Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

High-throughput analysis and protein engineering using microcapillary arrays.

We describe a multipurpose technology platform, termed µSCALE (microcapillary single-cell analysis and laser extraction), that enables massively parallel, quantitative biochemical and biophysical measurements on millions of protein variants expressed from yeast or bacteria. µSCALE spatially segregates single cells within a microcapillary array, enabling repeated imaging, cell growth and protein expression. We performed high-throughput analysis of cells and their protein products using a range of fluorescent assays, including binding-affinity measurements and dynamic enzymatic assays. A precise laser-based extraction method allows rapid recovery of live clones and their genetic material from microcapillaries for further study. With µSCALE, we discovered a new antibody against a clinical cancer target, evolved a fluorescent protein biosensor and engineered an enzyme to reduce its sensitivity to its inhibitor. These protein analysis and engineering applications each have unique assay requirements and different host organisms, highlighting the flexibility and technical capabilities of the µSCALE platform.

Point-of-Care Sexually Transmitted Infection Diagnostics: Proceedings of the STAR Sexually Transmitted Infection-Clinical Trial Group Programmatic Meeting.

The goal of the point-of-care (POC) sexually transmitted infection (STI) Diagnostics meeting was to review the state-of-the-art research and develop recommendations for the use of POC STI diagnostics. Experts from academia, government, nonprofit, and industry discussed POC diagnostics for STIs such as Chlamydia trachomatis, human papillomavirus, Neisseria gonorrhoeae, Trichomonas vaginalis, and Treponema pallidum. Key objectives included a review of current and emerging technologies, clinical and public health benefits, POC STI diagnostics in developing countries, regulatory considerations, and future areas of development. Key points of the meeting are as follows: (i) although some rapid point-of-care tests are affordable, sensitive, specific, easy to perform, and deliverable to those who need them for select sexually transmitted infections, implementation barriers exist at the device, patient, provider, and health system levels; (ii) further investment in research and development of point-of-care tests for sexually transmitted infections is needed, and new technologies can be used to improve diagnostic testing, test uptake, and treatment; (iii) efficient deployment of self-testing in supervised (ie, pharmacies, clinics, and so on) and/or unsupervised (ie, home, offices, and so on) settings could facilitate more screening and diagnosis that will reduce the burden of sexually transmitted infections; (iv) development of novel diagnostic technologies has outpaced the generation of guidance tools and documents issued by regulatory agencies; and (v) questions regarding quality management are emerging including the mechanism by which poor-performing diagnostics are removed from the market and quality assurance of self-testing is ensured.

Hybrid integrated PDMS microfluidics with a silica capillary.

To harness the properties of both PDMS and silica, we have demonstrated hybrid integrated PDMS microfluidic systems with fused silica capillaries. The hybrid integrated PDMS microfluidics and silica capillary (iPSC) modules exhibit a novel architecture and method for leakage free CE sample injection merely requiring a single high voltage source and one pair of electrodes. The use of the iPSC device is based on a modular approach which allows the capillary to be reused extensively whilst replacing the attached fluidic module for different experiments. Integrating fused silica capillaries with PDMS microfluidic modules allows the direct application of a wide variety of well established conventional CE protocols for separations of complex analytes. Furthermore it bears the potential for facile coupling to standard electro-spray ionization mass spectrometry (ESI-MS), letting users focus on the sample analysis rather than the development of new separation protocols. The fabrication of the iPSC module consists of a simple and quick three-step method that submerges a fused silica capillary in PDMS prepolymer. After cross linking the prepolymer and punching the inlets, the iPSC module layer can be mounted onto a microfluidic device for CE separation.

Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics.

We demonstrate the first integrated microfluidic tmRNA purification and nucleic acid sequence-based amplification (NASBA) device incorporating real-time detection. The real-time amplification and detection step produces pathogen-specific response in < 3 min from the chip-purified RNA from 100 lysed bacteria. On-chip RNA purification uses a new silica bead immobilization method. On-chip amplification uses custom-designed high-selectivity primers and real-time detection uses molecular beacon fluorescent probe technology; both are integrated on-chip with NASBA. Present in all bacteria, tmRNA (10Sa RNA) includes organism-specific identification sequences, exhibits unusually high stability relative to mRNA, and has high copy number per organism; the latter two factors improve the limit of detection, accelerate time-to-positive response, and suit this approach ideally to the detection of small numbers of bacteria. Device efficacy was demonstrated by integrated on-chip purification, amplification, and real-time detection of 100 E. coli bacteria in 100 microL of crude lysate in under 30 min for the entire process.

Engineering High Affinity Protein-Protein Interactions Using a High-Throughput Microcapillary Array Platform.

Affinity maturation of protein-protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed µSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity. Here, we demonstrate the first use of the µSCALE platform for affinity maturation of a protein-protein binding interaction. In this proof-of-concept study, we engineered an extracellular domain of the Axl receptor tyrosine kinase to bind tighter to its ligand Gas6. Within 2 weeks, two iterative rounds of library generation and screening resulted in engineered Axl variants with a 50-fold decrease in kinetic dissociation rate, highlighting the use of µSCALE as a new tool for directed evolution.

Profiling Melanoma Heterogeneity Using Microwell RNA Cytometry.

Unraveling heterogeneity of melanoma to discover new subpopulations of cells within the tumor has been fundamental to many advances in cancer biology, including identification of tumor initiating subsets and cells resisting immune-therapeutic approaches (Boiko et al., Nature 466:133-137, 2010; Civenni et al., Cancer Res 71:3098-3109, 2011; Schatton et al., Nature 451:345-349, 2008; Landsberg et al., Nature 490:412-416, 2012; Fang et al., Cancer Res 65:9328-9337, 2005). Traditionally, these discoveries were made possible due to the existence of well-characterized antibodies that enabled identification of cells homogeneous for the expression of specific cell surface antigen. However, further unwinding of heterogeneous cell populations into homogenous subsets in order to more precisely define their functional profile is limited by the availability of highly specific antibodies. Here we describe a technique capable of identifying homogeneous cell populations in heterogeneous sample based on the transcriptome profile. This approach enables semiquantitative measurement of gene expressions in hundreds to thousands of single cells in one step, paving the way to identify homogenous subpopulations of melanoma cells based on gene transcripts, independent of the availability of antibodies.

Dual transcript and protein quantification in a massive single cell array.

Recently, single-cell molecular analysis has been leveraged to achieve unprecedented levels of biological investigation. However, a lack of simple, high-throughput single-cell methods has hindered in-depth population-wide studies with single-cell resolution. We report a microwell-based cytometric method for simultaneous measurements of gene and protein expression dynamics in thousands of single cells. We quantified the regulatory effects of transcriptional and translational inhibitors on cMET mRNA and cMET protein in cell populations. We studied the dynamic responses of individual cells to drug treatments, by measuring cMET overexpression levels in individual non-small cell lung cancer (NSCLC) cells with induced drug resistance. Across NSCLC cell lines with a given protein expression, distinct patterns of transcript-protein correlation emerged. We believe this platform is applicable for interrogating the dynamics of gene expression, protein expression, and translational kinetics at the single-cell level - a paradigm shift in life science and medicine toward discovering vital cell regulatory mechanisms.

Real-time monitoring of cell migration, phagocytosis and cell surface receptor dynamics using a novel, live-cell opto-microfluidic technique.

We report an opto-microfluidic method for continuous and non-interfering monitoring of cell movement and dynamic molecular processes in living cells enabled by the microfluidic "Lab-in-a-Trench" (LiaT) platform. To demonstrate real-time monitoring of heterogeneous cell-cell interactions, cell tracking and agent-induced cell activation dynamics, we observe phagocytosis of Escherichia coli by murine macrophages, migration of active macrophages and LPS-induced CD86 expression in macrophages. The visualization of phagocytosis is facilitated through the loading of green fluorescent protein (GFP) expressing E. coli to the array of cell capture modules before the introduction of macrophages. Simple migration tracking of active macrophages is enabled by a spatio-temporal control of the environment conditions within the LiaT platform. Furthermore, we report an interference-free monitoring of non-modified, endogenous changes in protein expression on the surface of living cells using traditional, antibody immuno-reagents. Throughout the experiment, murine macrophages were captured in the LiaT device and exposed to sub-background levels of fluorescently labeled anti-CD86 antibody. Upon lipopolysaccharide (LPS) stimulation, CD86 changes were visualized in real-time by time-lapse microscopy. This novel opto-microfluidic effect is controlled by the equilibrium of convective-diffusive replenishment of fluorescently labeled antibodies and antibody affinity. Overall, our non-interfering analysis method allows the studying of active cellular processes and endogenous protein dynamics in live cells in a simple and cost-efficient manner.

Dynamic and social behaviors of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) can self-renew or differentiate to diverse cell types, thus providing a platform for basic and clinical applications. However, pluripotent stem cell populations are heterogeneous and functional properties at the single cell level are poorly documented leading to inefficiencies in differentiation and concerns regarding reproducibility and safety. Here, we use non-invasive time-lapse imaging to continuously examine hPSC maintenance and differentiation and to predict cell viability and fate. We document dynamic behaviors and social interactions that prospectively distinguish hPSC survival, self-renewal, and differentiation. Results highlight the molecular role of E-cadherin not only for cell-cell contact but also for clonal propagation of hPSCs. Results indicate that use of continuous time-lapse imaging can distinguish cellular heterogeneity with respect to pluripotency as well as a subset of karyotypic abnormalities whose dynamic properties were monitored.

Identification of multipotent progenitors that emerge prior to hematopoietic stem cells in embryonic development.

Hematopoiesis in the embryo proceeds in a series of waves, with primitive erythroid-biased waves succeeded by definitive waves, within which the properties of hematopoietic stem cells (multilineage potential, self-renewal, and engraftability) gradually arise. Whereas self-renewal and engraftability have previously been examined in the embryo, multipotency has not been thoroughly addressed, especially at the single-cell level or within well-defined populations. To identify when and where clonal multilineage potential arises during embryogenesis, we developed a single-cell multipotency assay. We find that, during the initiation of definitive hematopoiesis in the embryo, a defined population of multipotent, engraftable progenitors emerges that is much more abundant within the yolk sac (YS) than the aorta-gonad-mesonephros (AGM) or fetal liver. These experiments indicate that multipotent cells appear in concert within both the YS and AGM and strongly implicate YS-derived progenitors as contributors to definitive hematopoiesis.

Discriminating cellular heterogeneity using microwell-based RNA cytometry.

Discriminating cellular heterogeneity is important for understanding cellular physiology. However, it is limited by the technical difficulties of single-cell measurements. Here we develop a two-stage system to determine cellular heterogeneity. In the first stage, we perform multiplex single-cell RNA cytometry in a microwell array containing over 60,000 reaction chambers. In the second stage, we use the RNA cytometry data to determine cellular heterogeneity by providing a heterogeneity likelihood score (HLS). Moreover, we use Monte-Carlo simulation and RNA cytometry data to calculate the minimum number of cells required for detecting heterogeneity. We apply this system to characterize the RNA distributions of ageing-related genes in a highly purified mouse haematopoietic stem cell population. We identify genes that reveal novel heterogeneity of these cells. We also show that changes in expression of genes such as Birc6 during ageing can be attributed to the shift of relative portions of cells in the high-expressing subgroup versus low-expressing subgroup.

Identification of a colonial chordate histocompatibility gene.

Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from nonself. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self-nonself and determines "graft" outcomes in this organism. This gene is significantly up-regulated in colonies poised to undergo fusion and/or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition.

Digital LAMP in a sample self-digitization (SD) chip.

This paper describes the realization of digital loop-mediated DNA amplification (dLAMP) in a sample self-digitization (SD) chip. Digital DNA amplification has become an attractive technique to quantify absolute concentrations of DNA in a sample. While digital polymerase chain reaction is still the most widespread implementation, its use in resource-limited settings is impeded by the need for thermal cycling and robust temperature control. In such situations, isothermal protocols that can amplify DNA or RNA without thermal cycling are of great interest. Here, we accomplished the successful amplification of single DNA molecules in a stationary droplet array using isothermal digital loop-mediated DNA amplification. Unlike most (if not all) existing methods for sample discretization, our design allows for automated, loss-less digitization of sample volumes on-chip. We demonstrated accurate quantification of relative and absolute DNA concentrations with sample volumes of less than 2 µl. We assessed the homogeneity of droplet size during sample self-digitization in our device, and verified that the size variation was small enough such that straightforward counting of LAMP-active droplets sufficed for data analysis. We anticipate that the simplicity and robustness of our SD chip make it attractive as an inexpensive and easy-to-operate device for DNA amplification, for example in point-of-care settings.

Reactive deposition of nano-films in deep polymeric microcavities.

We report the controlled diffusion of gas-phase high-reactivity chemical species into long polymeric microcavities to form glass-like, low-permeability barrier films on the interior surfaces of the microcavities. Reactive species created from fragmentation of O(2) and hexamethyldisiloxane (HMDSO) in a radio-frequency (RF) plasma environment are allowed to diffuse into the microcavities of polydimethylsiloxane (PDMS), where surface reactions lead to the formation of an effective, glass-like thin-film barrier. Reactive species including silicon radicals and elemental oxygen maintain their reactivity for sufficient times (up to 7000 s) and survive the random diffusional walk through the microcavities to form glass barriers as much as 65 mm from the cavity entrance. The barrier thickness and the growth length can be controlled by the reaction time and chamber operating pressure. Increasing the cross sectional area of the cavity inlet and/or decreasing the mean free path was found to increase the thickness of the barrier film. Optical emission spectroscopic analysis was used to characterize the reactive fragments formed from HMDSO, and energy-dispersive X-ray analysis revealed that the barrier composition is consistent with oxides of silicon (SiO(x)). Formed inside PDMS microcavities, the glass barrier blocks the penetration or absorption of small molecules such as rhodamine B (RhB) and biotin, and also resists permeation of organic solvents such as toluene, preventing the PDMS microfluidic structures from swelling and deforming. Moreover, formation of glass-like thin films in PDMS microcavities enhances the stability of electroosmotic flow (EOF) relative to uncoated PDMS devices, in which EOF instabilities are significant; this enables separation by electrophoresis with reproducibility (relative standard deviation 3%, n = 5) and baseline peak resolution (R:1.3) comparable to that obtained in conventional fused-silica capillaries.

Systematic characterization of degas-driven flow for poly(dimethylsiloxane) microfluidic devices.

Degas-driven flow is a novel phenomenon used to propel fluids in poly(dimethylsiloxane) (PDMS)-based microfluidic devices without requiring any external power. This method takes advantage of the inherently high porosity and air solubility of PDMS by removing air molecules from the bulk PDMS before initiating the flow. The dynamics of degas-driven flow are dependent on the channel and device geometries and are highly sensitive to temporal parameters. These dependencies have not been fully characterized, hindering broad use of degas-driven flow as a microfluidic pumping mechanism. Here, we characterize, for the first time, the effect of various parameters on the dynamics of degas-driven flow, including channel geometry, PDMS thickness, PDMS exposure area, vacuum degassing time, and idle time at atmospheric pressure before loading. We investigate the effect of these parameters on flow velocity as well as channel fill time for the degas-driven flow process. Using our devices, we achieved reproducible flow with a standard deviation of less than 8% for flow velocity, as well as maximum flow rates of up to 3 nL∕s and mean flow rates of approximately 1-1.5 nL∕s. Parameters such as channel surface area and PDMS chip exposure area were found to have negligible impact on degas-driven flow dynamics, whereas channel cross-sectional area, degas time, PDMS thickness, and idle time were found to have a larger impact. In addition, we develop a physical model that can predict mean flow velocities within 6% of experimental values and can be used as a tool for future design of PDMS-based microfluidic devices that utilize degas-driven flow.

Integrated microfluidic array plate (iMAP) for cellular and molecular analysis.

Just as the Petri dish has been invaluable to the evolution of biomedical science in the last 100 years, microfluidic cell assay platforms have the potential to change significantly the way modern biology and clinical science are performed. However, an evolutionary process of creating an efficient microfluidic array for many different bioassays is necessary. Specifically for a complete view of a cell response it is essential to incorporate cytotoxic, protein and gene analysis on a single system. Here we present a novel cellular and molecular analysis platform, which allows access to gene expression, protein immunoassay, and cytotoxicity information in parallel. It is realized by an integrated microfluidic array plate (iMAP). The iMAP enables sample processing of cells, perfusion based cell culture, effective perturbation of biologic molecules or drugs, and simultaneous, real-time optical analysis for different bioassays. The key features of the iMAP design are the interface of on-board gravity driven flow, the open access input fluid exchange and the highly efficient sedimentation based cell capture mechanism (∼100% capture rates). The operation of the device is straightforward (tube and pump free) and capable of handling dilute samples (5-cells per experiment), low reagent volumes (50 nL per reaction), and performing single cell protein and gene expression measurements. We believe that the unique low cell number and triple analysis capabilities of the iMAP platform can enable novel dynamic studies of scarce cells.

Stand-alone self-powered integrated microfluidic blood analysis system (SIMBAS).

We present a self-powered integrated microfluidic blood analysis system (SIMBAS) that does not require any external connections, tethers, or tubing to deliver and analyze a raw whole-blood sample. SIMBAS only requires the user to place a 5 µL droplet of whole-blood at the inlet port of the device, whereupon the stand-alone SIMBAS performs on-chip removal of red and white cells, without external valving or pumping mechanisms, followed by analyte detection in platelet-containing plasma. Five complete biotin-streptavidin sample-to-answer assays are performed in 10 min; the limit of detection is 1.5 pM. Red and white blood cells are removed by trapping them in an integral trench structure. Simulations and experimental data show 99.9% to 100% blood cell retention in the passive structure. Powered by pre-evacuation of its PDMS substrate, SIMBAS' guiding design principle is the integration of the minimal number of components without sacrificing effectiveness in performing rapid complete bioassays, a critical step towards point-of-care molecular diagnostics.