Construction of a human cell landscape at single-cell level.

Single-cell analysis is a valuable tool for dissecting cellular heterogeneity in complex systems1. However, a comprehensive single-cell atlas has not been achieved for humans. Here we use single-cell mRNA sequencing to determine the cell-type composition of all major human organs and construct a scheme for the human cell landscape (HCL). We have uncovered a single-cell hierarchy for many tissues that have not been well characterized. We established a 'single-cell HCL analysis' pipeline that helps to define human cell identity. Finally, we performed a single-cell comparative analysis of landscapes from human and mouse to identify conserved genetic networks. We found that stem and progenitor cells exhibit strong transcriptomic stochasticity, whereas differentiated cells are more distinct. Our results provide a useful resource for the study of human biology.

An Ultramicroelectrode Electrochemistry and Surface Plasmon Resonance Coupling Method for Cell Exocytosis Study.

Exocytosis of a single cell has been extensively researched in recent years due to its close association with numerous diseases. However, current methods only investigate exocytosis at either the single-cell or multiple-cell level, and a method for simultaneously studying exocytosis at both levels has yet to be established. In this study, a combined device incorporating ultramicroelectrode (UME) electrochemistry and surface plasmon resonance (SPR) was developed, enabling the simultaneous monitoring of single-cell and multiple-cell exocytosis. PC12 cells were cultured directly on the SPR sensing Au film, with a carboxylated carbon nanopipette (c-CNP) electrode employed for electrochemical detection in the SPR reaction cell. Upon exocytosis, the released dopamine diffuses onto the inner wall of c-CNP, undergoing an electrochemical reaction to generate a current peak. Concurrently, exocytosis can also induce changes in the refractive index of the Au film surface, leading to the SPR signal. Consequently, the device enables real-time monitoring of exocytosis from both single and multiple cells with a high spatiotemporal resolution. The c-CNP electrode exhibited excellent resistance to protein contamination, high sensitivity for dopamine detection, and the capability to continuously monitor dopamine exocytosis over an extended period. Analysis of both SPR and electrochemical signals revealed a positive correlation between changes in the SPR signal and the frequency of exocytosis. This study introduces a novel method and platform for the simultaneous investigation of single-cell and multiple-cell exocytosis.

Optoelectronic Tweezers Micro-Well System for Highly Efficient Single-Cell Trapping, Dynamic Sorting, and Retrieval.

Single-cell arrays have emerged as a versatile method for executing single-cell manipulations across an array of biological applications. In this paper, an innovative microfluidic platform is unveiled that utilizes optoelectronic tweezers (OETs) to array and sort individual cells at a flow rate of 20 µL min-1. This platform is also adept at executing dielectrophoresis (DEP)-based, light-guided single-cell retrievals from designated micro-wells. This presents a compelling non-contact method for the rapid and straightforward sorting of cells that are hard to distinguish. Within this system, cells are individually confined to micro-wells, achieving an impressive high single-cell capture rate exceeding 91.9%. The roles of illuminating patterns, flow velocities, and applied electrical voltages are delved into in enhancing the single-cell capture rate. By integrating the OET system with the micro-well arrays, the device showcases adaptability and a plethora of functions. It can concurrently trap and segregate specific cells, guided by their dielectric signatures. Experimental results, derived from a mixed sample of HepG2 and L-O2 cells, reveal a sorting accuracy for L-O2 cells surpassing 91%. Fluorescence markers allow for the identification of sequestered, fluorescence-tagged HepG2 cells, which can subsequently be selectively released within the chip. This platform's rapidity in capturing and releasing individual cells augments its potential for future biological research and applications.

Glass nano/micron pipette-based ion current rectification sensing technology for single cell/in vivo analysis.

Glass nano/micron pipettes, owing to their easy preparation, unique confined space at the tip, and modifiable inner surface of the tip, can capture the ion current signal caused by a single entity, making them widely used in the construction of highly sensitive and highly selective electrochemical sensors for single entity analysis. Compared with other solid-state nanopores, their conical nano-tip causes less damage to cells when inserted into them, thereby becoming a powerful tool for the in situ analysis of important substances in cells. However, glass nanopipettes have some shortcomings, such as poor mechanical properties, difficulty in precise preparation (aperture less than 50 nm), and easy blockage during complex real sample detection, limiting their practicability. Therefore, in recent years, researchers have conducted a series of studies on glass micropipettes. Ionic current rectification technology is a novel electrochemical analysis technique. Compared with traditional electrochemical analysis methods, it does not generate redox products during the detection process; therefore, it can not only be used for the determination of non-electrochemically active substances, but also causes less damage to the cell/living body in situ analysis, becoming a powerful analysis technology for the in situ analysis of cells/in vivo in recent years. In this review, we summarize the preparation and functionalization of glass nano/micron pipettes and introduce the sensing mechanisms of two electrochemical sensing platforms constructed using glass nano/micron pipette-based ion current rectification sensing technology as well as their applications in single cell/in vivo analysis, existing problems, and future prospects.

Phenotyping of single plant cells on a microfluidic cytometry platform with fluorescent, mechanical, and electrical modules.

Compared to animal cells, phenotypic characterization of single plant cells on microfluidic platforms is still rare. In this work, we collated population statistics on the morphological, biochemical, physical and electrical properties of Arabidopsis protoplasts under different external and internal conditions, using progressively improved microfluidic platforms. First, we analyzed the different effects of three phytohormones (auxin, cytokinin and gibberellin) on the primary cell wall (PCW) regeneration process using a microfluidic flow cytometry platform equipped with a single-channel fluorescence sensor. Second, we correlated the intracellular reactive oxygen species (ROS) level induced by heavy metal stress with the concurrent PCW regeneration process by using a dual-channel fluorescence sensor. Third, by integrating contraction channels, we were able to effectively discriminate variations in cell size while monitoring the intensity of intracellular ROS signaling. Fourth, by combining an electrical impedance electrode with the contraction channel, we analyzed the differences in electrical and mechanical properties of wild-type and mutant plant cells before and after primary cell wall regeneration. Overall, our work demonstrates the feasibility and sensitivity of microfluidic flow cytometry in high-throughput phenotyping of plant cells and provides a reference for assessing metabolic and physiological indicators of individual plant cells in multiple dimensions.

High-resolution magnetic resonance spectroscopy using a solid-state spin sensor.

Quantum systems that consist of solid-state electronic spins can be sensitive detectors of nuclear magnetic resonance (NMR) signals, particularly from very small samples. For example, nitrogen-vacancy centres in diamond have been used to record NMR signals from nanometre-scale samples, with sensitivity sufficient to detect the magnetic field produced by a single protein. However, the best reported spectral resolution for NMR of molecules using nitrogen-vacancy centres is about 100 hertz. This is insufficient to resolve the key spectral identifiers of molecular structure that are critical to NMR applications in chemistry, structural biology and materials research, such as scalar couplings (which require a resolution of less than ten hertz) and small chemical shifts (which require a resolution of around one part per million of the nuclear Larmor frequency). Conventional, inductively detected NMR can provide the necessary high spectral resolution, but its limited sensitivity typically requires millimetre-scale samples, precluding applications that involve smaller samples, such as picolitre-volume chemical analysis or correlated optical and NMR microscopy. Here we demonstrate a measurement technique that uses a solid-state spin sensor (a magnetometer) consisting of an ensemble of nitrogen-vacancy centres in combination with a narrowband synchronized readout protocol to obtain NMR spectral resolution of about one hertz. We use this technique to observe NMR scalar couplings in a micrometre-scale sample volume of approximately ten picolitres. We also use the ensemble of nitrogen-vacancy centres to apply NMR to thermally polarized nuclear spins and resolve chemical-shift spectra from small molecules. Our technique enables analytical NMR spectroscopy at the scale of single cells.

The honeycomb maze provides a novel test to study hippocampal-dependent spatial navigation.

Here we describe the honeycomb maze, a behavioural paradigm for the study of spatial navigation in rats. The maze consists of 37 platforms that can be raised or lowered independently. Place navigation requires an animal to go to a goal platform from any of several start platforms via a series of sequential choices. For each, the animal is confined to a raised platform and allowed to choose between two of the six adjacent platforms, the correct one being the platform with the smallest angle to the goal-heading direction. Rats learn rapidly and their choices are influenced by three factors: the angle between the two choice platforms, the distance from the goal, and the angle between the correct platform and the direction of the goal. Rats with hippocampal damage are impaired in learning and their performance is affected by all three factors. The honeycomb maze represents a marked improvement over current spatial navigation tests, such as the Morris water maze, because it controls the choices of the animal at each point in the maze, provides the ability to assess knowledge of the goal direction from any location, enables the identification of factors influencing task performance and provides the possibility for concomitant single-cell recording.

Magnetic Stirring Device for Limiting the Sedimentation of Cells inside Microfluidic Devices.

In experiments considering cell handling in microchannels, cell sedimentation in the storage container is a key problem because it affects the reproducibility of the experiments. Here, a simple and low-cost cell mixing device (CMD) is presented; the device is designed to prevent the sedimentation of cells in a syringe during their injection into a microfluidic channel. The CMD is based on a slider crank device made of 3D-printed parts that, combined with a permanent magnet, actuate a stir bar placed into the syringe containing the cells. By using A549 cell lines, the device is characterized in terms of cell viability (higher than 95%) in different mixing conditions, by varying the oscillation frequency and the overall mixing time. Then, a dedicated microfluidic experiment is designed to evaluate the injection frequency of the cells within a microfluidic chip. In the presence of the CMD, a higher number of cells are injected into the microfluidic chip with respect to the static conditions (2.5 times), proving that it contrasts cell sedimentation and allows accurate cell handling. For these reasons, the CMD can be useful in microfluidic experiments involving single-cell analysis.

Cellular microarrays for assessing single-cell phenotypic changes in vascular cell populations.

Microengineering technologies provide bespoke tools for single-cell studies, including microarray approaches. There are many challenges when culturing adherent single cells in confined geometries for extended periods, including the ability of migratory cells to overcome confining cell-repellent surfaces with time. Following studies suggesting clonal expansion of only a few vascular smooth muscle cells (vSMCs) contributes to plaque formation, the investigation of vSMCs at the single-cell level is central to furthering our understanding of atherosclerosis. Herein, we present a medium throughput cellular microarray, for the tracking of single, freshly-isolated vSMCs as they undergo phenotypic modulation in vitro. Our solution facilitates long-term cell confinement (> 3 weeks) utilising novel application of surface functionalisation methods to define individual culture microwells. We demonstrate successful tracking of hundreds of native vSMCs isolated from rat aortic and carotid artery tissue, monitoring their proliferative capacity and uptake of oxidised low-density lipoprotein (oxLDL) by live-cell microscopy. After 7 days in vitro, the majority of viable SMCs remained as single non-proliferating cells (51% aorta, 78% carotid). However, a sub-population of vSMCs demonstrated high proliferative capacity (≥ 10 progeny; 18% aorta, 5% carotid), in line with reports that a limited number of medial SMCs selectively expand to populate atherosclerotic lesions. Furthermore, we show that, when exposed to oxLDL, proliferative cells uptake higher levels of lipoproteins, whilst also expressing greater levels of galectin-3. Our microwell array approach enables long-term characterisation of multiple phenotypic characteristics and the identification of new cellular sub-populations in migratory, proliferative adherent cell types.

Moving into the cell: single-molecule studies of molecular motors in complex environments.

Much has been learned in the past decades about molecular force generation. Single-molecule techniques, such as atomic force microscopy, single-molecule fluorescence microscopy and optical tweezers, have been key in resolving the mechanisms behind the power strokes, 'processive' steps and forces of cytoskeletal motors. However, it remains unclear how single force generators are integrated into composite mechanical machines in cells to generate complex functions such as mitosis, locomotion, intracellular transport or mechanical sensory transduction. Using dynamic single-molecule techniques to track, manipulate and probe cytoskeletal motor proteins will be crucial in providing new insights.

Inertial picobalance reveals fast mass fluctuations in mammalian cells.

The regulation of size, volume and mass in living cells is physiologically important, and dysregulation of these parameters gives rise to many diseases. Cell mass is largely determined by the amount of water, proteins, lipids, carbohydrates and nucleic acids present in a cell, and is tightly linked to metabolism, proliferation and gene expression. Technologies have emerged in recent years that make it possible to track the masses of single suspended cells and adherent cells. However, it has not been possible to track individual adherent cells in physiological conditions at the mass and time resolutions required to observe fast cellular dynamics. Here we introduce a cell balance (a 'picobalance'), based on an optically excited microresonator, that measures the total mass of single or multiple adherent cells in culture conditions over days with millisecond time resolution and picogram mass sensitivity. Using our technique, we observe that the mass of living mammalian cells fluctuates intrinsically by around one to four per cent over timescales of seconds throughout the cell cycle. Perturbation experiments link these mass fluctuations to the basic cellular processes of ATP synthesis and water transport. Furthermore, we show that growth and cell cycle progression are arrested in cells infected with vaccinia virus, but mass fluctuations continue until cell death. Our measurements suggest that all living cells show fast and subtle mass fluctuations throughout the cell cycle. As our cell balance is easy to handle and compatible with fluorescence microscopy, we anticipate that our approach will contribute to the understanding of cell mass regulation in various cell states and across timescales, which is important in areas including physiology, cancer research, stem-cell differentiation and drug discovery.

Metabolic cost of rapid adaptation of single yeast cells.

Cells can rapidly adapt to changing environments through nongenetic processes; however, the metabolic cost of such adaptation has never been considered. Here we demonstrate metabolic coupling in a remarkable, rapid adaptation process (1 in 1,000 cells adapt per hour) by simultaneously measuring metabolism and division of thousands of individual Saccharomyces cerevisiae cells using a droplet microfluidic system: droplets containing single cells are immobilized in a two-dimensional (2D) array, with osmotically induced changes in droplet volume being used to measure cell metabolism, while simultaneously imaging the cells to measure division. Following a severe challenge, most cells, while not dividing, continue to metabolize, displaying a remarkably wide diversity of metabolic trajectories from which adaptation events can be anticipated. Adaptation requires a characteristic amount of energy, indicating that it is an active process. The demonstration that metabolic trajectories predict a priori adaptation events provides evidence of tight energetic coupling between metabolism and regulatory reorganization in adaptation. This process allows S. cerevisiae to adapt on a physiological timescale, but related phenomena may also be important in other processes, such as cellular differentiation, cellular reprogramming, and the emergence of drug resistance in cancer.

Confined Nanopipet as a Versatile Tool for Precise Single Cell Manipulation.

The precise manipulation of single cells plays a fundamental role for single cell measurement, which is crucial for understanding the diverse cellular mechanisms. Unusual single cell behavior could thus be identified by integrating with advanced analytical methods such as single cell omics, unraveling the intrinsic cellular heterogeneity hidden in ensemble measurements. Herein, this technical note reports a nanopipet-based versatile method for manipulation of an ultrasmall volume of liquid, which further enables the precise manipulation of single cells. Femtoliter volumes of cytoplasm were extracted from single living cells and analyzed by time-of-flight secondary ion mass spectrometry. Moreover, several kinds of exogenous components were injected simultaneously into a cell, offering a delicate tool for multi-imaging in single living cells.

A discriminative learning approach to differential expression analysis for single-cell RNA-seq.

Single-cell RNA-seq makes it possible to characterize the transcriptomes of cell types across different conditions and to identify their transcriptional signatures via differential analysis. Our method detects changes in transcript dynamics and in overall gene abundance in large numbers of cells to determine differential expression. When applied to transcript compatibility counts obtained via pseudoalignment, our approach provides a quantification-free analysis of 3' single-cell RNA-seq that can identify previously undetectable marker genes.

Protein kinase sensors: an overview of new designs for visualizing kinase dynamics in single plant cells.

Protein kinase dynamics play key roles in regulation of cell differentiation, growth, development and in diverse cell signaling networks. Protein kinase sensors enable visualization of protein kinase activity in living cells and tissues in time and space. These sensors have therefore become important and powerful molecular tools for investigation of diverse kinase activities and can resolve long-standing and challenging biological questions. In the present Update, we review new advanced approaches for genetically encoded protein kinase biosensor designs developed in animal systems together with the basis of each biosensor's working principle and components. In addition, we review recent first examples of real time plant protein kinase activity biosensor development and application. We discuss how these sensors have helped to resolve how stomatal signal transduction in response to elevated CO2 merges with abscisic acid signaling downstream of a resolved basal SnRK2 kinase activity in guard cells. Furthermore, recent advances, combined with the new strategies described in this Update, can help deepen the understanding of how signaling networks regulate unique functions and responses in distinct plant cell types and tissues and how different stimuli and signaling pathways can interact.

Toward a translationally independent RNA-based synthetic oscillator using deactivated CRISPR-Cas.

In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nt guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging approximately nine generations, a dCas12a oscillator design with 40-nt CRISPR RNAs performed much better, having a strongly repressed off-state, distinct autocorrelation function peaks, and an average peak-to-peak period length of ∼7.5 generations. Along with free-running oscillator circuits, we measure repression response times in open-loop systems with inducible RNA steps to compare with oscillator period times. We track thousands of cells for 24+ h at the single-cell level using a microfluidic device. In creating a circuit with nearly translationally independent behavior, as the RNAs control each others' transcription, we present the possibility for a synthetic oscillator generalizable across many organisms and readily linkable for transcriptional control.

The optoelectronic microrobot: A versatile toolbox for micromanipulation.

Microrobotics extends the reach of human-controlled machines to submillimeter dimensions. We introduce a microrobot that relies on optoelectronic tweezers (OET) that is straightforward to manufacture, can take nearly any desirable shape or form, and can be programmed to carry out sophisticated, multiaxis operations. One particularly useful program is a serial combination of "load," "transport," and "deliver," which can be applied to manipulate a wide range of micrometer-dimension payloads. Importantly, microrobots programmed in this manner are much gentler on fragile mammalian cells than conventional OET techniques. The microrobotic system described here was demonstrated to be useful for single-cell isolation, clonal expansion, RNA sequencing, manipulation within enclosed systems, controlling cell-cell interactions, and isolating precious microtissues from heterogeneous mixtures. We propose that the optoelectronic microrobotic system, which can be implemented using a microscope and consumer-grade optical projector, will be useful for a wide range of applications in the life sciences and beyond.

Imaging microphysiological systems: a review.

Microphysiological systems (MPS), often referred to as "organ-on-chips," are microfluidic-based in vitro models that aim to recapitulate the dynamic chemical and mechanical microenvironment of living organs. MPS promise to bridge the gap between in vitro and in vivo models and ultimately improve the translation from preclinical animal studies to clinical trials. However, despite the explosion of interest in this area in recent years, and the obvious rewards for such models that could improve R&D efficiency and reduce drug attrition in the clinic, the pharmaceutical industry has been slow to fully adopt this technology. The ability to extract robust, quantitative information from MPS at scale is a key requirement if these models are to impact drug discovery and the subsequent drug development process. Microscopy imaging remains a core technology that enables the capture of information at the single-cell level and with subcellular resolution. Furthermore, such imaging techniques can be automated, increasing throughput and enabling compound screening. In this review, we discuss a range of imaging techniques that have been applied to MPS of varying focus, such as organoids and organ-chip-type models. We outline the opportunities these technologies can bring in terms of understanding mechanistic biology, but also how they could be used in higher-throughput screens, widening the scope of their impact in drug discovery. We discuss the associated challenges of imaging these complex models and the steps required to enable full exploitation. Finally, we discuss the requirements for MPS, if they are to be applied at a scale necessary to support drug discovery projects.

Reconstitution of cell migration at a glance.

Single cells migrate in a myriad of physiological contexts, such as tissue patrolling by immune cells, and during neurogenesis and tissue remodeling, as well as in metastasis, the spread of cancer cells. To understand the basic principles of single-cell migration, a reductionist approach can be taken. This aims to control and deconstruct the complexity of different cellular microenvironments into simpler elementary constrains that can be recombined together. This approach is the cell microenvironment equivalent of in vitro reconstituted systems that combine elementary molecular players to understand cellular functions. In this Cell Science at a Glance article and accompanying poster, we present selected experimental setups that mimic different events that cells undergo during migration in vivo These include polydimethylsiloxane (PDMS) devices to deform whole cells or organelles, micro patterning, nano-fabricated structures like grooves, and compartmentalized collagen chambers with chemical gradients. We also outline the main contribution of each technique to the understanding of different aspects of single-cell migration.