Small Methods

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.

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

Single-molecule localization microscopy (SMLM) enables nanoscale imaging but remains limited in three-dimensional (3D), high-speed, and high-density applications due to background fluorescence, photon inefficiency, and large point-spread function (PSF) footprints. Here, we present single-objective light-sheet microscopy with PSF-engineering enhanced biplane detection (SoLiD-3D), a versatile imaging platform that integrates dual-wavelength light-sheet illumination with dual-color, multi-configuration biplane imaging for parallel acquisition with PSF engineered detection for high-speed volumetric SMLM. Parallelized single-objective light-sheet excitation combined with PSF engineering overcomes key limitations of conventional wide-field and biplane approaches. Independent control of two excitation wavelengths for optical sectioning enables simultaneous dual-target imaging and single-target dual-color imaging with improved contrast and temporal resolution utilizing dynamically displaced light sheets for volumetric coverage. Using SoLiD-3D, we demonstrate high-speed single- and dual-target dual-color imaging that doubles localization density without sacrificing photon efficiency and continuous volumetric imaging via PSF-engineering enhanced biplane detection for whole-cell 3D imaging with improved axial localization performance over extended depth ranges. We further demonstrate improved speed by utilizing the Hummus PSF, a compact engineered PSF that enables high-precision 3D localization with a substantially reduced spatial footprint, for the first time for super-resolution imaging applications. Taken together, SoLiD-3D mitigates the trade-off between axial range and localization precision and offers improved speed compared to conventional 3D SMLM approaches.

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.

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

Laser lithotripsy (LL) is the gold standard for urinary stone management yet maximizing ablation efficiency while maintaining procedural safety remains clinically challenging. Here, we present a visibly transparent, near-infrared (NIR)-absorbing ITO@SiO2 nanofluid irrigation strategy that significantly enhances LL efficiency without compromising endoscopic visibility. By spectrally matching the absorption profile of ITO@SiO2 with the clinical Holmium:YAG laser wavelength, ablation efficiency improved by >200% in the bench-top spot treatments and >100% in the hydrogel kidney model. Mechanistic investigations revealed that the enhanced optical absorption of the nanofluid modifies bubble dynamics and synergistically amplifies photothermal/microexplosion effects and cavitation damage. Importantly, both in-vitro hydrogel and in-vivo porcine kidney models demonstrated a substantial thermal safety margin (maximum temperatures <35 {degrees}C) and excellent acute biocompatibility, with no evidence of thermal tissue injury. Integrating seamlessly into established clinical workflows without requiring stone pretreatment, this strategy offers a highly translatable, safe, and efficient platform for next-generation endoscopic lithotripsy.

Expanding genetic engineering beyond model microorganisms is critical to unlocking novel applications in biotechnology, yet the low efficiency of DNA delivery methods like conjugation, remains a major bottleneck in non-model and environmental microbes. Here, we present an automated, high-throughput droplet microfluidic platform that enhances conjugation by encapsulating donor and recipient microbes in picoliter-scale water-in-oil microdroplets, stabilizing cell-cell contact and DNA transfer. Optimization of incubation time, donor to recipient ratio, and plasmid type yielded over a 100-fold increase in conjugation efficiency compared to conventional methods and enabled delivery of complex DNA libraries in low reaction volumes, demonstrating scalability for pooled plasmid library delivery. We further utilized a synthetic biology circuit for donor removal within microdroplets without antibiotic selection, eliminating the need for host-specific selection markers or engineered auxotrophs. When applied to a soil microbial community, this platform improved community-level conjugation, preserving microbial diversity and enabling the identification of genetically accessible chassis. Collectively, this platform establishes a scalable, generalizable solution for high throughput DNA delivery in previously inaccessible microbial hosts. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/721591v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@c7a8d4org.highwire.dtl.DTLVardef@1d1fbaorg.highwire.dtl.DTLVardef@e1faforg.highwire.dtl.DTLVardef@14234dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chronic infections persist in large part thanks to protection that biofilms afford their bacterial creators. The extracellular polymeric substance of biofilms is a hydrated matrix of DNA, polysaccharides, and structural proteins, amongst other components, through which nutrients, signalling molecules, and antimicrobial agents must diffuse to reach the bacteria within. Quantitative measurement of transport on the nanoscale within in vivo biofilms remains challenging due to optical heterogeneity, autofluorescence, active remodelling of biofilms and the ambiguity in trajectory reconstruction during single-particle tracking (SPT). Here, we present a methodological framework for measuring molecular transport in defined minimal extracellular matrix models using quantum dots as fluorescent nanoscale probes imaged with high-speed SlimVar microscopy. To establish conditions in which high-diffusivity particle trajectories can be reliably reconstructed, upper limits to quantum dot concentrations were estimated from Brownian motion. The 99th-percentile inter-frame jump distance was estimated from the three-dimensional Brownian jump distance distribution and used to define a target average nearest neighbour distance, and therefore a per-particle volume, used for calculating a concentration which minimises the probability of trajectory collision during data acquisition. Quantum dot movement was imaged at sub-millisecond frame rates and diffusion coefficients were calculated in a 20% glycerol control and in DNA nanostar hydrogels modelling minimal extracellular matrix scaffolds assembled at 250 M and 500 M. Median diffusion coefficients decreased from 94.9 m2*s-1 in glycerol to 15.9 m2*s-1 and 8.3 m2*s-1 in the 250 M and 500 M hydrogels, respectively. More broadly, this work establishes a workflow for quantitative SPT in minimal biofilm models. Rather than attempting to reproduce the full biological complexity of native biofilms, this approach provides the basis of a modular experimental framework in which individual extracellular matrix components can be incorporated sequentially and their effects on molecular transport quantified.

Intestinal organoids are powerful in vitro models, but their use in large-scale analyses remains constrained by the low throughput, labor-intensive handling, and high reagent consumption of conventional Matrigel dome culture. Here, we present Organoid-in-Bead (OrB), a vortex-based compartmentalization workflow that partitions organoid fragments into thousands of discrete Matrigel microbeads, enabling scalable, high-density culture from a single batch preparation. OrB maintains dome-comparable organoid growth and epithelial polarity, supports passaging-based culture expansion, yields more than 5,000 organoids in the final 10 cm dish format, and reduces Matrigel and medium consumption by approximately 70% on a per-organoid basis. OrB therefore provides a practical and scalable upstream workflow for generating screening-scale intestinal organoids. HighlightsO_LIOrB generates Matrigel microcompartments by vortexing without microfluidics C_LIO_LIOrB enables scalable, high-density intestinal organoid culture in one batch C_LIO_LIOrB maintains dome-comparable growth and epithelial polarity and supports passaging C_LIO_LIOrB yields >5,000 organoids per batch with [~]70% less Matrigel/medium per organoid C_LI

Adoptive T-cell therapies rely on the identification and expansion of rare tumor-reactive T cells, yet current enrichment strategies are limited by the low abundance of these cells and complexity of their functional enrichment. Here, we present a microfluidic platform that exploits hydrodynamic shear as a controllable parameter for enriching antigen-specific T cells through peptide-major histocompatibility complex (pMHC)-mediated capture. An eight-channel microfluidic device was engineered to simultaneously interrogate a range of wall shear stresses while maintaining uniform cell delivery, enabling systematic identification of shear conditions that maximize antigen-specific enrichment. Using engineered MART-1-specific Jurkat cells, we demonstrate that T-cell capture is jointly regulated by wall shear stress and pMHC density, with intermediate shear preferentially enriching antigen-specific cells over nonspecific binders. Translation of the optimal operating condition to a high-throughput single-shear device enabled approximately 35-fold enrichment of antigen-specific T cells from peripheral blood mononuclear cells containing only 0.05% target cells. We further show that peptide-MHC complexes isolated directly from melanoma whole-cell lysates support shear-dependent enrichment comparable to recombinant pMHCs. Finally, primary MART-1-specific CD8 T cells enriched using tumor-derived pMHCs retained the ability to recognize melanoma cells and upregulated the activation marker CD137 following antigen-specific stimulation. Together, these findings establish hydrodynamic shear as an orthogonal parameter for antigen-specific T-cell enrichment and provide a framework for integrating force-based selection with tumor-derived pMHCs to isolate functional antigen-specific T cells using tumor-derived pMHCs.

Label-free tracking of adherent cell migration could enable important insights into biological processes such as tissue repair, inflammatory response, or cancer progression. Nevertheless, visualizing unlabeled animal cells using optical microscopy remains challenging due to low contrast as well as frequent changes in cell shape and number. A promising alternative uses electrical capacitance measurements, which are sensitive to cell adhesion to electrode surfaces. However, prior examples often utilized electrodes with areas larger than single cells, resulting in averaged readouts over multiple cells. Here, we demonstrate label-free, live-cell tracking using a capacitance sensor array with more than 1 million pixels on a 10 micron pitch across an area larger than 1 square centimeter. We show that single cell morphology can be clearly segmented, and then used to reconstruct migration and proliferation dynamics using optical flow. We further track the spreading of multicellular spheroids, revealing fast-moving peripheral regions led by a collective leader cell "front." Finally, we demonstrate label-free imaging of millimeter-scale honeycomb-shaped tissues without the multi-image stitching often required for conventional microscopy. We utilize mutual capacitance measurements with electrically-programmable electrode spacing to reconstruct topographical features of these engineered tissues. Overall, CMOS capacitance imaging arrays enables label-free imaging spanning from single cells to large tissues, in a portable and scalable format for settings where optical microscopy may be difficult to access.

In vitro fertilization (IVF) is essential for many couples facing infertility, e.g. in cases of low sperm count (oligospermia), where natural fertilization is unlikely. Medical microrobotics, making use of microscopic devices designed to perform targeted tasks inside the body under imaging guidance and controlled actuation, represents a promising strategy to guide sperm cells toward the oocyte. This approach may significantly reduce the time, invasiveness, and patient burden of conventional IVF, with long-term potential for in vivo assisted reproduction. Here, we report the first successful in vitro fertilization (IVF) using magnetically functionalized spermatozoa, termed magnetotactic sperm cells (MSCs), as a step toward in vivo microrobotic guidance of sperm cells for targeted artificial insemination. We present a protocol for the preparation of MSCs for their use in IVF, resulting in samples largely free of non-functionalized sperm cells (99.69% purity). We systematically evaluate the effect of particle functionalization on sperm health, including acrosome integrity, DNA fragmentation, mitochondrial membrane potential, oxidative stress, and epithelial interactions, and observe no adverse effects. Notably, MSCs showed improved mitochondrial membrane integrity compared to the control samples after two hours of incubation. Using MSCs, we successfully performed complete IVF cycles that resulted in embryos developing to the blastocyst stage at a comparable rate as non-functionalized sperm cells of the same concentration. Lower concentrations of non-functionalized sperm cells (comparable to those remaining in the MSC sample after purification) did not result in any development of embryos to blastocysts. To facilitate manipulation and translation, we implemented automated image-based recognition, magnetic manipulation, and pre-clustering routines that increased guidance efficiency and are compatible with standard IVF workflows. Together, these results demonstrate that magnetic functionalization can be applied without compromising key sperm quality metrics and can enable directed sperm guidance for assisted oocyte fertilization. This work provides a practical framework for integrating microrobotic sperm manipulation into assisted-reproduction workflows and supports further development toward automated in vitro and eventual in vivo applications.

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

Extracellular vesicles (EVs) carry molecular signatures of their originating cells and have thus emerged as promising biomarkers. However, their clinical utility remains limited due to their low abundance and the modest sensitivity of current EV detection methods in complex biological environments. Here, we present a quantum well defect functionalized carbon nanotube sensor coupled with integrin-recognition RGD tripeptide for EV detection in human plasma. Leveraging the abundance of integrins on EV surfaces, we targeted 5{beta}1, V{beta}1, and V{beta}3 subtypes. The nanosensor exhibited robust hypsochromic shifts in defect emission upon integrin binding, achieving sub-picomolar detection limits for integrin subunits and quantifying EVs at concentrations as low as 104 EVs{middle dot}mL-1 for glioblastoma, ovarian cancer, and fibroblast cell-derived EV types. Molecular dynamics simulation indicated that integrin docking at the RGD-coupled quantum defect can substantially reshape the interfacial environments of the quantum defects, explaining the high sensitivity in EV detection in complex biological media. Finally, transmembrane protein analysis validated the expression of surface integrins across the tested EV types. The modular nanosensor construct can be targeted to detect disease-associated EV subpopulations, advancing EV-based diagnostics.

We present a user-friendly correlative second harmonic generation (SHG) and atomic force microscopy (AFM) imaging workflow for quantifying the nanomechanical properties of collagen in unfixed, unlabeled tissue sections. SHG Aligned Profiling for Elasticity and Segmentation or SHAPES utilizes SHG imaging to guide AFM force mapping, enabling label-free, anatomically specific selection of regions of interest, facilitating spatially resolved characterization of fibrous collagen morphology and local stiffness. Compared to brightfield or confocal contrast-guided AFM mapping, SHG improves anatomical specificity without fixation or staining, enabling downstream analysis on the same tissue section while preserving spatial correspondence between structural (SHG) and mechanical (AFM) data. Rapid identification of regions of interest also reduces the risk of costly AFM tip breakage, improving throughput and reducing operator burden. Utilizing only standard turn-key commercial systems common in many user facilities and clinical laboratories, multimodal image coregistration and automated identification of anatomical regions of interest are employed to integrate SHG and AFM datasets across complex sample topographies. Coregistration of SHG and AFM images substantially increases the number of usable datasets per measurement session, facilitating translation of these complementary modalities and bridging nanomechanical imaging with clinical research practice.

Single-particle tracking (SPT) over time enables direct observation of molecular transport and interactions in living cells. Fluorescence-based SPT has provided insights into intracellular processes such as endocytosis, receptor signaling, and drug delivery. Extending the observation window to several hours and beyond is critical for capturing slow intracellular dynamics, including the full course of endosomal trafficking, the long-term accumulation of particles within subcellular compartments, and transitions between transport modes that occur on hour-scale timescales. However, long-term intracellular SPT under visible-wavelength excitation remains challenging because fluorescence probes generally suffer from photobleaching and phototoxicity. While near-infrared (NIR) excitation can simultaneously mitigate these issues, generally weak emission of NIR-emitting dyes has hindered its wide application in long-term SPT. Here, we demonstrate long-term NIR SPT using atomically precise gold quantum needles, Au42(PET)32 (PET = 2-phenylethanethiolate). Continuous tracking of intracellular particles in living HEK293 cells was achieved for up to 12 h. Trajectory analysis revealed temporal transitions between directional and diffusive transport, as well as the accumulation of multiple particles within localized intracellular domains over several-hour timescales. The high photostability of Au42, combined with low phototoxicity of NIR excitation, enables visualization of intracellular transport dynamics over timescales difficult to access using conventional visible fluorescent probes. These results establish Au42-based NIR imaging as a platform for long-term, low-phototoxicity intracellular SPT and provide a framework for investigating slow intracellular dynamics in living systems.

High-quality cellular imaging, especially in live cells, remains constrained by the trade-off among signal-to-noise ratio, phototoxicity, and instrumentation complexity. Here, we report a scalable plasmonic metasurface that generates a spatially ordered array of fluorescence-enhancing near-field hotspots and enables self-supervised denoised, cellular imaging with improved feature readability on a conventional wide-field microscope. The registered hotspot lattice serves as a physics-derived functional prior that identifies where fluorescence amplification is physically grounded and steers neural-network training accordingly, reducing reliance on paired ground truth, large external pretrained models, or extensive supervised datasets. We demonstrate two labeling-density-dependent operating regimes: dense labeling for cytoskeleton structural imaging and sparse labeling for multiplexed sensing of plasma-membrane-associated dynamics across the hotspot array. Our work unites scalable nanophotonic hardware and self-supervised computational imaging into a practical platform for structural bioimaging and on-chip live-cell biosensing under simple wide-field imaging conditions.

Urinary extracellular vesicles (uEVs) represent a promising source of non-invasive biomarkers; however, their clinical translation is still limited by suboptimal isolation strategies, which often suffer from poor scalability, co-isolation of abundant urinary proteins, and bias toward specific EV subpopulations. Here, we employ a membrane-sensing peptide (MSP)-based affinity approach for uEVs isolation, that exploits the highly lipid membranes curvature of EV as universal target, enabling pan-specific capture independent of surface marker expression. MSP-functionalized beads were applied to minimally processed urine samples and benchmarked against differential ultracentrifugation (dUC) and size-exclusion chromatography (SEC). Comprehensive characterization by nanoparticle tracking analysis, transmission electron microscopy, high-sensitivity flow cytometry, single-molecule array (SiMoA), and fluorescence nanoparticle tracking analysis, demonstrated that MSP-based isolation preserves vesicle integrity and maintains the native distribution of canonical tetraspanins (CD9, CD63, CD81), without evidence of subpopulation bias. Notably, MSP-based isolation significantly reduced co-isolated contaminants, such as uromodulin, resulting in improved sample purity. By combining high recovery, improved purity, and operational simplicity, MSP workflow offers practical advantages, including reduced processing time, scalability, and compatibility with standard laboratory equipment, without the need for extensive pre-processing. These properties characterize MSP-based affinity capture as a robust and versatile alternative to conventional uEVs isolation approaches, with strong potential for translational and clinical applications.

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.

Tissue phantoms that mimic microvasculature and perfusion are essential for modelling vascular function, guiding interventions, and calibrating imaging systems, which require faithful replication of vascular geometry and flow. Conventional fabrication strategies, including wire-based molding, lithographic micromachining, and additive manufacturing, offer useful capabilities but remain constrained by predefined designs, rectangular channel cross-sections, limited scalability, and high production costs. Reliance on predefined digital vascular models restricts design flexibility and limits the ability to capture the natural variability and complexity of real vascular systems. Here, we present a lithography-free, fractal-generating approach based on a modified Lifted Hele-Shaw Cell (LHSC) technique, in which vascular networks emerge spontaneously via interfacial fluid instabilities. Unlike pre-designed methods, these structures are governed by fluid properties and flow conditions, enabling adaptive, physiologically relevant geometries with smooth Gaussian cross-sections and natural diameter tapering. We demonstrate four phantom designs: a planar vascular tree, an anatomically guided cerebral network, a retinal vascular model, and a conformable curved substrate phantom. Validation using Laser Speckle Contrast Imaging confirms structural fidelity and physiologically relevant flow consistent with Murrays law. This platform uniquely integrates realistic vascular architecture with emergent, fractal driven formation, highlighting its potential as a reproducible and biologically relevant alternative to conventional vascular phantom fabrication. Furthermore, the availability of such realistic in vitro vascular models can reduce reliance on animal experiments and contribute towards more ethical and sustainable preclinical research.