics, offering a powerful platform for advancing research in histopathology, developmental biology, and neuroscience.

Extracellular vesicles (EVs) are critical mediators of intercellular communication, carrying molecular cargos such as small noncoding RNAs (ncRNAs) that reflect the physiological and pathological state of their cells of origin. However, studying brain-derived EVs has been challenging due to the blood-brain barrier. Here, we optimized and validated an open-flow microdialysis (OFM) protocol for sampling EVs directly from brain interstitial fluid (ISF) in wild-type and APP/PS1 transgenic mice. Ex-vivo validation using plasma EVs demonstrated that OFM effectively captures the full EV population. In-vivo cerebral OFM (cOFM) enabled successful collection of brain ISF EVs, which were characterized by nanoparticle tracking analysis (NTA), electron microscopy, and western blotting, confirming their similarity to EVs isolated directly from brain tissue and plasma. Identification of small ncRNA cargos revealed that EVs sampled from brain ISF by cOFM were enriched in brain-specific signatures, many of which are associated with neuronal cell populations and biological functions. Furthermore, we observed a unique small ncRNA signature from the brain ISF EVs in the Alzheimers disease preclinical model compared to wild-type mice. These small ncRNAs were associated with genes considered important in biological functions associated with neurodegeneration. Our findings demonstrate that cOFM is a powerful tool for in-vivo sampling of brain EVs and highlight the unique molecular landscape of ISF EV small ncRNA cargos. This study offers new opportunities for biomarker discovery and mechanistic insights into neurodegenerative diseases, such as Alzheimers disease.

Extracellular vesicles (EVs) are cell-secreted biological nanoparticles that play a crucial role in intercellular communication and are gaining increasing attention as diagnostic biomarkers, therapeutic agents, and drug delivery vehicles. Consequently, the development of robust and sensitive methods for their characterization is essential. Herein we present the use of a microscope-mounted nanofluidic device for direct size determination and multi-parametric (3-color) fluorescence-based phenotyping of single biological nanoparticles that are in the size range of 20-200 nm in a method we denote Nano-SMF (SMF; size and multiplexed fluorescence). We demonstrate that it is possible to accurately determine the size of nanoparticles by analyzing their one-dimensional Brownian motion during directional flow through nanochannels, achieving size distributions for monodisperse nanoparticle solutions that are on par with TEM analysis, and size discrimination of nanoparticle mixtures that is significantly improved compared to conventional nanoparticle tracking analysis (NTA). Furter, we demonstrate that the method can be applied to analyze EVs directly in minute volumes of cell supernatant, avoiding pre-isolation or concentration steps. The method was applied to phenotype CD63- and CD81-positive EVs from a human embryonic kidney cell model, demonstrating that vesicle sub-populations defined by these two tetraspanin biomarkers differ significantly in size.

The ability to encode and reliably read nanoscale information is increasingly important for multiplexed biomolecular detection and super-resolution imaging. DNA origami provides a uniquely programmable platform for arranging structural and functional elements with nanometer precision, enabling the creation of identifiable nanoscale patterns. In this context, DNA origami-based barcodes that incorporate gold nanoparticles (AuNPs) to encode either origami geometry or the identity of specific biological targets within defined nanoparticle patterns have been paired with transmission electron microscopy imaging for decoding. However, surface-bond AuNPs may detach during handling, purification, or biological incubation, leading to misidentification or decoding errors in barcode analysis. Here we report a rational design for the controlled encapsulation of AuNPs within DNA origami tubes to enhance nanoparticle retention and structural integrity. We engineered curvature-inducing modifications in a flat rectangular DNA origami scaffold to promote inward folding and confinement of AuNPs. These barcodes can be further functionalized on the outer surface with bioactive aptamers and/or fluorescence dyes, enabling targeted interactions with cells and optical readout. Programable dimerization further expands multiplexing capacity. This design provides a robust framework for structurally stable origami barcodes and advances the development of high-resolution, multiplexed labeling and diagnostic platforms. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/725969v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@686c1aorg.highwire.dtl.DTLVardef@1914c4eorg.highwire.dtl.DTLVardef@28ad47org.highwire.dtl.DTLVardef@8847ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

We developed a cost-effective human alveolus-on-chip based on 3D printing molds (3DP-Lung) to study early events of Mycobacterium tuberculosis (Mtb) infection in a physiologically human relevant microenvironment. This organ-on-chip platform is compatible with advanced imaging and recreates the alveolar-capillary interface by co-culturing primary human alveolar epithelial cells, endothelial cells and macrophages. We show that epithelial-only models display limited susceptibility to Mtb infection, whereas the integration of macrophages significantly enhances infection levels of the alveolar barrier and supports intracellular bacterial replication. Quantitative imaging reveals that macrophages act as a permissive niche, promoting Mtb infection of both epithelial and endothelial compartments. This accessible organ-on-chip platform enables robust modeling of early events of host-respiratory pathogen interactions and provides a valuable tool for studying tuberculosis pathogenesis in human-relevant conditions. More broadly, it lowers technical and economic barriers to accelerate the adoption of organ-on-chip technologies for studying human specific infection. SummaryA cost-effective human alveolus-on-chip enables physiologically relevant modeling of early host pathogen interaction and revealing a key role of macrophages in Mycobacterium tuberculosis infection

We present a tunable microfabrication pipeline for creating robust, reflective inserts that adapt conventional commercial imaging chambers for single-objective light sheet (LS) illumination. This system reduces the complexity associated with dual-objective LS setups and specialized LS chambers while retaining the native functionality and biocompatibility of the original chambers. The fabricated insert features a metalized, 3D nanoprinted micromirror with an angled reflective surface, enabling alignment of a thin LS for sectioning and imaging throughout mammalian cells. Using this pipeline, we demonstrate that single-objective LS illumination achieves an over 4X improvement in the signal-to-background ratio compared with conventional widefield epi-illumination in both fixed and live cell samples. Furthermore, we show substantial resolution enhancement for single-molecule localization microscopy compared to epi-illumination for improved imaging at the nanoscale. The versatile and scalable design offers an easily implemented approach to bring the benefits of single-objective LS microscopy to a wide array of biological studies.

Droplet sorting technology has the potential to revolutionize the biotechnology sector as it provides massive high-throughput screening capacity, but the technology remains not accessible for a wider audience yet. There is a need for more affordable droplet sorting platforms to design cell factories and screen cell libraries. In here we demonstrate our droplet cytometry/sorter platform for single-cell screening of yeast cells based on their fluorescence signal.

The superior stealth properties and high information density make DNA a sought-after candidate in the field of molecular steganography. Here, we developed the InfinMark end-to-end DNA steganography framework for anti-counterfeiting applications by combining the characteristics of both the Internet of Things (IoT) and DNA-of-Things (DoT). InfinMark includes five modules: Information Transcoding, Fingerprint Writing, Nano-encapsulation, Invisible Marking, and Multi-level Rapid Authentication. It ensures precise anti-counterfeiting information reading and writing through a dynamic DNA-compatible transcoding algorithm, achieves seamless embedding by developing scalable nanoparticle manufacture methods, and supports cross-scenario on-site verification, ultimately granting it comprehensive anti-counterfeiting capabilities spanning from source labeling to terminal tracing. By addressing the bottlenecks in IoT and DoT integration, lifecycle tracking, as well on-site product authentication, this research constructs a full-chain bimodal anti-counterfeiting system, thereby showcasing the practical application of informational DNA nanoparticles in various aspects of production and daily life.

Immune receptor profiling enables tracking of individual T or B cell clones across time and tissues, providing insight into immune responses, cancer, and autoimmunity. When combined with single-cell transcriptomics, it links clonotype identity to cellular function, revealing the diversity and dynamics of immune cell populations. This study presents a head-to-head benchmarking comparison of two single-cell immune profiling technologies: droplet-based microfluidics from 10x Genomics (10x) and combinatorial barcoding from Parse Biosciences (Parse). Using matched human samples from PBMCs, the analysis evaluates performance across transcriptomic and T cell immune receptor features to assess data quality, reproducibility, and chain-specific recovery. The findings provide a framework for interpreting single-cell immune profiling platforms and emphasize the importance of accounting for technology-specific biases in bioinformatic analyses.

Cerebral blood volume (CBV) and blood flow (CBF) constitute key metrics for cerebrovascular monitoring, enabling assessment of stroke severity and risk-prediction, aging-related changes, and neurological diseases. CBF and CBV monitoring are key aspects in diagnosis, treatment triage, and clinical outcome of ischemic and hemorrhagic strokes. In recent years, there have been ongoing efforts toward the development of optical devices for noninvasive monitoring of CBV and CBF. Speckle contrast optical spectroscopy (SCOS) has recently emerged as a strong candidate for clinical translation in monitoring CBF and CBV, due to its affordability, compact and wearable design, and noninvasive nature. However, experimental demonstrations that SCOS can effectively monitor brain hemodynamics remain sparse. This is primarily due to challenges in design experiments that isolate cerebral blood dynamics from those in the scalp and skull. In this paper, we report experiments using SCOS to monitor cerebral hemodynamics in rats during intracerebral blood flow modulation. To modify cerebral blood dynamics, a surgical procedure was performed to insert a catheter for direct injection of flow modulation fluids into the brain. Using the SCOS device, we monitored changes in CBV during deliberate CBF interventions into the brains of five rats. A saline solution was also injected as a sham control of the flow intervention. The results show a significant decrease in CBV during injection, followed by a return to baseline. This behavior is consistent with physiological expectations, as the injected fluids dilute the blood, leading to a transient reduction in blood volume. Notably, the CBV decrease induced by the flow modulation fluid solution required more than twice as long to recover to baseline compared with the saline solution, which is consistent with the delayed clearance of the flow modulation fluid by design. These experimental results demonstrate the effectiveness of SCOS for monitoring cerebral hemodynamics in animal models and highlight its potential for translation to human studies. Moreover, this work paves the way for the testing and characterization of cerebral therapeutic agents intended for blood flow modulation in animal models.

Double-stranded RNA (dsRNA) is a potent immunogenic impurity and its detection is a critical quality attribute in characterizing mRNA therapeutics. Standard analytical methods (e.g., sandwich ELISA) are only able to resolve the bulk presence of dsRNA and cannot characterize the different sub-species that may be present within a mRNA sample.. In this study, we use mass photometry (MP) as a single-molecule analytical platform for the simultaneous detection and characterization of dsRNA impurities in mRNA samples. We demonstrate how ionic strength can interfere with the stability of the mAb/dsRNA complex and measure the binding affinity (1 nM) under a set of parameters for reproducible characterization of the complex. We then leverage the J2 antibody to identify antibody/dsRNA complexes that then resolve dsRNA-positive species within an mRNA sample based on discrete molecular weight profiles. Furthermore, we introduce a novel MP assay that harnesses the repulsive surface chemistry of uncoated glass to exclude the bulk mRNA analyte to enable the use of higher loading concentrations to sensitively profile trace dsRNA impurities as antibody-bound species. This work establishes MP as a valuable next generation mRNA analytical tool for analyzing dsRNA byproducts within mRNA samples.

Extracellular vesicles (EVs) carry microRNAs (miRNAs) that mediate intercellular communication and have strong potential as disease biomarkers, yet the roles of miRNA isoforms (isomiRs) in EVs remain poorly understood. Here, we analyzed 96 human EV and corresponding source samples from nine public datasets. We found that EV samples consistently contained substantially higher proportions of isomiR reads than their corresponding source samples, indicating widespread isomiR enrichment in EVs. Although individual isomiRs showed limited reproducibility across biological replicates and limited sharing between EVs and their corresponding source samples, the parent miRNAs that generated these isomiRs remained highly reproducible across replicates and strongly shared between EV-source pairs. Despite extensive isomiR diversification, EV-source pairs retained highly correlated miRNA expression profiles. Using integrated miRNA- and isomiR-related features, we further developed a random forest model that successfully associated EV samples with their corresponding source samples, with improved performance when isomiR information was included. Together, our results demonstrate that EVs are enriched for biologically meaningful isomiRs while preserving source-associated miRNA landscapes, highlighting the importance of incorporating isomiRs into future EV studies.

Many experiments rely on expensive or scarce liquids, such as costly reagents, or biological samples available only in limited quantities. Droplet microarrays are an especially promising approach to conserving these materials because they support highly parallelized reactions in small volumes. However, existing droplet microarray loading methods based on discontinuous dewetting suffer from loading inconsistencies and large dead volumes. In this work, we present the Small Volume Loader (SVL) for the Surface Patterned Omniphobic Tiles (SPOTs) platform that enables precise deposition on droplet microarrays while minimizing reagent waste. By establishing a physical model of the loading process, we identified that deposition volume is governed by the sum of hydrostatic and Laplace pressures at the reservoir outlet. To optimize performance, we engineered a pressure-compensating flared reservoir geometry that maintains constant total pressure regardless of the remaining liquid level. This design ensures that the deposited volume is independent of reservoir volume and reduces dead volume to 5 L. We demonstrated the platforms utility through high-throughput elicitor screening for natural antimicrobial production from Streptomyces venezuelae. The resulting assays used 100-fold less material than conventional methods, allowing us to conduct over 32,000 assays with modest quantities of starting material. This enabled us to identify specific stressors that optimize the production of the antibiotics chloramphenicol and jadomycin B. Together, we demonstrated improved loading performance for droplet microarray platforms, allowing precise, accessible, and high-throughput assays using only minimal volumes of scarce materials.

Multiplex staining is a technique that allows the identification of cell types within a single tissue section by simultaneously detecting multiple molecular markers. Generally, multiplex staining is performed using several combinations of probes, including specific antibodies, nucleic acid probes, and lectins. Here, a novel multiplex staining strategy that relies exclusively on lectin probes that target glycans is presented. Glycans have a vast variety of structural forms that vary depending on cell type-specific modifications. Furthermore, an enormous number of glycan-binding molecules, collectively known as lectins, exist in the biological world. Each lectin displays specificity for a particular glycan motif while maintaining broad affinity. Although lectin-based cell staining has been used in various applications, the partial and limited specificity of lectins has hindered the use of glycan-targeted multiplex staining with lectins. In addition, lectin probes have largely been avoided for cell-type identification because of the absence of strict cell-type-specific glycans. Here, a novel staining method, Glycan Painting, is introduced. Rather than viewing the partial specificity of lectins and the broad, non-cell-type-specific distribution of glycans as drawbacks, this approach turns these features into advantages by generating distinct color patterns that comprehensively visualize cell-type-specific glycan combinations and enable full-color imaging of tissues.

A critical challenge in endocrine neurosurgery is intraoperative discrimination between normal pituitary tissue and pituitary neuroendocrine tumors (PitNETs). Suggesting the universal persistence of near-infrared autofluorescence (NIRAF) in endocrine organs and inspired by routine clinical use of NIRAF for parathyroid gland identification, we discovered that pituitary NIRAF can be employed for label-free transsphenoidal surgery guidance. Ex vivo confocal spectral imaging of 33 specimens identified secretory granules as the dominant long-wavelength fluorescence source and showed that normal pituitary had higher granule content than PitNETs. For the first time, we made use of the pituitary NIRAF during surgery and assessed its performance for pituitary/adenoma separation in vivo for 27 surgeries and showed near-perfect separability between pituitary and non-pituitary measurement sites with ROC-AUC of 0.98. The obtained results clearly demonstrate that the suggested method, based on the solid microscopic background, has the potential for clinical translation and paves the way for enhanced gland preservation during resection.

The optimization of mRNA-lipid nanoparticles (mRNA-LNPs) for therapeutic applications is limited in part by the inadequate characterization of mRNA payload heterogeneity. One current challenge is accurately measuring the number of mRNA copies within individual LNPs, where the standard method of intensity-based mRNA number determination is sensitive to fluorescent dye-dye interactions and heterogeneity of mRNA labeling. Here we present a single-particle microscopy method that combines direct counting of the mRNA copies per LNP with LNP size measurements. While confined in microwells, individual mRNA-LNPs are lysed to release their cargo and stained with a dye such that the number of mRNA molecules in each well can be directly counted using fluorescence microscopy. Since the method stains the mRNA cargo in situ, it enables characterization of LNPs formulated with therapeutic grade (e.g., unlabeled) mRNA. We applied this approach to two Onpattro(R)-based LNP formulations prepared using different formulation buffers, where the two formulations had different average mRNA copy number, particle size, and fraction of LNPs lacking mRNA. The ability to directly count the number of mRNA molecules in LNPs establishes a complimentary method to intensity-based mRNA number determination and supports the characterization and screening of clinically relevant LNP formulations.

Apohas Liquid State Intelligence Platform (LSIP) records ellipsometric waveforms from injections depositing sub-microgram quantities of antibody drop-by-drop onto a liquid reservoir. We previously showed that a behavioural feature extracted from the waveforms, VIBE1, identified antibodies carrying multiple biophysical liabilities in an industrial dataset of 71 monoclonal antibodies, and enriched for clinical failure across a larger dataset of 235 therapeutic antibodies [1]. Here, we use an auxiliary coalescence-sensor channel to decode VIBE1 by separating the coalescence event from its propagation through the substrate. The pertitration drop-to-drop standard deviation of pinch-off time,{sigma}{tau} , explains most of VIBE1s variance across the dataset (R2 = 0.92, n = 1182). High-speed imaging at 10,000 frames per second reveals that all imaged drops initially thin at the same Newtonian capillary-inertial rate while the neck remains wide. In drops from certain antibodies, the thinning bridge then decelerates as internal strain builds in the narrowing neck. This elasto-capillary stiffening response has a timescale{lambda} that decreases as pinch-off time{tau} i increases across the imaged set.{sigma}{tau} is therefore a readout of the antibodys propensity to undergo a transient gel-like stiffening response during coalescence, and that variability is what VIBE1 captures. The signal is concentration dependent, and absent in bovine serum albumin (BSA) tested at up to an order of magnitude higher molarity than the antibodies, despite BSA being a strongly surface-active globular protein. The instrument is configured so that complex behaviours of this kind appear in its recorded waveforms; the gel-like coalescence response we identify here is one such phenomenon.

With the introduction of dedicated nanoscale flow cytometers, the need for suitable compensation beads has emerged. Here, we present a rapid and cost-effective method to generate [~]100 nm antibody-binding compensation beads compatible with a wide range of antibody species for use in nanoscale flow cytometry. This approach may provide a practical interim solution until commercial alternatives become available.

Extracellular vesicles (EVs) carry molecular cargo that can reflect the real-time state of parental cells, yet most in vitro EV analyses rely on bulk approaches and therefore average over pronounced heterogeneity in both cell and EV populations. Here, we present a semi-open microfluidic platform that enables multi-marker profiling of EVs released from single-cell-derived clones, allowing EV signatures to be linked to clonal progeny originating from a single parental cell. The platform integrates aligned cell and EV arrays containing 17,305 wells, assembled with a 3D-printed housing to capture released EVs in one-to-one matched wells. Captured EVs are immunolabeled for canonical tetraspanin markers (CD9, CD63, CD81) and EpCAM, imaged by high-resolution fluorescence microscopy, and quantified using an automated image-analysis pipeline. Applying the platform to single-cell-derived PC3 clones revealed substantial heterogeneity in EV marker co-expression, with hierarchical clustering identifying four distinct tetraspanin co-expression profiles. The fraction of EpCAM-positive EVs increased with PC3 cell proliferation, as assessed by endpoint cell number, whereas free (non-EV-associated) EpCAM showed no correlation. This platform enables near single-EV-level, multi-marker profiling from single-cell lineages and provides a practical approach to simultaneously dissect both cellular and EV heterogeneity.

Paper-based diagnostics such as lateral flow assays (LFAs) and microfluidic paper-based analytical devices ({micro}PADs) have attracted considerable attention because of their low cost, portability, and ease of use. Currently, to enable fabrication of {micro}PADs and improve LFA performance, hydrophobic blocks are patterned on paper substrates. However, fabrication of high-resolution hydrophobic barriers remains a major challenge. In this work, we developed a novel silicone extrudable ink for the fabrication of hydrophobic features on paper substrates. The ink was formulated using a vinyl-terminated polydimethylsiloxane (vPDMS) and polymethylhydrosiloxane (PMHS) system crosslinked through platinum-catalyzed hydrosilylation, and its rheological properties were tailored by incorporating silica fillers, obtaining a shear-thinning gel suitable for extrusion. The resulting formulation provided tunable properties, controlled deposition, and stable feature formation, enabling simple, low-cost, rapid, and robust fabrication of high-resolution hydrophobic barriers. Using this approach, we demonstrated improved fluid confinement and pattern fidelity on paper substrates, fabricated high-resolution paper microfluidic devices down to 150 {micro}m channel width, and enhanced the sensitivity of an LFA for a malaria diagnostic test. These results highlight the potential of this silicone ink platform as a practical and scalable strategy for advancing high-performance paper-based diagnostic technologies.