High-throughput fluorescence lifetime imaging flow cytometry.

Flow cytometry is a vital tool in biomedical research and laboratory medicine. However, its accuracy is often compromised by undesired fluctuations in fluorescence intensity. While fluorescence lifetime imaging microscopy (FLIM) bypasses this challenge as fluorescence lifetime remains unaffected by such fluctuations, the full integration of FLIM into flow cytometry has yet to be demonstrated due to speed limitations. Here we overcome the speed limitations in FLIM, thereby enabling high-throughput FLIM flow cytometry at a high rate of over 10,000 cells per second. This is made possible by using dual intensity-modulated continuous-wave beam arrays with complementary modulation frequency pairs for fluorophore excitation and acquiring fluorescence lifetime images of rapidly flowing cells. Moreover, our FLIM system distinguishes subpopulations in male rat glioma and captures dynamic changes in the cell nucleus induced by an anti-cancer drug. FLIM flow cytometry significantly enhances cellular analysis capabilities, providing detailed insights into cellular functions, interactions, and environments.

High-throughput mechanical phenotyping and transcriptomics of single cells.

The molecular system regulating cellular mechanical properties remains unexplored at single-cell resolution mainly due to a limited ability to combine mechanophenotyping with unbiased transcriptional screening. Here, we describe an electroporation-based lipid-bilayer assay for cell surface tension and transcriptomics (ELASTomics), a method in which oligonucleotide-labelled macromolecules are imported into cells via nanopore electroporation to assess the mechanical state of the cell surface and are enumerated by sequencing. ELASTomics can be readily integrated with existing single-cell sequencing approaches and enables the joint study of cell surface mechanics and underlying transcriptional regulation at an unprecedented resolution. We validate ELASTomics via analysis of cancer cell lines from various malignancies and show that the method can accurately identify cell types and assess cell surface tension. ELASTomics enables exploration of the relationships between cell surface tension, surface proteins, and transcripts along cell lineages differentiating from the haematopoietic progenitor cells of mice. We study the surface mechanics of cellular senescence and demonstrate that RRAD regulates cell surface tension in senescent TIG-1 cells. ELASTomics provides a unique opportunity to profile the mechanical and molecular phenotypes of single cells and can dissect the interplay among these in a range of biological contexts.

Opto-combinatorial indexing enables high-content transcriptomics by linking cell images and transcriptomes.

We introduce a simple integrated analysis method that links cellular phenotypic behaviour with single-cell RNA sequencing (scRNA-seq) by utilizing a combination of optical indices from cells and hydrogel beads. With our method, the combinations, referred to as joint colour codes, enable the link via matching the optical combinations measured by conventional epi-fluorescence microscopy with the concatenated DNA molecular barcodes created by cell-hydrogel bead pairs and sequenced by next-generation sequencing. We validated our approach by demonstrating an accurate link between the cell image and scRNA-seq with mixed species experiments, longitudinal cell tagging by electroporation and lipofection, and gene expression analysis. Furthermore, we extended our approach to multiplexed chemical transcriptomics, which enabled us to identify distinct phenotypic behaviours in HeLa cells treated with various concentrations of paclitaxel, and determine the corresponding gene regulation associated with the formation of a multipolar spindle.

Replicating shear-mediated self-assembly of spider silk through microfluidics.

The development of artificial spider silk with properties similar to native silk has been a challenging task in materials science. In this study, we use a microfluidic device to create continuous fibers based on recombinant MaSp2 spidroin. The strategy incorporates ion-induced liquid-liquid phase separation, pH-driven fibrillation, and shear-dependent induction of ß-sheet formation. We find that a threshold shear stress of approximately 72 Pa is required for fiber formation, and that ß-sheet formation is dependent on the presence of polyalanine blocks in the repetitive sequence. The MaSp2 fiber formed has a ß-sheet content (29.2%) comparable to that of native dragline with a shear stress requirement of 111 Pa. Interestingly, the polyalanine blocks have limited influence on the occurrence of liquid-liquid phase separation and hierarchical structure. These results offer insights into the shear-induced crystallization and sequence-structure relationship of spider silk and have significant implications for the rational design of artificially spun fibers.

Microfluidic vascular formation model for assessing angiogenic capacities of single islets.

Pancreatic islet transplantation presents a promising therapy for individuals suffering from type 1 diabetes. To maintain the function of transplanted islets in vivo, it is imperative to induce angiogenesis. However, the mechanisms underlying angiogenesis triggered by islets remain unclear. In this study, we introduced a microphysiological system to study the angiogenic capacity and dynamics of individual islets. The system, which features an open-top structure, uniquely facilitates the inoculation of islets and the longitudinal observation of vascular formation in in vivo like microenvironment with islet-endothelial cell communication. By leveraging our system, we discovered notable islet-islet heterogeneity in the angiogenic capacity. Transcriptomic analysis of the vascularized islets revealed that islets with high angiogenic capacity exhibited upregulation of genes related to insulin secretion and downregulation of genes related to angiogenesis and fibroblasts. In conclusion, our microfluidic approach is effective in characterizing the vascular formation of individual islets and holds great promise for elucidating the angiogenic mechanisms that enhance islet transplantation therapy.

Secretory GFP reconstitution labeling of neighboring cells interrogates cell-cell interactions in metastatic niches.

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

A SINC-Seq Protocol for the Analysis of Subcellular Gene Expression in Single Cells.

Microfluidic devices offer precise control of single cells and molecules by liquid flows, downsizing tools to allow us to perform single-cell assays at unprecedented resolutions and minimizing contamination. In this chapter, we introduce an approach, called single-cell integrated nuclear and cytoplasmic RNA-sequencing (SINC-seq), which enables precise fractionation of cytoplasmic and nuclear RNA of single cells. This approach uses electric field control in microfluidics to manipulate single cells and RNA sequencing to dissect gene expression and RNA localization in subcellular compartments. The microfluidic system for SINC-seq exploits a hydrodynamic trap (a constriction in a microchannel) to isolate a single cell, selectively lyses its plasma membrane via a focused electric field, and retains the nucleus at the hydrodynamic trap during the electrophoretic extraction of cytoplasmic RNA. Here, we provide a step-by-step protocol from microfluidic RNA fractionation to off-chip preparation of RNA-sequencing libraries for full-length cDNA sequencing using both a short-read sequencer (Illumina) and a long-read sequencer (Oxford Nanopore Technologies).

The mechanosensitive ion channel PIEZO1 promotes satellite cell function in muscle regeneration.

Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscles, play an essential role in muscle regeneration. After skeletal muscle injury, quiescent MuSCs are activated to enter the cell cycle and proliferate, thereby initiating regeneration; however, the mechanisms that ensure successful MuSC division, including chromosome segregation, remain unclear. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel activated by membrane tension, mediates spontaneous Ca2+ influx to control the regenerative function of MuSCs. Our genetic engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs and that Piezo1 deletion in these cells delays myofibre regeneration after injury. These results are, at least in part, due to a mitotic defect in MuSCs. Mechanistically, this phenotype is caused by impaired PIEZO1-Rho signalling during myogenesis. Thus, we provide the first concrete evidence that PIEZO1, a bona fide mechanosensitive ion channel, promotes proliferation and regenerative functions of MuSCs through precise control of cell division.

Extreme deformability of insect cell membranes is governed by phospholipid scrambling.

Organization of dynamic cellular structure is crucial for a variety of cellular functions. In this study, we report that Drosophila and Aedes have highly elastic cell membranes with extremely low membrane tension and high resistance to mechanical stress. In contrast to other eukaryotic cells, phospholipids are symmetrically distributed between the bilayer leaflets of the insect plasma membrane, where phospholipid scramblase (XKR) that disrupts the lipid asymmetry is constitutively active. We also demonstrate that XKR-facilitated phospholipid scrambling promotes the deformability of cell membranes by regulating both actin cortex dynamics and mechanical properties of the phospholipid bilayer. Moreover, XKR-mediated construction of elastic cell membranes is essential for hemocyte circulation in the Drosophila cardiovascular system. Deformation of mammalian cells is also enhanced by the expression of Aedes XKR, and thus phospholipid scrambling may contribute to formation of highly deformable cell membranes in a variety of living eukaryotic cells.

NanoSINC-seq dissects the isoform diversity in subcellular compartments of single cells.

Alternative mRNA isoforms play a key role in generating diverse protein isoforms. To dissect isoform usage in the subcellular compartments of single cells, we introduced an novel approach, nanopore sequencing coupled with single-cell integrated nuclear and cytoplasmic RNA sequencing, that couples microfluidic fractionation, which separates cytoplasmic RNA from nuclear RNA, with full-length complementary DNA (cDNA) sequencing using a nanopore sequencer. Leveraging full-length cDNA reads, we found that the nuclear transcripts are notably more diverse than cytoplasmic transcripts. Our findings also indicated that transcriptional noise emanating from the nucleus is regulated across the nuclear membrane and then either attenuated or amplified in the cytoplasm depending on the function involved. Overall, our results provide the landscape that shows how the transcriptional noise arising from the nucleus propagates to the cytoplasm.

Targeted permeabilization of the cell wall and extraction of charged molecules from single cells in intact plant clusters using a focused electric field.

The extraction of cellular contents from plant cells covered with cell walls remains a challenge, as it is physically hindered by the cell wall. We present a new microfluidic approach that leverages an intense pulsed electric field for permeabilizing the cell wall and a focused DC electric field for extracting the cellular contents selectively from a few targeted cells in a cluster of intact plant cells. We coupled the approach with on-chip fluorescence quantification of extracted molecules leveraging isotachophoresis as well as off-chip reverse transcription-quantitative polymerase chain reaction detecting extracted mRNA molecules. Our approach offers a workflow of about 5 min, isolating a cluster of intact plant cells, permeabilizing the cell wall, selectively extracting cytosolic molecules from a few targeted cells in the cluster, and outputting them to off-chip analyses without any enzymatic reactions. We anticipate that this approach will create a new opportunity to explore plant biology through less biased data realized by the rapid extraction of molecules from intact plant clusters.

Development of a sequencing system for spatial decoding of DNA barcode molecules at single-molecule resolution.

Single-cell transcriptome analysis has been revolutionized by DNA barcodes that index cDNA libraries, allowing highly multiplexed analyses to be performed. Furthermore, DNA barcodes are being leveraged for spatial transcriptomes. Although spatial resolution relies on methods used to decode DNA barcodes, achieving single-molecule decoding remains a challenge. Here, we developed an in-house sequencing system inspired by a single-molecule sequencing system, HeliScope, to spatially decode DNA barcode molecules at single-molecule resolution. We benchmarked our system with 30 types of DNA barcode molecules and obtained an average read length of ~20 nt with an error rate of less than 5% per nucleotide, which was sufficient to spatially identify them. Additionally, we spatially identified DNA barcode molecules bound to antibodies at single-molecule resolution. Leveraging this, we devised a method, termed "molecular foot printing", showing potential for applying our system not only to spatial transcriptomics, but also to spatial proteomics.

Different motilities of microtubules driven by kinesin-1 and kinesin-14 motors patterned on nanopillars.

Kinesin is a motor protein that plays important roles in a variety of cellular functions. In vivo, multiple kinesin molecules are bound to cargo and work as a team to produce larger forces or higher speeds than a single kinesin. However, the coordination of kinesins remains poorly understood because of the experimental difficulty in controlling the number and arrangement of kinesins, which are considered to affect their coordination. Here, we report that both the number and spacing significantly influence the velocity of microtubules driven by nonprocessive kinesin-14 (Ncd), whereas neither the number nor the spacing changes the velocity in the case of highly processive kinesin-1. This result was realized by the optimum nanopatterning method of kinesins that enables immobilization of a single kinesin on a nanopillar. Our proposed method enables us to study the individual effects of the number and spacing of motors on the collective dynamics of multiple motors.

Distinct Kinetics in Electrophoretic Extraction of Cytoplasmic RNA from Single Cells.

The physical fractionation of cytoplasmic versus nuclear components of cells is a key step for studying the subcellular localization of molecules. The application of an electric field is an emerging method for subcellular fractionation of proteins and nucleic acids from single cells. However, the multibiophysical process that involves electrical lysis of cytoplasmic membranes, electrophoresis, and diffusion of charged molecules remains unclear. Here we study RNA dynamics in single cells during the electrophoretic extraction via a microfluidic system that enables stringent fractionation of the subcellular components leveraging a focused electric field. We identified two distinct kinetics in the extraction of RNA molecules, which were respectively associated with soluble RNA and mitochondrial RNA. We show that the extraction kinetics of soluble RNA is dominated by electrophoresis over diffusion and has a time constant of 0.15 s. Interestingly, the extraction of mitochondrial RNA showed unexpected heterogeneity in the extraction with slower kinetics (3.8 s), while reproducibly resulting in the extraction of 98.9% ± 2% after 40 s. Together, we uncover that the microfluidic system uniquely offers length bias-free fractionation of RNA molecules for quantitative analysis of correlations among subcellular compartments by exploiting the homogeneous electrophoretic properties of RNA.

Spatial Patterning of Kinesin-1 and Dynein Motor Proteins in an In Vitro Assay using Aqueous Two-Phase Systems (ATPS).

Cooperativity of motor proteins is essential for intracellular transport. Although their motion is unidirectional, they often cause bidirectional movement by different types of motors as seen in organelles. However, in vitro assessments of such cellular functions are still inadequate owing to the experimental limitations in precisely patterning multiple motors. Here, we present an approach to immobilize two motor proteins, kinesin-1 and dynein, using the aqueous two-phase system (ATPS) made of poly(ethylene glycol) and dextran polymers. The negligible influence of polymer solutions on the attachment and velocity of motor proteins ensures the compatibility of using ATPS as the patterning technique. The selective fixation of kinesin and dynein was assessed using polarity-marked microtubules (PMMTs). Our experimental results show that on a patterned kinesin surface, 72% of PMMTs display minus-end leading motility, while on a dynein surface, 79% of PMMTs display plus-end leading motility. This work offers a universal and biocompatible method to pattern motor proteins of different classes at the nanoscale, providing a new route to study different cellular functions performed by molecular motors such as the formation of mitotic spindles.

Transport of microtubules according to the number and spacing of kinesin motors on gold nano-pillars.

Motor proteins function in in vivo ensembles to achieve cargo transport, flagellum motion, and mitotic cell division. Although the cooperativity of multiple motors is indispensable for physiological function, reconstituting the arrangement of motors in vitro is challenging, so detailed analysis of the functions of motor ensembles has not yet been achieved. Here, we developed an assay platform to study the motility of microtubules driven by a defined number of kinesin motors spaced in a definite manner. Gold (Au) nano-pillar arrays were fabricated on a silicon/silicon dioxide (Si/SiO2) substrate with spacings of 100 nm to 500 nm. The thiol-polyethylene glycol (PEG)-biotin self-assembled monolayer (SAM) and silane-PEG-CH3 SAM were then selectively formed on the pillars and SiO2 surface, respectively. This allowed for both immobilization of kinesin molecules on Au nano-pillars in a precise manner and repulsion of kinesins from the SiO2 surface. Using arrayed kinesin motors, we report that motor number and spacing do not influence the motility of microtubules driven by kinesin-1 motors. This assay platform is applicable to all kinds of biotinylated motors, allows the study of the effects of motor number and spacing, and is expected to reveal novel behaviors of motor proteins.

Simultaneous Observation of Kinesin-Driven Microtubule Motility and Binding of Adenosine Triphosphate Using Linear Zero-Mode Waveguides.

Single-molecule fluorescence observation of adenosine triphosphate (ATP) is a powerful tool to elucidate the chemomechanical coupling of ATP with a motor protein. However, in total internal reflection fluorescence microscopy (TIRFM), available ATP concentration is much lower than that in the in vivo environment. To achieve single-molecule observation with a high signal-to-noise ratio, zero-mode waveguides (ZMWs) are utilized even at high fluorescent molecule concentrations in the micromolar range. Despite the advantages of ZMWs, the use of cytoskeletal filaments for single-molecule observation has not been reported because of difficulties in immobilization of cytoskeletal filaments in the cylindrical aperture of ZMWs. Here, we propose linear ZMWs (LZMWs) to visualize enzymatic reactions on cytoskeletal filaments, specifically kinesin-driven microtubule motility accompanied by ATP binding/unbinding. Finite element method simulation revealed excitation light confinement in a 100 nm wide slit of LZMWs. Single-molecule observation was then demonstrated with up to 1 µM labeled ATP, which was 10-fold higher than that available in TIRFM. Direct observation of binding/unbinding of ATP to kinesins that propel microtubules enabled us to find that a significant fraction of ATP molecules bound to kinesins were dissociated without hydrolysis. This highlights the advantages of LZMWs for single-molecule observation of proteins that interact with cytoskeletal filaments such as microtubules, actin filaments, or intermediate filaments.

Electrical Lysis and RNA Extraction from Single Cells Fixed by Dithiobis(succinimidyl propionate).

We present a microfluidic method for electrical lysis and RNA extraction from single fixed cells leveraging reversible cross-linker dithiobis(succinimidyl propionate) (DSP). Our microfluidic system captures a single DSP-fixed cell at a hydrodynamic trap, reverse-cross-links the DSP molecules on a chip with dithiothreitol, lyses the plasma membrane via electrical field, and extracts cytoplasmic RNA with isotachophoresis-aided nucleic acids extraction. All of the on-chip processes complete in less than 5 min. We demonstrated the method using K562 leukemia cells and benchmarked the performance of RNA extraction with reverse transcription quantitative polymerase chain reaction. We also demonstrated the integration of our method with single-cell RNA sequencing.

Intelligent Image-Activated Cell Sorting.

A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.