Small Methods

SIMIT-seq is a bead-free, scalable microfluidic platform designed for high-efficiency single-cell mRNA sequencing. Conventional microfluidic-based single-cell RNA sequencing platforms rely heavily on barcoded beads and intricate co-encapsulation schemes, often constrained by double Poisson limitations and the complexities of bead synthesis. In contrast, SIMIT-seq eliminates the need for beads entirely by employing a deterministic, orthogonal barcoding strategy within a two-dimensional micro-well array. This platform achieves an impressive single-cell indexing rate of 96.6% without the need for complex microfluidic operations. Here, we describe the design and fabrication of the SIMIT-seq platform, outline its workflow for transcript capture and library preparation, and demonstrate its application in profiling K562 cells. Our results validate both the high fidelity of molecular barcode immobilization and the systems capability to support downstream single-cell mRNA sequencing. SIMIT-seq offers a cost-effective and scalable alternative for single-cell transcriptomics, providing a promising foundation for future single-cell omics applications.

Experiments with gradients of soluble bioactive species have significantly advanced with microfluidic developments that enable cell observation and stringent control of environmental conditions. While some methodologies rely on flow to establish gradients, other opt for flow-free conditions, which is particularly beneficial for studying non-adherent and/or shear-sensitive cells. In flow-free devices, bioactive species diffuse either through resistive microchannels in "microchannel-based" devices, a porous membrane in "membrane-based" devices, or a hydrogel in "gel-based" devices. However, despite significant advancements over traditional methods such as "Boyden chambers", these technologies have not widely disseminated in biological laboratories, arguably due to entrenched practices and the intricate skills required for conducting microfluidic assays. Here, we integrated Quake-type pneumatic microvalves in place of microgrooves, membranes, or gels, and developed devices with precise control over residual flow, establishment initial gradient, and long-term stability of gradients. The "Microvalve-based" approach enables the generation of the automatization of delicate microfluidic manipulations, which paves the way for routine applications of controlled and tunable flow-free gradients in academic laboratories and biomedical units.

Spatial transcriptomics can reveal molecular signatures of tissue at spatial scales, but current technologies cannot integrate excellent accessibility and easy data-decoding. Here, we use oligonucleotides whose number is the square root of the number of spots to generate cross-amplified spatial barcodes on slides using microfluidic technology. This method can obtain microarrays with well-defined barcodes easily and at low cost, without post-decoding, which contributes to the popularization of spatial transcriptomics.

This study utilizes a novel inducible X-CreERT2/Ai9 system to comprehensively delineate the spatial expression landscape of Fermt2, which encodes a key focal adhesion protein Kindlin-2, across organs in mice. We can confirm all known Kindlin-2-expressing cells and identify any previously unknown Kindlin-2-expressing cells and those that do not express Kindlin-2 in all tissues/organs in mice. We have developed this cost-effective, rapid, and high-fidelity system and implemented its first whole-organism application for comprehensive gene expression profiling. This work not only provides unprecedented insights into roles of Kindlin-2 in diverse physiologies and pathologies, but also establishes the inducible X-CreERT2/Ai9 system as a powerful paradigm-shifting platform for organism-wide pan-organ spatiotemporal expression profiling of any target genes encoding any proteins and micro-RNAs. By enabling high-resolution mapping of drug target expression, this system may lay critical groundwork for elucidating previously unknown adverse and beneficial effects of targeted therapies, offering transformative potential for precision medicine.

Single-cell technologies are becoming increasingly widespread and have been revolutionizing our understanding of cell identity, state, diversity and function. However, current platforms can be slow to apply to large-scale studies and resource-limited clinical arenas due to a variety of reasons including cost, infrastructure, sample quality and requirements. Here we report DNBelab C4 (C4), a negative pressure orchestrated, portable and cost-effective device that enables high-throughput single-cell transcriptional profiling. C4 system can efficiently allow discrimination of species-specific cells at high resolution and dissect tissue heterogeneity in different organs, such as murine lung and cerebral cortex. Finally, we show that the C4 system is comparable to existing platforms but has huge benefits in cost and portability and, as such, it will be of great interest for the wider scientific community.

Polydimethylsiloxane (PDMS) is widely used in academic microfluidics due to its favorable biocompatible properties and compatibility with soft lithography. Moreover, the recent developments of reconfigurable microfluidics rely on the microfabrication of sliding elements, which are 3D objects insertable inside a microfluidic chip to provide a given function. However, the complexity of microfluidic device geometries or sliding elements remains largely limited by the traditional microfabrication methods such as, among others, on SU-8 photolithography or dry epoxy films. Such methods are suited for simple, planar "2.5D" structures with uniform depths, yet struggle to produce more advanced architectures due to material and alignment constraints. To address these limitations, we propose to investigate advanced laser-based approaches, such as Direct Laser Writing (DLW) and Selective Laser Etching (SLE), which enable the construction of high-resolution, 3D designs. We further explore the use of SLE to fabricate sliding elements that enhance chip functionality, including chamber reconfiguration and biological sample manipulation. As a proof of concept, we show that these elements can be functionalized into pH microsensors, paving the way to reusable and reconfigurable electrochemistry-on-chip.

Immunoblotting typically requires protein immobilization onto a membrane surface for quantification. Particularly, in real-time single-molecule blotting, stringent surface passivation is essential to prevent false-positive signals from nonspecific binding. In this study, we introduce a novel surface-free single-molecule blotting platform, termed the DNA Hanger. This platform employs a 3D structure of a quartz slide. Modified{lambda} -phage DNA molecules are randomly biotinylated along their bases and suspended between 4 m-high thin barriers, positioned away from the quartz slide surface. A light sheet, produced within the 3D structure, illuminates fluorophore-conjugated proteins bound to the biotinylated DNA, enabling single-molecule detection. The DNA Hanger assay significantly reduces nonspecific binding and enhances sensitivity to sub-picomolar concentrations, suggesting that this platform provides a novel, surface-condition-independent, and real-time approach in a wide range of single-molecule blotting.

In situ hybridization (ISH) is a powerful tool for investigating the spatial arrangement of nucleic acid targets in fixed samples. ISH is typically visualized using fluorophores to allow high sensitivity and multiplexing or with colorimetric labels to facilitate co-visualization with histopathological stains. Both approaches benefit from signal amplification, which makes target detection effective, rapid, and compatible with a broad range of optical systems. Here, we introduce a unified technical platform, termed pSABER, for the amplification of ISH signals in cell and tissue systems. pSABER decorates the in situ target with concatemeric binding sites for a horseradish peroxidase-conjugated oligonucleotide which can then catalyze the massive localized deposition of fluorescent or colorimetric substrates. We demonstrate that pSABER effectively labels DNA and RNA targets, works robustly in cultured cells and challenging formalin fixed paraffin embedded (FFPE) specimens. Furthermore, pSABER can achieve 25-fold signal amplification over conventional signal amplification by exchange reaction (SABER) and can be serially multiplexed using solution exchange. Therefore, by linking nucleic acid detection to robust signal amplification capable of diverse readouts, pSABER will have broad utility in research and clinical settings.

Imaging mass cytometry (IMC) is a powerful multiplexed tissue imaging technology that allows simultaneous detection of more than 30 makers on a single slide. It has been increasingly used for singlecell-based spatial phenotyping in a wide range of samples. However, it only acquires a small, rectangle field of view (FOV) with a low image resolution that hinders downstream analysis. Here, we reported a highly practical dual-modality imaging method that combines high-resolution immunofluorescence (IF) and high-dimensional IMC on the same tissue slide. Our computational pipeline uses the whole slide image (WSI) of IF as a spatial reference and integrates small FOVs IMC into a WSI of IMC. The high-resolution IF images enable accurate single-cell segmentation to extract robust high-dimensional IMC features for downstream analysis. We applied this method in esophageal adenocarcinoma of different stages, identified the single-cell pathology landscape via reconstruction of WSI IMC images, and demonstrated the advantage of the dual-modality imaging strategy. MotivationHighly multiplexed tissue imaging allows visualization of the spatially resolved expression of multiple proteins at the single-cell level. Although imaging mass cytometry (IMC) using metal isotope-conjugated antibodies has a significant advantage of low background signal and absence of autofluorescence or batch effect, it has a low resolution that hampers accurate cell segmentation and results in inaccurate feature extraction. In addition, IMC only acquires mm2-sized rectangle regions, which limits its application and efficiency when studying larger clinical samples with non-rectangle shapes. To maximize the research output of IMC, we developed the dual-modality imaging method based on a highly practical and technical improvement requiring no extra specialized equipment or agents and proposed a comprehensive computational pipeline that combines IF and IMC. The proposed method greatly improves the accuracy of cell segmentation and downstream analysis and is able to obtain whole slide image IMC to capture the comprehensive cellular landscape of large tissue sections.

Spatial transcriptomic technologies are promising tools to reveal fine anatomical profiles of tissues. As for the methodologies based on barcoded probe arrays, improving the balance among probe barcoding complexity and cost, gene capture sensitivity, and spatial resolution can accelerate the spreading of spatial transcriptomic in basic science and clinical work. Here, based on miniaturized microfluidic and microarray technologies, we constructed a spatially cellular-level RNA-capture probe arrays. Owing to the predetermined and cost-effective probe fixation characteristics of the methodology, the consumable cost and fabrication time of the probe array can be reduced to $1.21/mm2 and approximately 2 hours, and the preparation process does not rely on large precision instruments. Moreover, the efficiency of the transcript captured by the probe array is even comparable to conventional single-cell RNA sequencing. Based on this technology, we achieved the spatial transcriptome expression mapping and gained insight into spatial cell heterogeneity of the mouse hippocampus.

Single-cell analysis is essential for uncovering heterogeneous biological functions that arise from intricate cellular interaction. Microfluidic droplet arrays enable precise dynamic data collection through cell encapsulation in picoliter volumes. The time-lapse imaging of these arrays can reveal functional kinetics and cellular fates, but accurate tracking of cell identities across time frames remains challenging when droplets move significantly. Specifically, existing machine learning methods often depend on labeled data or require neighboring cells as reference; without them, these methods struggle to track identical objects across long distances with complex movements. To address these limitations, we developed a pipeline combining visual object detection, feature extraction via contrastive learning, and optimal transport-based object matching, which minimizes reliance on labeled training data. Our approach was validated across various experimental conditions and was able to track thousands of water-in-oil microfluidic droplets over large distances and long (> 30 min) time-separated frames. We achieved high precision in previously untraceable scenarios, tracking small, medium and large movements (corresponding to ~126, ~800 and ~10,000 {micro}m, respectively) with a success rate of correctly tracked droplets of > 90% for average movements within 212 object diameters, and > 60% for average movements of > 100 object diameters. This workflow lays the foundation for high-resolution, dynamic analysis of droplets and cells in both spatial and temporal dimensions without relying on visual labeling, allowing high-accuracy tracking in samples, where the uniqueness of the sample makes repeating experiments infeasible.

We present a high-throughput method using standard laboratory equipment and microfluidics to produce cellular force microscopy probes with controlled size and elastic modulus. Mechanical forces play crucial roles in cell biology but quantifying these forces in physiologically relevant systems remains challenging due to the complexity of the native cell environment. Polymerized hydrogel microspheres offer great promise for interrogating the mechanics of processes inaccessible to classic force microscopy methods. However, despite significant recent advances, their small size and large surface-to-volume ratio impede the high-yield production of probes with tunable, monodisperse distributions of size and mechanical properties. To overcome these limitations, we use a flow-focusing microfluidic device to generate large quantities of droplets with highly reproducible, adjustable radii. These droplets contain acrylamide gel precursor and the photoinitiator Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a source of free radicals. LAP provides fine control over microsphere polymerization due to its high molar absorptivity at UV wavelengths and moderate water solubility. The polymerized microspheres can be functionalized with different conjugated extracellular matrix proteins and embedded with fluorescent nanobeads to promote cell attachment and track microsphere deformation. As proof of concept, we measure the mechanical forces generated by a monolayer of vascular endothelial cells engulfing functionalized microspheres. Individual nanobead motions are tracked in 3D and analyzed to determine the 3D traction forces within seconds and without the need for solving an ill-posed inverse problem. These results reveal that the cell monolayer collectively exerts strong radial compression and subtle lateral distortions on the encapsulated probe.

Volumetric interrogating of a large population of live specimens at high throughput is a challenging task that necessitates new technology. We propose vertical-aligned multi-sheet array (VAMSA) illumination PSF that enables interrogation of specimens flowing simultaneously through multiple microfluidic channels. The very geometry of PSF enables high quality cross-sectional imaging, and facilitates volumetric interrogation of specimens flowing through commercial microfluidic chip (consists of multiple flow-channels), which is a step towards large population screening. The SMMIC technique employs a unique combination of transmission grating, beam-expander and high NA objective system in a specific optical configuration to generate diffraction-limited illumination PSF (VAMSA-PSF). However, the detection is accomplished by a large field-of-view widefield 4f-system that consists of low NA objective lens, high performance fluorescence filters, and tube lens. Studies show high quality sectional images (resolution [~] 2.5m, and SBR [~] 4.8dB) of HeLa cancerous cells at high flow throughput (flow-rate of, 2500 nl/min). A cell count of > 1k and volume reconstruction efficiency of [~] 121 cells/min is noteworthy. In addition, SMMIC system demonstrate organelle-level resolution with a SBR comparable to that of confocal especially at low flow-rates. It is hoped that the proposed system may accelerates drug-treatment studies for a large population of live specimens to advance the evolving field of translational medicine and health-care.

Barcoding strategies are fundamental to droplet-based single-cell sequencing, and understanding the biases and caveats between approaches is essential. Here, we comprehensively evaluated both short and long reads of the cDNA obtained through the two marketed approaches from 10x Genomics, the "3 assay" and the "5 assay", which attach barcodes at different ends of the mRNA molecule. Although the barcode detection, cell-type identification, and gene expression profile are similar in both assays, the 5 assay captured more exonic molecules and fewer intronic molecules compared to the 3 assay. We found that 13.7% of genes sequenced have longer average read lengths and are more complete (spanning both polyA-site and TSS) in the long reads from the 5 assay compared to the 3 assay. These genes are characterized by long average transcript length, high intron number, and low expression overall. Despite these differences, cell-type-specific isoform profiles observed from the two assays remain highly correlated. This study provides a benchmark for choosing the single-cell assay for the intended research question, and insights regarding platform-specific biases to be mindful of when analyzing data, particularly across samples and technologies.

Imaging flow cytometry enables the detailed analysis of cell morphology and internal structures through high-throughput cell imaging, and quantitative phase imaging (QPI)-based microfluidic approaches have extended this by providing label-free measures such as dry mass and refractive index (RI). Building on these developments, we present quantitative phase deformability cytometry (QP-DC), which integrates QPI with deformability cytometry to simultaneously measure morphology, mechanics, and intrinsic biophysical parameters such as mass density and dry mass. Numerical refocusing ensures in-focus images independent of axial position, improving precision in contour detection and feature extraction. Using microspheres and whole blood, we validated QP-DC and then applied it to neutrophils under lipopolysaccharide (LPS) stimulation and from patients with systemic lupus erythematosus (SLE). QP-DC revealed LPS-induced reductions in neutrophil mass density and identified heterogeneous subpopulations in SLE. These results demonstrate the capability of QP-DC for precise biophysical and mechanical characterization, offering significant potential for research and clinical diagnostics.

Recent years have witnessed profound advances in spatial transcriptomics (ST). However, current ST strategies are limited to tissue sections, unable to directly profile spatiotemporal transcriptome landscapes of individual living animals. To overcome this limitation, we present acupuncture-extracted in vivo spatiotemporal transcriptomics (aivST). It is mainly based on engineering clinical-grade acupuncture gold microneedles as a spatial-barcoding array patch with high-performance DNA recognition interfaces. These interfaces first utilize nanoparticles surface deposition to increase the density of DNA probes. A freeze-thawing method is further developed to regulate interfaced DNA probes with laterally uniform distribution and vertically upright conformation, mainly by loose stretching and local concentration of these DNA probes. It increased mRNA capture with 250% and speeded up reaction (from 60 min to 15 min), compared to that without freeze-thawing surface engineering. Using this method, we explored in vivo spatiotemporal transcriptome dynamics during skin wound healing of individual mice. We found four distinct expression changes of transcripts, including continuous upregulation, continuous downregulation, initial upregulation then downregulation, and initial downregulation then upregulation. Our aivST method advances the field from sparse tissue section sampling toward in vivo measurements of biological stereotypy and variability in individual living animals.

Cost-effective and scalable production is critical for advancing the clinical translation of adeno-associated virus (AAV)-mediated gene therapy. The widely used transient transfection method using plasmid DNA (pDNA)-loaded transfection particles for AAV production faces technical challenges due to instability of the particles and the concentration limits for particle preparation, hindering reproducibility and scalability. Here, we report a streamlined and scalable strategy to generate shelf-stable, highly concentrated pDNA/poly(ethylenimine) (PEI) transfection particles. By incorporating trivalent citrate ions in the dilution buffers, we kinetically modulate electrostatic complexation to achieve uniform nanoparticle assembly and prevent aggregation at high concentrations. This enables a tenfold increase in pDNA concentration in stabilized transfection particles from a typical range of 10-20 g/mL to 200 g/mL, while reducing the required dosing volume from 5-10% to 0.5% of the cell culture medium. The particle assembly process is robust to changes in mixing scale and timing and is compatible with standard workflows. We demonstrate equivalent AAV production efficiencies to standard methods and consistent performance in various production scales, which confirms the practical utility of this assembly method in developing robust, scalable, and cost-effective AAV manufacturing processes.

Although many lab-on-chip applications require inch-sized devices with microscale feature resolution, achieving this via current 3D printing methods remains challenging due to inherent tradeoffs between print resolution, design complexity, and build sizes. Inspired by microscopes that can switch objectives to achieve multiscale imaging, we report a new optical printer coined as Multipath Projection Stereolithography (MPS) specifically designed for printing microfluidic devices. MPS is designed to switch between high-resolution (1xmode, [~]10{micro}m) and low-resolution (3x mode, [~]30{micro}m) optical paths to generate centimeter sized constructs (3cm x 6cm) with a feature resolution of [~]10{micro}m. Illumination and projection systems were designed, resin formulations were optimized, and slicing software was integrated with hardware with the goal of ease of use. Using a test-case of micromixers, we show user-defined CAD models can be directly input to an automated slicing software to define printing of low-resolution features via the 3x mode with embedded microscale fins via 1x mode. A new computational model, validated using experimental results, was used to simulate various fin designs and experiments were conducted to verify simulated mixing efficiencies. New 3D out-of-plane micromixer designs were simulated and tested. To show broad applications of MPS, multi-chambered chips and microfluidic devices with microtraps were also printed. Overall, MPS can be a new fabrication tool to rapidly print a range of lab-on-chip applications.

Large-scale nanoarrays of single biomolecules enable high-throughput assays while unmasking the underlying heterogeneity within ensemble populations. Until recently, creating such grids which combine the unique advantages of microarrays and single-molecule experiments (SMEs) has been particularly challenging due to the mismatch between the size of these molecules and the resolution of top-down fabrication techniques. DNA Origami Placement (DOP) combines two powerful techniques to address this issue: (i) DNA origami, which provides a [~] 100-nm self-assembled template for single-molecule organization with 5 nm resolution, and (ii) top-down lithography, which patterns these DNA nanostructures, transforming them into functional nanodevices via large-scale integration with arbitrary substrates. Presently, this technique relies on state-of-the-art infrastructure and highly-trained personnel, making it prohibitively expensive for researchers. Here, we introduce a bench-top technique to create meso-to-macro-scale DNA origami nanoarrays using self-assembled colloidal nanoparticles, thereby circumventing the need for top-down fabrication. We report a maximum yield of 74%, two-fold higher than the statistical limit of 37% imposed on non-specific molecular loading alternatives. Furthermore, we provide a proof-of-principle for the ability of this nanoarray platform to transform traditionally low-throughput, stochastic, single-molecule assays into high-throughput, deterministic ones, without compromising data quality. Our approach has the potential to democratize single-molecule nanoarrays and demonstrates their utility as a tool for biophysical assays and diagnostics.

DNA-Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) enables multiplexed super-resolution imaging of biological samples. We expand the repertoire of speed-optimized DNA sequences for DNA-PAINT imaging to drive visualization of as many as twelve targets in a sequential manner with molecular resolution. By implementing Exchange-PAINT protocol, we demonstrate 12-plex super-resolved imaging of docking strand patterned DNA origami nanostructures within four hours with a localization precision of 3 to 5 nm. Using these sequences, we demonstrate 9-plex super-resolution imaging of diverse nuclear targets within four hours. Further, we present a comprehensive analysis pipeline to quantify nanoscale chromatin in single cells. The combination of multiplexed imaging and analysis pipeline enabled us to reveal the loss of chromatin contacts with nuclear speckles upon global transcription inhibition. This work highlights the versatility of our approach to simultaneously image multiple targets at accelerated speeds while maintaining precise spatial localization for each target, enabling in depth mapping of the nuclear landscape. These speed-optimized imager sequences for high multiplexed super-resolution imaging will drive its further adoption for diverse cellular imaging applications.