Rapid and signal crowdedness-robust in situ sequencing through hybrid block coding.

Spatial transcriptomics technology has revolutionized our understanding of cell types and tissue organization, opening possibilities for researchers to explore transcript distributions at subcellular levels. However, existing methods have limitations in resolution, sensitivity, or speed. To overcome these challenges, we introduce SPRINTseq (Spatially Resolved and signal-diluted Next-generation Targeted sequencing), an innovative in situ sequencing strategy that combines hybrid block coding and molecular dilution strategies. Our method enables fast and sensitive high-resolution data acquisition, as demonstrated by recovering over 142 million transcripts using a 108-gene panel from 453,843 cells from four mouse brain coronal slices in less than 2 d. Using this advanced technology, we uncover the cellular and subcellular molecular architecture of Alzheimer's disease, providing additional information into abnormal cellular behaviors and their subcellular mRNA distribution. This improved spatial transcriptomics technology holds great promise for exploring complex biological processes and disease mechanisms.

Open-top light-sheet imaging of CLEAR emulsion for high-throughput loss-free analysis of massive fluorescent droplets.

Digital droplet PCR (ddPCR) is classified as the third-generation PCR technology that enables absolute quantitative detection of nucleic acid molecules and has become an increasingly powerful tool for clinic diagnosis. We previously established a CLEAR-dPCR technique based on the combination of CLEAR droplets generated by micro-centrifuge-based microtubule arrays (MiCA) andinsitu3D readout by light-sheet fluorescence imaging. This CLEAR-dPCR technique attains very high readout speed and dynamic range. Meanwhile, it is free from sample loss and contamination, showing its advantages over commercial d-PCR technologies. However, a conventional orthogonal light-sheet imaging setup in CLEAR d-PCR cannot image multiple centrifuge tubes, thereby limiting its widespread application to large-scale, high-speed dd-PCR assays. Herein, we propose an in-parallel 3D dd-PCR readout technique based on an open-top light-sheet microscopy setup. This approach can continuously scan multiple centrifuge tubes which contain CLEAR emulsions with highly diverse concentrations, and thus further boost the scale and throughput of our 3D dd-PCR technique.

A virtual sequencer reveals the dephasing patterns in error-correction code DNA sequencing.

An error-correction code (ECC) sequencing approach has recently been reported to effectively reduce sequencing errors by interrogating a DNA fragment with three orthogonal degenerate sequencing-by-synthesis (SBS) reactions. However, similar to other non-single-molecule SBS methods, the reaction will gradually lose its synchronization within a molecular colony in ECC sequencing. This phenomenon, called dephasing, causes sequencing error, and in ECC sequencing, induces distinctive dephasing patterns. To understand the characteristic dephasing patterns of the dual-base flowgram in ECC sequencing and to generate a correction algorithm, we built a virtual sequencer in silico. Starting from first principles and based on sequencing chemical reactions, we simulated ECC sequencing results, identified the key factors of dephasing in ECC sequencing chemistry and designed an effective dephasing algorithm. The results show that our dephasing algorithm is applicable to sequencing signals with at least 500 cycles, or 1000-bp average read length, with acceptably low error rate for further parity checks and ECC deduction. Our virtual sequencer with our dephasing algorithm can further be extended to a dichromatic form of ECC sequencing, allowing for a potentially much more accurate sequencing approach.

Rotational scan digital LAMP for accurate quantitation of nucleic acids.

Digital quantitation of nucleic acids is precise and sensitive because of its molecular-level resolution. However, only several quantitation formats are common, especially pertaining to how one obtains digital signals from multiple droplets. Here we present rotational scan digital loop-mediated amplification, termed RS-dLAMP. Droplets generated by centrifugation undergo isothermal loop-mediated amplification (LAMP), and self-tile by gravitation into a tubular space between two coaxial cylinders, which are then rotated and scanned to acquire droplet fluorescence signals. RS-dLAMP is quantitatively comparable to commercial digital PCR, yet has higher throughput. Moreover, by sealing the sample throughout analysis, RS-dLAMP eliminates contamination, facilitating point-of-care diagnosis and other applications.

Three-dimensional digital PCR through light-sheet imaging of optically cleared emulsion.

The realization of the vast potential of digital PCR (dPCR) to provide extremely accurate and sensitive measurements in the clinical setting has thus far been hindered by challenges such as assay robustness and high costs. Here we introduce a lossless and contamination-free dPCR technology, termed CLEAR-dPCR, which addresses these challenges by completing the dPCR sample preparation, PCR, and readout all in one tube. Optical clearing of the droplet dPCR emulsion was combined with emerging light-sheet fluorescence microscopy, to acquire a three-dimensional (3D) image of a half million droplets sealed in a tube in seconds. CLEAR-dPCR provides ultrahigh-throughput readout results in situ and fundamentally eliminates the possibility of either sample loss or contamination. This approach exhibits improved accuracy over existing dPCR platforms and enables a greatly increased dynamic range to be comparable to that of real-time quantitative PCR.

Surfactant and oil formulations for monodisperse droplet emulsion PCR.

Emulsion PCR has become a popular and widely applied method in biological research and clinical diagnostics to provide evenly amplified products and perform highly quantitative counting of target sequences. However, there is still a lack of information to support further development of appropriate water-in-oil emulsion formulations, which need to be both thermally and mechanically stable for digital amplification reactions. Here, we present a systematic survey of the oil and surfactant components of stable monodisperse w/o emulsions suitable for use with our previously developed micro-capillary array (MiCA)-based centrifugal emulsion generation method. Our findings show that a binary formula consisting of isopropyl palmitate and a silicone copolymer demonstrated the best performance, and provided a general guideline for the development of emulsion systems for digital PCR and emulsion amplification applications.

Highly accurate fluorogenic DNA sequencing with information theory-based error correction.

Eliminating errors in next-generation DNA sequencing has proved challenging. Here we present error-correction code (ECC) sequencing, a method to greatly improve sequencing accuracy by combining fluorogenic sequencing-by-synthesis (SBS) with an information theory-based error-correction algorithm. ECC embeds redundancy in sequencing reads by creating three orthogonal degenerate sequences, generated by alternate dual-base reactions. This is similar to encoding and decoding strategies that have proved effective in detecting and correcting errors in information communication and storage. We show that, when combined with a fluorogenic SBS chemistry with raw accuracy of 98.1%, ECC sequencing provides single-end, error-free sequences up to 200 bp. ECC approaches should enable accurate identification of extremely rare genomic variations in various applications in biology and medicine.

Centrifugal micro-channel array droplet generation for highly parallel digital PCR.

Stable water-in-oil emulsion is essential to digital PCR and many other bioanalytical reactions that employ droplets as microreactors. We developed a novel technology to produce monodisperse emulsion droplets with high efficiency and high throughput using a bench-top centrifuge. Upon centrifugal spinning, the continuous aqueous phase is dispersed into monodisperse droplet jets in air through a micro-channel array (MiCA) and then submerged into oil as a stable emulsion. We performed dPCR reactions with a high dynamic range through the MiCA approach, and demonstrated that this cost-effective method not only eliminates the usage of complex microfluidic devices and control systems, but also greatly suppresses the loss of materials and cross-contamination. MiCA-enabled highly parallel emulsion generation combines both easiness and robustness of picoliter droplet production, and breaks the technical challenges by using conventional lab equipment and supplies.

Spinning micropipette liquid emulsion generator for single cell whole genome amplification.

Many on-chip approaches that use flow-focusing to pinch the continuous aqueous phase into droplets have become the most popular methods that provide monodisperse emulsion droplets. However, not every lab can easily adapt a microfluidic workflow into their familiar protocols. We develop an off-chip approach, spinning micro-pipette liquid emulsion (SiMPLE) generator, to produce highly stable monodisperse water-in-oil emulsions using a moving micropipette to disperse the aqueous phase in an oil-filled microcentrifuge tube. This method provides a simple way to produce picoliter-size droplets in situ with no dead volume during emulsification. With SiMPLE, single-cell emulsion whole genome amplification was performed to demonstrate that this novel method can seamlessly be integrated with experimental operations and supplies that most researchers are familiar with. The SiMPLE generator has effectively lowered the technical difficulties in applications relying on emulsion droplets.

Fluorogenic sequencing using halogen-fluorescein-labeled nucleotides.

Fluorogenic sequencing is a sequencing-by-synthesis technology that combines the advantages of pyrosequencing and fluorescence detection. With native duplex DNA as the major product, we employ polymerase to incorporate the complement- arily matched terminal phosphate-labeled fluorogenic nucleotides into the DNA template and release halogen-fluorescein as the reporter. This red-emitting fluorophore successfully avoids spectral overlap with the autofluorescence background of the flow chip. We fully characterized the enzymatic reaction kinetics of the new substrates, and performed a 35-base sequencing experiment with 60 reaction cycles. Our achievement expands the substrate repertoire for fluorogenic sequencing, and extends the spectral range to obtain better signal-to-background performance.

A valve-less microfluidic peristaltic pumping method.

We demonstrate a valve-less microfluidic peristaltic pumping method which enables the delivery of continuous nanoliter-scale flow with high precision. The fluid is driven by squeezing the microchannels embedded in a poly(dimethylsiloxane) device with rolling cams or bearings. We achieve continuous and uniform flow with velocity range from 1 to 500 nl/s, with outflow volume error within 3 nl. The devices show enhanced backpressure resistance up to 340 kPa. This method also shows great flexibility. By altering the channels' layout, emulsions and plugs can be generated easily. These low-cost and easy-to-fabricate micro-pumps offer novel approaches for liquid actuation in various microfluidic applications.

Design, preparation, and selection of DNA-encoded dynamic libraries.

We report a method for the preparation and selection of DNA-encoded dynamic libraries (DEDLs). The library is composed of two sets of DNA-linked small molecules that are under dynamic exchange through DNA hybridization. Addition of the protein target shifted the equilibrium, favouring the assembly of high affinity bivalent binders. Notably, we introduced a novel locking mechanism to stop the dynamic exchange and "freeze" the equilibrium, thereby enabling downstream hit isolation and decoding by PCR amplification and DNA sequencing. Our DEDL approach has circumvented the limitation of library size and realized the analysis and selection of large dynamic libraries. In addition, this method also eliminates the requirement for modified and immobilized target proteins.

A DNA-templated synthesis of encoded small molecules by DNA self-assembly.

We report a novel method for the synthesis of DNA-encoded libraries without the need for discrete DNA template. Reactant DNAs self-assemble to enable chemical reactions and photo-cleavage transfers the product to the DNA terminus, making it suitable for the subsequent affinity-based selection and hit deconvolution.

Selection of DNA-encoded small molecule libraries against unmodified and non-immobilized protein targets.

The selection of DNA-encoded libraries against biological targets has become an important discovery method in chemical biology and drug discovery, but the requirement of modified and immobilized targets remains a significant disadvantage. With a terminal protection strategy and ligand-induced photo-crosslinking, we show that iterated selections of DNA-encoded libraries can be realized with unmodified and non-immobilized protein targets.

A compact optofluidic cytometer with integrated liquid-core/PDMS-cladding waveguides.

We developed a simple method to construct liquid-core/PDMS-cladding optical waveguides through pressurized filling of dead-ended micro-channels with optical fluids. The waveguides are in the same layer as microfluidic channels which greatly simplifies device fabrication. With proper contrast between the refractive index of the core and cladding, the transmission loss of the waveguides is less than 5 dB cm(-1). We also developed a method to create flat and optically clear surfaces on the sides of PDMS devices in order to couple light between free-space and the waveguides embedded inside the chip. With these newly developed techniques, we make a compact flow cytometer and demonstrate the fluorescence counting of single cells at a rate of up to ~50 cell s(-1) and total sample requirement of a few microlitres. This method of making liquid-core optical waveguides and flat surfaces has great potential to be integrated into many PDMS-based microsystems.

A high-throughput imaging system to quantitatively analyze the growth dynamics of plant seedlings.

Most current methods for analyzing the growth rate of plant seedlings are limited to low-throughput experimental configurations. We have developed an automatic system to investigate the dynamics of the growth of hypocotyls using Arabidopsis as model. This system is able to capture time-lapse infrared images of 24 seedlings automatically, with a spatial resolution of 2 µm per pixel and temporal interval of 5 min. Seedling length is rapidly calculated using automated geometric image-processing algorithms. With this high-throughput platform, we have investigated the genotype dependent difference of growth patterns, as well as the response to plant hormone - ethylene. Our analyses suggest that cytoskeleton function is not required in ethylene-induced hypocotyl inhibition. This novel integrative method can be applied to large-scale dynamic screening of plants, as well as any other image-based biological studies related to dynamic growth.

Live cell imaging analysis of the epigenetic regulation of the human endothelial cell migration at single-cell resolution.

Epigenetic regulation plays an important role in cell migration. Although many methods have been developed to measure the motility of mammalian cells, accurate quantitative assessments of the migration speed of individual cells remain a major challenge. It is difficult for conventional scratch assays to differentiate proliferation from migration during the so-called wound-healing processes because of the long experimental time required. In addition, it is also challenging to create identical conditions for evaluating cell migration by conventional methods. We developed a microfluidic device with precisely created blanks allowing for robust and reproducible cell migration inside accurately-controlled microenvironments to study the regulatory effect of the epigenetic regulator histone deacetylase 7 (HDAC7) on cell migration. Through analyzing time-lapse imaging of the cells migrating into individual blank regions, we can measure the migration speed parameter for human primary cells within a few hours, eliminating the confounding effect of cell proliferation. We also developed an automatic image analysis and a numeric model-based data fitting to set up an integrated cell migration analysis system at single-cell resolution. Using this system, we measured the motility of primary human umbilical vein endothelial cells (HUVECs) and the migration speed reduction due to the silencing of HDAC7 and various other genes. We showed that the migration behaviour of these human primary cells are clearly regulated by epigenetic mechanisms, demonstrating the great potential of this accurate and robust assay in the fields of quantitatively migration studies and high-throughput screening.

Digital polymerase chain reaction in an array of femtoliter polydimethylsiloxane microreactors.

We developed a simple, compact microfluidic device to perform high dynamic-range digital polymerase chain reaction (dPCR) in an array of isolated 36-femtoliter microreactors. The density of the microreactors exceeded 20000/mm(2). This device, made from polydimethylsiloxane (PDMS), allows the samples to be loaded into all microreactors simultaneously. The microreactors are completely sealed through the deformation of a PDMS membrane. The small volume of the microreactors ensures a compact device with high reaction efficiency and low reagent and sample consumption. Future potential applications of this platform include multicolor dPCR and massively parallel dPCR for next generation sequencing library preparation.

Squeeze-chip: a finger-controlled microfluidic flow network device and its application to biochemical assays.

We designed and fabricated a novel microfluidic device that can be operated through simple finger squeezing. On-chip microfluidic flow control is enabled through an optimized network of check-valves and squeeze-pumps. The sophisticated flow system can be easily constructed by combining a few key elements. We implemented this device to perform quantitative biochemical assays with no requirement for precision instruments.