Small Methods

Uniform, reproducible blood smears are critical for reliable hematological evaluation. Manual smear preparation, however, is user-dependent and introduces variability that limits quantitative microscopy. Here we developed BUsmear, a low-cost, 3D-printed, motorized blood smear device that prepares two smears simultaneously from a printed stage and a micro-motor drive with tunable linear velocity, controlled through a joystick-operated driver module. By spreading two slides in parallel with fully repeatable slide-to-slide motion, the device doubles throughput while eliminating operator-dependent motion artifacts, and can be fabricated on any benchtop 3D printer in under one day. To validate smear quality, we analyzed blood films from three donors together with a manual smear prepared by an expert from the blood of one of the same donors, giving a matched device-versus-manual pair. Automated Cellpose segmentation of 14,046 red blood cells across 12 bright-field fields showed that cell diameter was preserved and closely matched the expert smear (6.7-7.8 um across groups, within the 6.2-8.2 um human reference range; 6.7 vs 6.9 um in the matched pair), and that all films formed non-aggregated monolayers (Clark-Evans index of aggregation 1.04-1.24). Critically, red blood cells in the device films were markedly more circular than in the expert manual smear (mean eccentricity 0.405 vs 0.502; 0.443 vs 0.502 in the matched pair), with complete separation between the two methods at the level of whole fields of view. Because eccentricity reports smear-induced cell distortion, this indicates that a constant, mechanically controlled spreading velocity preserves red blood cell morphology better than skilled manual technique. BUsmear offers an accessible route to standardized smear geometry for quantitative analysis, including AI-based morphometry, and its low cost may be particularly advantageous in low-income countries with a high prevalence of malaria.

The ultrastructure of endothelial cells (ECs) "in situ" is of great interest due to their involvement in many physiological processes. In some organs, these cells form transcellular pores or fenestrae, allowing for the rapid exchange of molecules between blood and interstitium. Despite their importance, no optical images of these dynamic morphological structures have yet been acquired in situ. Major obstacles to their in-situ imaging are the lack of specifical labels for fenestrae and their size well below the optical diffraction limit. Here, we report how we have overcome these challenges and managed to visualize the EC ultrastructure in situ in 25 {micro}m thick liver sections. To enable this, a lipophilic, fluorescent membrane dye was infused into the portal vein of murine livers to stain the sinusoidal ECs before the organ was harvested. Tissue sections were subsequently imaged using a novel, super-resolution optical-sectioning structured illumination microscope (OS-SIM), providing approx. 170 nm spatial resolution with significantly faster image acquisition compared to confocal microscopy.

Ovarian cancer (OC) remains a leading cause of gynecologic cancer mortality due to late-stage diagnosis and limited early detection strategies. Ascites fluid, a pathological hallmark of OC, is a rich source of tumor-derived extracellular vesicles (EVs) that reflect the tumor microenvironment and hold promise for biomarker discovery. However, isolating EVs from minimal ascites volumes (<100 {micro}L) poses technical challenges using conventional methods like ultracentrifugation or size-exclusion chromatography (SEC). This study explores the application of strong anion exchange (SAX) magnetic beads (Mag-Net) for efficient EV isolation from as little as 2 {micro}L of ascites fluid from both murine models and a human patient with mucinous borderline tumor. We demonstrate that SAX achieves robust EV capture at 10{micro}l of input volume, enabling comprehensive proteomic profiling and single-EV surface-enhanced Raman spectroscopy (SERS) with a >2-fold increase in proteomic depth compared to raw ascites. Notably, this study was able to identify 1000 proteins not previously annotated in Vesiclepedia for OC-derived EVs, alongside distinct SERS signatures, highlighting the potential for multiomic analysis. Comparative analysis with UC revealed enhanced proteomic depth obtained with SAX beads, albeit we also observed differential detection of canonical markers (e.g., CD9, CD81) between input volumes of ascites fluid. These findings establish SAX as a scalable, low-input platform for EV-based biomarker discovery, paving the way for improved early detection and molecular insights into OC progression.

Automation of organoid and cell culture processes is essential for achieving scalable and standardized experimentation in regenerative medicine and stem cell research. However, existing microfluidic platforms often rely on complex setups, limiting their integration within standard incubator environments. To address these challenges, we developed a compact, scalable multi-well platform featuring 3D-printed, servo-actuated disposable microvalves for fully automated media and drug exchange. This design eliminates the need for external pressure sources and control channels, providing a simplified and cost-effective solution for organoid culture. The platform integrates an internet-connected microscopy module with a motorized XYZ stage, allowing continuous, real-time imaging of individual wells directly within the incubator. It supports precise and reliable fluid handling under physiological conditions, improving throughput, reproducibility, and accessibility. We validate the platform through bench-top testing and in both mouse and human organoid models. Morphological analysis, immunohistochemistry (IHC), and qPCR demonstrate comparable viability, growth, and gene expression profiles between automated and manual culture conditions. These results establish a robust and scalable framework for fully automated organoid culture, offering a simplified and accessible alternative to conventional microfluidic systems with broad applications in regenerative medicine, drug discovery, and scalable biological screening. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/732526v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@5efd07org.highwire.dtl.DTLVardef@3600d0org.highwire.dtl.DTLVardef@16f85f5org.highwire.dtl.DTLVardef@c39fbd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Non-obstructive azoospermia (NOA) represents the most severe form of male infertility, severely limiting a patient's prospects for biological fatherhood when surgical retrieval fails. However, the true biological limits of NOA remain obscured by the inherent limitations of conventional gamete recovery protocols: standard centrifugation frequently causes substantial cell loss, masking extremely rare sperm, while surgical interventions are constrained by spatial sampling biases. Here we report SpermSeek, an integrated AI-guided microfluidic platform for real-time, non-destructive isolation of single sperm directly from semen. Operating at scalable throughput (0.36 mL/h), the system achieves 98.3% detection precision and a 95.5% target encapsulation efficiency, suppressing background debris. In a 59-patient NOA cohort, SpermSeek detected morphologically identifiable sperm in 64.4% (38/59) of cases, spanning diverse genetic etiologies, including AZFb/c microdeletions, and severe histopathological phenotypes, such as Sertoli-cell-only syndrome (SCOS). Notably, among a sub-cohort of 41 patients who remained consistently sperm-negative despite prior medical or micro-TESE interventions, our platform identified gametes in 53.7% (22/41) of these cases. Comprehensive safety profiling in healthy human donors and wild-type mice confirmed that processed sperm retain high DNA integrity and epigenomic concordance (r=0.98), supporting transgenerational developmental stability in mice. Furthermore, in a 26-patient validation cohort, SpermSeek recovered rare sperm in 11 cases. Utilizing gametes from a subset (n=5), we demonstrated their capacity to support early human embryogenesis, yielding high-quality cleavage-stage embryos with confirmed genomic euploidy. This work establishes a highly sensitive framework for re-examining the biological limits of human spermatogenesis, laying the foundation to expand autologous reproductive options for patients refractory to conventional retrieval protocols.

Tissue monolayers are active living matter whose three-dimensional (3D) architecture and mechanical remodeling regulate tissue function, yet fast and continuous topographic monitoring remains difficult. Here, we present FLuorescence EXclusion microscopy fOr Monolayers (FLEXOM), a microfluidic platform that combines multilayer micropillar arrays with negative-staining optics to convert wide-field images into self-calibrated 3D height maps. Geometry-anchored reference cavities maintain in-frame calibration standards even in fully confluent monolayer fields, enabling continuous 3D profiling from isolated cells to confluent sheets with sub-second temporal resolution ([~]250 ms), sub-micrometer axial precision ([~]0.37 {micro}m), and multi-day biocompatibility and operational stability (>96 h). FLEXOM reveals two distinct anisotropic signatures in tissue monolayers. First, monolayers of different cell types show distinct vertical thickness profiles despite comparable lateral footprints. Only two vertical descriptors, mean height and height variability, distinguished cell types more accurately than nine 2D lateral descriptors, including area, circularity, and convexity (95% vs. 76%). Second, under acute osmotic shock, confluent monolayers exhibit geometrically anisotropic and temporally decoupled vertical-lateral remodeling: height changes dominate the overall volume response and precede lateral remodeling. Surprisingly, cyclic isotonic-hypotonic pulses every 3 min confine remodeling entirely to the vertical axis. Overall, our work provides a high-resolution 3D profiling tool and a framework for axis-resolved analysis of topographic responses in active living matter.

Treatment with chimeric antigen receptor (CAR) T cells has emerged as a promising immune therapy for relapsed and refractory hematologic malignancies. The CAR T cells are manufactured in a series of steps that involve isolating T cells from the patients leukapheresis product, genetically modifying them to express the CAR against the target antigen, and reinfusing them into the patient. Efficient T-cell enrichment from leukapheresis products is critical to the success of these therapies. Current methods for T-cell sorting on a clinical scale involve several washing steps to remove red blood cells and platelets, followed by T-cell selection and activation. These multi-step processes result in cell loss during processing and involve several handling steps. Here, we utilize fluidically assembled micromagnetic lenses to develop a high-throughput, continuous-flow microfluidic T-cell sorter, designated as the T-Chip, for sorting magnetic bead-labeled CD3+ T cells in a single step. Our approach allows direct sorting of T cells in expansion media from leukopaks without any washing steps, effectively removing 99.999% of RBCs and platelets from the leukapheresis product. A single 1-inch x 3-inch T-Chip can process leukapheresis product at a throughput of 60 mL/hr and 2.56 {+/-} 0.12 billion cells/hr. Using this optimized workflow, we demonstrate clinical-scale enrichment of highly pure CD3+ T cells (97.7 {+/-} 1.3%) with high viability (97.0 {+/-} 1.1%) and recovery (87.3 {+/-} 14.8%) in a functionally closed manner. Downstream processing of T cells isolated using the T-Chip yielded potent anti-mesothelin CAR T cells with demonstrated anti-tumor efficacy. Overall, by exploiting precisely engineered magnetic forces and laminar flow, the microfluidic T-Chip overcomes bottlenecks caused by low throughput and enables single-step large-scale T-cell purification for the rapid development of CAR T cells.

High-throughput droplet microfluidics can compartmentalize bacterial interactions, but recovering droplets displaying phenotypes of interest often requires custom fluorescence-activated droplet-sorting instrumentation. Here, we introduce post-assay photogelation to decouple the material requirements of bacterial coculture from those of commercial flow sorting. Bacteria are cocultured in initially aqueous water-in-oil droplets containing photoreactive polymer precursors. After interaction phenotypes develop, ultraviolet exposure converts the droplets into mechanically stable hydrogel particles that can be transferred to an aqueous carrier and sorted using a commercial benchtop cell sorter. The sorted particles can subsequently be degraded enzymatically to release the encapsulated bacteria. We show that the timing of gelation alters bacterial growth and spatial distribution within droplets, with post-assay gelation supporting greater and more uniformly distributed growth than culture in preformed hydrogels. Using two fluorescent bead-encoded hydrogel-particle populations, we demonstrate sorting to greater than 99% purity. As an end-to-end demonstration, we cocultured sfGFP-expressing Escherichia coli Nissle 1917 with a cultured human nasal bacterial community and found that E. coli Nissle became the predominant detectable population under the tested conditions with possible inhibition of the cultured nasal bacteriome. This liquid-to-solid transition provides an accessible interface between aqueous bacterial droplet assays, commercial particle sorting, and downstream microbial analysis.

Epithelial tissues continuously remodel their curvature during morphogenesis, homeostasis, regeneration, and disease, yet experimental access to time-varying curvature remains limited. Here, we introduce CurvoChip, a pneumatically actuated microsystem that reversibly deforms confluent epithelial monolayers cultured on a 20-m elastic membrane into concave or convex geometries. The device operates either in a standard incubator or on a microscope stage and provides programmable control over pressure amplitude, direction, and cycling. Analytical scaling, finite-element simulations, and confocal profilometry establish predictable membrane deformation across the operating range, whereas cycling between -400 and +400 mbar for 120 cycles produces stable deflection without detectable drift or residual deformation. We further implement a three-dimensional surface-reconstruction and segmentation workflow to quantify cell and nuclear morphology on curved monolayers. Acute curvature induction produces a marked polarity-dependent response: convex deformation causes greater cell spreading and epithelial thinning than concave deformation, while nuclear projected area, thickness, and volume change in a direction- and position-dependent manner. These results show that epithelial architecture is sensitive not only to curvature magnitude but also to its orientation relative to the apico-basal axis. CurvoChip therefore provides an accessible platform for dissecting how epithelial tissues integrate dynamic geometric cues.

Tracking suspended cells over multiple time points at the single-cell level remains challenging because existing flow-based methods cannot preserve cell identity while maintaining high throughput. Here, we present RASPBerry, a hydrogel-based spatial barcoding platform for time-lapse flow cytometry. RASPBerry generates unique barcodes by randomly co-encapsulating fluorescent beads with individual cells in hydrogel droplets, eliminating the need for predefined barcode patterns or specialized optical instrumentation. We integrate RASPBerry with acoustofluidic imaging flow cytometry to enable time-lapse imaging flow cytometry of suspended cells. The platform identifies more than 17,000 hydrogel droplets with 99.8% matching accuracy. We further demonstrate time-lapse tracking of more than 10,000 suspended cells and quantify stress-induced nuclear morphological changes in more than 5,000 individual cells. RASPBerry provides a simple, scalable, and broadly accessible strategy for time-lapse imaging flow cytometry, expanding the capability for dynamic single-cell analysis of suspended cells.

Precise, non-invasive manipulation of individual living cells remains a central challenge in biomedical science, with far-reaching implications for single-cell analysis, tissue engineering, and the study of cell-cell interactions. Here, we report the first demonstration of single-cell control using bulk acoustic standing-wave acoustofluidics with closed-loop feedback. We introduce VeLO (Vector-based Local Optimization), a model-free, reinforcement learning-inspired algorithm that enables programmable two-dimensional manipulation of individual cells using a single piezoelectric transducer. Without prior calibration or physical modeling, VeLO learns system dynamics online from acoustically induced cell displacements and automatically adapts to nonlinear, time-varying conditions. We achieve robust control across multiple cell types (DU-145, Jurkat, K-562) and independent manipulation of multiple cells, including controlled cell-cell contact. By combining simplicity of hardware with autonomous, adaptive control, this approach establishes multimodal acoustofluidics as a versatile tool for label-free, high-precision single-cell manipulation.

Kidney-function assessment relies on blood urea as a clinically informative metabolic marker; however, its dependence on venipuncture and centralised laboratory testing limits high-frequency monitoring and delays timely clinical intervention. Here, we report an integrated platform combining a wearable buffered microfluidic patch with a physiology-informed, data-driven calibration framework for real-time, non-invasive estimation of blood urea from microlitre-scale sweat volumes (4.79 L). By precisely regulating the release kinetics of internal buffer salts, the device stabilises the local reaction microenvironment, mitigating variability in sweat pH and flow to ensure reproducible measurement. The resulting signals are processed through an artificial intelligence (AI)-enabled analysis pipeline that integrates sweat urea with patient-specific physiological information to generate clinically interpretable outputs. In multicentre studies, sweat urea shows a strong association with blood urea across diverse cohorts, but with nonlinear and time-lagged relationships that limit direct use. The AI-enabled calibration model compensates for these effects, enabling high-fidelity estimation of blood urea (r = 0.945 versus gold-standard measurements) at clinically relevant concordance levels. The platform further identifies kidney injury with 89.1% accuracy and stratifies disease severity with 83.2% accuracy. Notably, these results demonstrate that the integration of physicochemical stabilisation and AI-enabled data-driven translation establishes sweat as a clinically actionable surrogate for renal monitoring, supporting population-level estimation and highlighting the potential for personalised longitudinal assessment, and enabling a scalable, non-invasive strategy for high-frequency kidney disease management.

Perfusable vascular microphysiological systems are increasingly used to model angiogenesis, tissue crosstalk, and disease. However, many platforms still rely on oscillatory, discontinuous, or poorly controlled perfusion regimes, limiting the study of sustained flow-dependent vascular remodeling. Here, we establish a tunable, unidirectional laminar flow workflow for long-term perfusion of angiogenic vasculature-on-chip cultures and use it to investigate endothelial, perivascular, and immune cell responses to sustained flow. Using an AIM Biotech microfluidic platform containing 14-day-old human umbilical vein endothelial cell-derived angiogenic sprouts and pericytes, continuous perfusion enabled intraluminal transport of 1 m tracer beads through vessels, demonstrating stable flow across the vascular bed. Sustained laminar flow induced endothelial remodeling at both the mother vessel and sprout levels, with cellular alignment evident in both compartments. Quantitative analysis of the mother vessel further revealed Golgi polarization against the direction of flow. Sustained perfusion also increased pericyte recruitment to angiogenic sprouts and reduced endothelial proliferation within the mother vessel, consistent with flow-driven vascular maturation and quiescence. Live-cell imaging further captured directional endothelial migration against the flow, lumen remodeling, and dynamic pericyte behavior under continuous perfusion. In immune-cell assays performed under continuous-flow conditions, interactions with untreated endothelium were limited, whereas inflammatory activation increased immune-cell adhesion and crawling. These observations suggest that sustained flow supports a quiescent endothelial phenotype and demonstrate the suitability of the platform for studying inflammatory activation and immune-vascular communication under controlled hemodynamic conditions. Beyond its biological relevance, the workflow provides practical advantages for live-cell imaging, low medium consumption, and downstream perturbation studies. Moreover, the modular design of the platform makes it well suited for vascular-organ crosstalk applications. Collectively, these results establish laminar flow angiogenic vasculature-on-chip as an experimentally tractable model for studying vascular mechanobiology, vascular maturation, and dynamic cell interactions under defined hemodynamic conditions.

All-gold electron microscopy (EM) grids reduce beam-induced motion relative to conventional holey carbon supports and provide biocompatible substrates for cellular cryo-EM. However, placing customizable all-gold grid fabrication in the hands of researchers requires accessible processes based on standard microfabrication tools. We report a wafer-scale process using microfabrication techniques available in most academic cleanrooms such as lift-off metallization, electroplating, and sacrificial layer release to fabricate 594 all-gold grids per 4-inch wafer without individual grid handling. A numerical electroplating model provides a quantitative framework to relate gold deposition, grid-bar thickness, and tilt-compatible grid geometry. We show that oval 2 {micro}m x 6 {micro}m foil holes bias on-grid actin organization by substrate geometry alone, without chemical micropatterning. The EM grids supported a 2.15 [A] apoferritin single-particle reconstruction on a 200 kV cryo-TEM and are compatible with protein micropatterning and cell culture. This platform establishes an accessible route to programmable, application-specific all-gold cryo-EM supports that couple high-resolution structural imaging with engineered control of cellular organization.

Three-dimensional (3D) imaging represents the development of next generation of fluorescence microscopy. However, routine axial down-sampling makes isotropic resolution unrealistic. Here, we propose DeepUI, a physical zero-shot framework designed to achieve isotropic 3D fluorescence images from a low axial sampling rate. DeepUI fully leverages the intrinsic characteristics of 3D images through physics-guided degradation, which incorporates spatial-frequency joint learning to generate a scaled optical transfer function, combined with noise degradation and an up-sampling branch. Typically requiring just 5 minutes for training and 0.5 minutes for high-throughput and fast prediction, we demonstrate the superior performance of DeepUI to get isotropic results, and the exclusivity to axial down-sampling conditions, even in more challenging conditions, including defocused background, noise, and resolution blur.

Despite the significant technical advancement in spatial transcriptomics, its clinical usage is largely untapped. Here, we develop an integrated system, ENDO-Genome, for minimally invasive in-body transcript sampling to facilitate live spatial transcriptomic analysis of human internal organs. This is achieved by integrating a nanoarrayed biochip with existing endoscope to perform pressure-sensor-calibrated "Touch & Go" RNA extraction directly from human internal organs, including the highly vascularized liver or kidney, without the need for tissue biopsy, voiding any bleeding risks. By a demonstration using gastrointestinal endoscopy, multiplexed landscape of 55 mRNA transcripts was obtained from multiple locations of human intestinal tract via a 5-minute operation in routine examinations. Benefiting from a sequencing-free approach, each assay costs less than 10 US dollars. For the clinical study involving 15 Crohn' s disease (CD) patients, no complication case was reported out of 47 ENDO-Genome operations, showcasing the gentle deposition and excellent safety of the technique. The live spatial transcriptomics provides direct in vivo pictures of the heterogenous spatial transcriptional programs underlying CD pathological response at different intestinal locations, revealing distinct ileal phenotypes. This is manifested by unique microscale scattering of inflammation gene clusters, along with the discovery of a tissue-specific cooperative mechanisms between inflammation and RNA methylation regulations at single- or multi-cell scales.

Multicellular spheroids better recapitulate native cardiac tissue than two-dimensional systems, preserving cell-cell and cell-matrix interactions and relevant signalling. However, analytical tools for extracting quantitative data from these complex models remain limited. Here, we present an optimised holotomography (HT) workflow for fixed spheroids, enabling label-free quantification of protein concentration and dry mass across conditions. Using a hypoxia-reperfusion injury model to mimic myocardial infarction, HT measurements reveal a statistically significant reduction in the protein concentration of cardioids, reflecting impaired structural integrity and declining viability, subtle changes often missed by conventional approaches. These findings establish HT as a robust, scalable method for quantitative analysis of 3D cardiac models, with direct relevance for disease modelling and preclinical research.

DNA-based technologies rely on short, transient hybridization events, but selecting sequences with desired kinetic properties remains largely empirical because hybridization kinetics are difficult to predict from sequence and slow to measure one sequence at a time. Here, we introduce SPARXS-Hyb, an implementation of SPARXS (Single-molecule Parallel Analysis for Rapid eXploration of Sequence space) for multiplexed sequence-resolved screening of DNA hybridization. Using a surface-immobilized docking-strand library and a quencher-labelled imager-strand library, we screened 128 different DNA sequences in a single kinetic measurement, exposing all sequences to identical experimental conditions. This multiplexed approach removes a major confounding factor of serial measurements, allowing sequence-dependent differences to be compared directly. The resulting dataset reveals sequence-dependent transient binding behaviours and enabled us to identify a sequence with which an order-of-magnitude higher sampling rate can be achieved in DNA-PAINT (DNA points accumulation for imaging in nanoscale topography), a super-resolution microscopy technique based on DNA hybridization. By enabling multiplexed screening across a sequence library, SPARXS-Hyb provides a route to kinetics-guided sequence selection for programmable transient interactions in DNA nanotechnology.

Cardiac blood flow is a regulator of several important developmental and remodelling processes in the heart, including through fluid shear forces sensed by the endothelial cells lining the heart. However, optically mapping these flow fields in the complex 3D geometry of the heart is challenging even in transparent animal models such as the zebrafish. One of the main challenges is the difficulty in measuring the out-of-plane (axial) velocity component, preventing accurate mapping of the complete 3-component-3-dimension (3C-3D) blood flow velocity field; image-based techniques such as microscopic particle image velocimetry ({micro}PIV) traditionally only provide the in-plane flow components. Here we present a computational approach to achieve full time-varying 3C-3D blood flow vector mapping using a standard selective plane illumination microscope (SPIM), based on robust cardiac phase assignment, precise measurement-driven registration of sequentially acquired z-stacks, and PIV data fusion from multiple sample orientations. Our approach holds the key to understanding the complex dynamic flow fields within the developing heart, and their role in shaping cardiac development.

Minimally invasive surgery is a powerful technique that enables operations deep within the body while minimizing patient trauma and recovery time. Optical endoscopes are key to providing intraoperative vision but still face challenges due to the loss of essential senses, including depth perception and tactile feedback for tissue evaluation. Thus, it is critical to develop endoscopic imaging technologies that can augment operators with critical information. In this work, we explore a prototype multimodal 3D imaging endoscope that integrates volumetric light-field imaging with laser-speckle contrast imaging to simultaneously capture 3D structure and blood-flow information in a clinically relevant form factor.