Analysis of subcellular surface structure, function and dynamics

Analytics of single biological cells allows quantitative investigation from a structural, functional and dynamical point of view and opens novel possibilities to an unamplified subcellular analysis. In this article, we report on three different experimental methods and their applications to single cellular systems with a subcellular sensitivity down to the single molecule level. First, the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic force microscopy (AFM) at the resolution of individual surface layer (S-layer) proteins; discrimination of bacterial strains that lack the expression of hexagonally packed surface layer proteins was possible. Second, quantitative measurement of individual recognition events of membrane-bound receptors on living B-cells was achieved in single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy. And third, intracellular dynamics of translocating photoactivatable GFP in plant protoplasts (Nicotiana tabacum BY-2) was quantitatively monitored by two-photon laser scanning microscopy (2PLSM).     

Intracellular injection of phospholipids directly alters exocytosis and the fraction of chemical release in chromaffin cells as measured by nano-electrochemistry

Using a nano-injection method, we introduced phospholipids having different intrinsic geometries into single secretory cells and used single cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC) with nanotip electrodes to monitor the effects of intracellular incubation on the exocytosis process and vesicular storage. Combining tools, this work provides new information to understand the impact of intracellular membrane lipid engineering on exocytotic release, vesicular content and fraction of chemical release. We also assessed the effect of membrane lipid alteration on catecholamine storage of isolated vesicles by implementing another amperometric technique, vesicle impact electrochemical cytometry (VIEC), outside the cell. Exocytosis analysis reveals that the intracellular nano-injection of phosphatidylcholine and lysophosphatidylcholine decreases the number of released catecholamines, whereas phosphatidylethanolamine shows the opposite effect. These observations support the emerging hypothesis that lipid curvature results in membrane remodeling through secretory pathways, and also provide new evidence for a critical role of the lipid localization in modulating the release process. Interestingly, the IVIEC data imply that total vesicular content is also affected by in situ supplementation of the cells with some lipids, while, the corresponding VIEC results show that the neurotransmitter content in isolated vesicles is not affected by altering the vesicle membrane lipids. This suggests that the intervention of phospholipids inside the cell has its effect on the cellular machinery for vesicle release rather than vesicle structure, and leads to the somewhat surprising conclusion that modulating release has a direct effect on vesicle structure, which is likely due to the vesicles opening and closing again during exocytosis. These findings could lead to a novel regulatory mechanism for the exocytotic or synaptic strength based on lipid heterogeneity across the cell membrane.

Amperometry and intracellular vesicle impact electrochemical cytometry with nanotip electrodes were used to monitor the effects on exocytosis and vesicular storage after nano-injection of phospholipids with different geometries into secretory cells.     

模板法合成纳米通道在微流控系统中的应用

纳流控芯片作为研究单分子水平上分子行为的一种新工具,人们期待纳流控在生物技术等领域有更广泛的应用.纳米结构的制作,作为纳流控芯片应用的前提之一,正逐渐引起人们的重视.本文综述了近几年来纳流控芯片中模板法制作纳米结构的研究进展,主要是以氧化铝膜,多孔硅,以及其它一些带有纳米通道的物质为模板,制作纳米结构应用于纳流控生化分析.     

纳流控-电化学技术在生化分析领域的研究进展

纳流控作为一个崭新的研究领域正受到越来越多的关注,并且已被成功应用到纳米尺度分离、生化传感、能量转化等诸多领域. 纳流控的发展与电化学紧密相连,一方面,电化学可以为纳米孔道中的物质传输特性的研究提供驱动力;另一方面,纳米孔道可以为限域电化学研究提供微环境. 纳流控和电化学技术相辅相成,催生了许多单分子、单粒子分析以及纳米流体操控的新理念与新技术. 本综述从纳米孔道与电极的结合方式出发,对纳流控-电化学相关研究进行了总结与展望.     

Analysis of affinity maps of membrane proteins on individual human embryonic stem cells

The heterogeneity found in many cell types has greatly inspired research in single-cell gene and protein profiling for discovering the origin of heterogeneity and its role in cell fate decisions. Among the existing techniques to probe heterogeneity, atomic force microscopy (AFM) utilizes an antibody/ligand-modified tip to explore the distribution of a target membrane protein on individual cells in their native environment. In this paper, we establish a practical model to analyze the data systematically, and attempt the quantification of membrane protein abundance on single cells by taking account issues, such as the level of nonspecific interaction, the probe resolution, and the reproducibility of detecting protein distribution. We demonstrated the application in examining the heterogeneous distribution and the local protein abundance of TRA-1-81 antigen on human embryonic stem (hES) cells at the subcellular level. Heterogeneity in TRA-1-81 expression was also detected at the single cell level, suggesting the presence of subpopulation cells within an undifferentiated hES cell colony. The method provides a platform to unveiling the correlation between heterogeneity of membrane proteins and cell development in a complex cell community.     

Quantitative measurement of quantum dot uptake at the cell population level using microfluidic evanescent-wave-based flow cytometry

The intracellular uptake of nanoparticles (NPs) is an important process for molecular and cellular labeling, drug/gene delivery and medical imaging. The vast majority of investigations into NP uptake have been conducted using confocal imaging that is limited to observation of a small number of cells. Such data may not yield quantitative information about the cell population due to the tiny sample size and the potential heterogeneity. Flow cytometry is the technique of choice for studying cell populations with single cell resolution. Unfortunately, classic flow cytometry detects fluorescence from whole cells and does not shed light on subcellular dynamics. In this report, we demonstrate the use of microfluidics-based total internal reflection fluorescence flow cytometry (TIRF-FC) for examining initial quantum dot (QD) entry into cells and the associated subcellular movement at the single cell level with a rate of ～200 cells s(-1). Our cytometric tool allows extraction of quantitative data from a large cell population and reveals details about the QD transport in the periphery of the cell membrane (～100 nm deep into the cytosol). Our data indicate that the fluorescence density at the membrane vicinity decreases after initial QD dosage due to the decline in the density of QDs in the evanescent field and the transport into the cytosol is very rapid.     

Fluorescent Triazole Urea Activity‐Based Probes for the Single‐Cell Phenotypic Characterization of Staphylococcus aureus

Phenotypically distinct cellular (sub)populations are clinically relevant for the virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Herein, we introduce fluorescent activity‐based probes as chemical tools for the single‐cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3‐triazole urea library to identify selective inhibitors of fluorophosphonate‐binding serine hydrolases and lipases in S. aureus and synthesized target‐selective activity‐based probes. Molecular imaging and activity‐based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single‐cell phenotypic heterogeneity. We propose the labeling of enzymatic activities by chemical probes as a generalizable method for the phenotyping of bacterial cells at the population and single‐cell level.     

Single cell analysis at the nanoscale

The fundamental life processes such as signal transduction, intracellular trafficking, protein degradation, and DNA repair often occur in nanometric subcellular compartments. It is essential to conduct single cell analysis specifically at the nanoscale to fully understand the critical cellular processes while providing important medical applications. However, there are great challenges in achieving high spatial resolution in single cells for uncovering spatial heterogeneity, high sensitivity for biomolecule detections and high specificity in complicated cellular environment. In this tutorial review, we survey recent progress toward single cell analysis at the nanoscale by emphasizing how the advancement in nanotechnology has brought a plethora of nanotools to interrogate single cells with high spatiotemporal resolutions. In particular, analysis principle, nanoscale probe fabrication, high resolution cellular analysis, data collection and processing are introduced. New cell biochemistry and biology insights revealed by the unique single cell analysis methods are highlighted. The perspectives on future opportunities and unsolved challenges are also discussed.     

In Situ Scatheless Cell Detachment Reveals Correlation between Adhesion Strength and Viability at Single‐Cell Resolution

Single‐cell biology provides insights into some of the most fundamental processes in biology and promotes the understanding of life's mysteries. As the technologies to study single‐cells expand, they will require sophisticated analytical tools to make sense of various behaviors and components of single‐cells as well as their relations in the adherent tissue culture. In this paper, we revealed cell heterogeneity and uncovered the connections between cell adhesion strength and cell viability at single‐cell resolution by extracting single adherent cells of interest from a standard tissue culture by using a microfluidic chip‐based live single‐cell extractor (LSCE). We believe that this method will provide a valuable new tool for single‐cell biology.     

Investigating cellular signaling reactions in single attoliter vesicles

Understanding cellular signaling mediated by cell surface receptors is key to modern biomedical research and drug development. The discovery of a growing number of potential molecular targets and therapeutic compounds requires downscaling and accelerated functional screening. Receptor-mediated cellular responses are typically investigated on single cells or cell populations. Here, we show how to monitor cellular signaling reactions at a yet unreached miniaturization level. On the basis of our observations, cytochalasin induces mammalian cells to extrude from their plasma membrane submicrometer-sized native vesicles. They comprise functional cell surface receptors correctly exposing their extracellular ligand binding sites on the outer vesicle surface and retaining cytosolic proteins in the vesicle interior. As a prototypical example, ligand binding to the ionotropic 5-HT(3) receptor and subsequent transmembrane Ca(2+) signaling were monitored in single attoliter vesicles. Thus, native vesicles are the smallest autonomous containers capable of performing cellular signaling reactions under physiological conditions. Because a single cell delivers about 50 native vesicles, which can be isolated and addressed as individuals, our concept allows multiple functional analyses of individual cells having a limited availability and opens new vistas for miniaturized bioanalytics.     

Rapid protein concentration, efficient fluorescence labeling and purification on a micro/nanofluidics chip

Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency.     

Visually precise,low-damage,single-cell spatial manipulation with single-pixel resolution

The analysis of single living cells, including intracellular delivery and extraction, is essential for monitoring their dynamic biochemical processes and exploring intracellular heterogeneity. However, owing to the 2D view in bright-field microscopy and optical distortions caused by the cell shape and the variation in the refractive index both inside and around the cells, achieving spatially undistorted imaging for high-precision manipulation within a cell is challenging. Here, an accurate and visual system is developed for single-cell spatial manipulation by correcting the aberration for simultaneous bright-field triple-view imaging. Stereo information from the triple view enables higher spatial resolution that facilitates the precise manipulation of single cells. In the bright field, we resolved the spatial locations of subcellular structures of a single cell suspended in a medium and measured the random spatial rotation angle of the cell with a precision of ±5°. Furthermore, we demonstrated the visual manipulation of a probe to an arbitrary spatial point of a cell with an accuracy of <1 pixel. This novel system is more accurate and less destructive for subcellular content extraction and drug delivery.

We achieved the low-damage spatial puncture of single cells at specific visual points with an accuracy of <65 nm.     

Analysis of single mammalian cells on-chip

A goal of modern biology is to understand the molecular mechanisms underlying cellular function. The ability to manipulate and analyze single cells is crucial for this task. The advent of microengineering is providing biologists with unprecedented opportunities for cell handling and investigation on a cell-by-cell basis. For this reason, lab-on-a-chip (LOC) technologies are emerging as the next revolution in tools for biological discovery. In the current discussion, we seek to summarize the state of the art for conventional technologies in use by biologists for the analysis of single, mammalian cells, and then compare LOC devices engineered for these same single-cell studies. While a review of the technical progress is included, a major goal is to present the view point of the practicing biologist and the advances that might increase adoption by these individuals. The LOC field is expanding rapidly, and we have focused on areas of broad interest to the biology community where the technology is sufficiently far advanced to contemplate near-term application in biological experimentation. Focus areas to be covered include flow cytometry, electrophoretic analysis of cell contents, fluorescent-indicator-based analyses, cells as small volume reactors, control of the cellular microenvironment, and single-cell PCR.     

Imaging methods for elemental, chemical, molecular, and morphological analyses of single cells

Combining elemental, chemical, molecular, and morphological imaging information from individual cells with a lateral resolution well below 1?×?1 μm² is the current technological challenge for investigating the smallest dimensions of living systems. In the race for such analytical performance, several techniques have been successfully developed; some use probes to determine given cellular contents whereas others use possible interactions between cellular matter with light or elements for characterization of contents. Morphological techniques providing information about cell dimensions have, when combined with other techniques, also opened the way to quantitative studies. New analytical opportunities are now being considered in cell biology, combining top-performance imaging techniques, applied to the same biosystem, with microscopy (nm–μm range) techniques providing elemental (micro-X-ray fluorescence, particle-induced X-ray emission, secondary-ion mass spectrometry), chemical (Raman, coherent anti-stokes Raman, Fourier-transform infrared, and near-field), molecular (UV–visible confocal and multiphoton), and morphological (AFM, ellipsometry, X-ray phase contrast, digital holography) information. Dedicated cell-culture methods have been proposed for multimodal imaging in vitro and/or ex vivo. This review shows that in addition to UV–fluorescent techniques, the imaging modalities able to provide interesting information about a cell, with high spatial and time resolution, have grown sufficiently to envisage quantitative analysis of chemical species inside subcellular compartments.     

Monitoring casein kinase II at subcellular level via bio-bar-code-based electrochemiluminescence biosensing method

A highly sensitive electrochemiluminescence (ECL) biosensing method was developed for monitoring casein kinase II (CK2) at subcellular level via bio-bar-code assay. A bio-bar-code probe (h-DNA/AuNPs/p-DNA) prepared by conjugating phosphorylated DNA (p-DNA) and hairpin DNA (h-DNA) onto gold nanoparticles (AuNPs) was used as a carrier for ECL signal reagent (Ru(phen)₃²⁺) while a specific peptide was used as a recognition substance. A gold ultramicroelectrode with a diameter of 400 nm was fabricated and then modified with the specific peptide via self-assembly technique to obtain peptide modified gold ultramicroelectrode. The peptide on gold ultramicroelectrode was phosphorylated in the presence of CK2 and adenosine 5′-triphosphate, and then the phosphorylated peptide was integrated with the h-DNA/AuNPs/p-DNA through a process mediated by zirconium cations (Zr⁴⁺), and finally Ru(phen)₃²⁺ was intercalated into h-DNA. A “signal on” ECL method was developed for the detection of CK2 in the range of 0.005–0.2 U/mL with a detection limit of 0.001 U/mL. Additionally, combined efficient subcellular phosphorylation in vivo with bio-bar-code-based ECL biosensing method, the ECL method was further applied to monitor CK2 at subcellular level without tedious subcellular fractionation. It was found that the concentration of CK2 by inserting the peptide modified gold ultramicroelectrode into the nucleus was higher than that into cytoplasm of HeLa cells. A distinct heterogeneity among CK2 concentrations in single cells was observed for cellular heterogeneity assessment.     

Microfabricated analytical systems for integrated cancer cytomics

Tracking and understanding cell-to-cell variability is fundamental for systems biology, cytomics and computational modelling that aids e.g. anti-cancer drug discovery. Limitations of conventional cell-based techniques, such as flow cytometry and single cell imaging, however, make the high-throughput dynamic analysis on cellular and subcellular processes tedious and exceedingly expensive. The development of microfluidic lab-on-a-chip technologies is one of the most innovative and cost-effective approaches towards integrated cytomics. Lab-on-a-chip devices promise greatly reduced costs, increased sensitivity and ultrahigh throughput by implementing parallel sample processing. The application of laminar fluid flow under low Reynolds numbers provides an attractive analytical avenue for the rapid delivery and exchange of reagents with exceptional accuracy. Under these conditions, the fluid flow has no inertia, enabling the precise dosing of drugs, both spatially and temporally. In addition, by confining the dimensions of the microfluidic structure, it is possible to facilitate the precise sequential delivery of drugs and/or functional probes into the cellular systems. As only low cell numbers and operational reagent volumes are required, high-throughput integrated cytomics on a single cell level finally appears within the reach of clinical diagnostics and drug screening routines. Lab-on-a-chip microfluidic technologies therefore provide new opportunities for the development of content-rich personalized clinical diagnostics and cost-effective drug discovery. It is largely anticipated that advances in microfluidic technologies should aid in tailoring of investigational therapies and support the current computational efforts in systems biology.     

Ultrasmall Nanopipette: Toward Continuous Monitoring of Redox Metabolism at Subcellular Level

Ionic current rectification (ICR) based nanopipettes allow accurate monitoring of cellular behavior in single living cells. Herein, we proposed a 30 nm nanopipette functionalized with G‐quadruplex DNAzyme as an efficient biomimetic recognizer for ROS generation at subcellular level via the changes of current–voltage relationship. Taking advantages of the ultra‐small tip, the nanopipette could penetrate into a single living cell repeatedly or keep measuring for a long time without compromising the cellular functions. Coupled with precision nanopositioning system, generation of ROS in mitochondria in response to cell inflammation was determined with high spatial resolution. Meanwhile, the changes of aerobic metabolism in different cell lines under drug‐induced oxidative stress were monitored continuously. We believe that the ICR‐nanopipette could be developed as a powerful approach for the study of cellular activities via electrochemical imaging in living cells.     

Discerning the Chemistry in Individual Organelles with Small‐Molecule Fluorescent Probes

Principle has it that even the most advanced super‐resolution microscope would be futile in providing biological insight into subcellular matrices without well‐designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small‐molecule fluorescent probes that not only allow cellular‐level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle‐anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology.