Integrating fluorescence computed tomography with optical sheet illumination for imaging of live single cells.

We present a new approach for three-dimensional (3D) live single-cell imaging with isotropic sub-micron spatial resolution using fluorescence computed tomography (fCT). A thin, highly inclined and laminated optical (HILO) sheet of light is used for fluorescence excitation in live single cells that are rotated around an axis perpendicular to the optical axis. During a full rotation, 400-500 two-dimensional (2D) projection images of the cell are acquired from multiple viewing perspectives by rapidly scanning the HILO light sheet along the optical axis. We report technical characteristics of the HILO approach and the results of a quantitative comparison with conventional epi fCT, demonstrating that HILO fCT offers significantly (about 17 times) reduced photobleaching and a two-fold improvement in 3D imaging contrast. We discuss potential application areas of the method for cell structure studies in live single cells with isotropic 3D spatial resolution.

Parallel fabrication of macroporous scaffolds.

Scaffolds generated from naturally occurring and synthetic polymers have been investigated in several applications because of their biocompatibility and tunable chemo-mechanical properties. Existing methods for generation of 3D polymeric scaffolds typically cannot be parallelized, suffer from low throughputs, and do not allow for quick and easy removal of the fragile structures that are formed. Current molds used in hydrogel and scaffold fabrication using solvent casting and porogen leaching are often single-use and do not facilitate 3D scaffold formation in parallel. Here, we describe a simple device and related approaches for the parallel fabrication of macroporous scaffolds. This approach was employed for the generation of macroporous and non-macroporous materials in parallel, in higher throughput and allowed for easy retrieval of these 3D scaffolds once formed. In addition, macroporous scaffolds with interconnected as well as non-interconnected pores were generated, and the versatility of this approach was employed for the generation of 3D scaffolds from diverse materials including an aminoglycoside-derived cationic hydrogel ("Amikagel"), poly(lactic-co-glycolic acid) or PLGA, and collagen. Macroporous scaffolds generated using the device were investigated for plasmid DNA binding and cell loading, indicating the use of this approach for developing materials for different applications in biotechnology. Our results demonstrate that the device-based approach is a simple technology for generating scaffolds in parallel, which can enhance the toolbox of current fabrication techniques.

Improved Performance of Loop-Mediated Isothermal Amplification Assays via Swarm Priming.

This work describes an enhancement to the loop-mediated isothermal amplification (LAMP) reaction which results in improved performance. Enhancement is achieved by adding a new set of primers to conventional LAMP reactions. These primers are termed "swarm primers" based on their relatively high concentration and their ability to create new amplicons despite the theoretical lack of single-stranded annealing sites. The primers target a region upstream of the FIP/BIP primer recognition sequences on opposite strands, substantially overlapping F1/B1 sites. Thus, despite the addition of a new primer set to an already complex assay, no significant increase in assay complexity is incurred. Swarm priming is presented for three DNA templates: Lambda phage, Synechocystis sp. PCC 6803 rbcL gene, and human HFE. The results of adding swarm primers to conventional LAMP reactions include increased amplification speed, increased indicator contrast, and increased reaction products. For at least one template, minor improvements in assay repeatability are also shown. In addition, swarm priming is shown to be effective at increasing the reaction speed for RNA amplification via RT-LAMP. Collectively, these results suggest that the addition of swarm primers will likely benefit most if not all existing LAMP assays based on state-of-the-art, six-primer reactions.

Laser-fabricated cell patterning stencil for single cell analysis.

Precise spatial positioning and isolation of mammalian cells is a critical component of many single cell experimental methods and biological engineering applications. Although a variety of cell patterning methods have been demonstrated, many of these methods subject cells to high stress environments, discriminate against certain phenotypes, or are a challenge to implement. Here, we demonstrate a rapid, simple, indiscriminate, and minimally perturbing cell patterning method using a laser fabricated polymer stencil. The stencil fabrication process requires no stencil-substrate alignment, and is readily adaptable to various substrate geometries and experiments.

The oxindole Syk inhibitor OXSI-2 blocks nigericin-induced inflammasome signaling and pyroptosis independent of potassium efflux.

The inflammasome is a caspase-1-activating complex that is implicated in a growing number of acute and chronic pathologies. Interest has increased in identifying small molecular inhibitors of inflammasome signaling because of its role in clinically relevant diseases. It was recently reported that the protein tyrosine kinase, Syk, regulates pathogen-induced inflammasome signaling by phosphorylating a molecular switch on the adapter protein ASC. However, several aspects of the role of Syk in inflammasome signaling and the effects of its inhibition remain unclear. The aim of the present study is to explore in detail the effects of the oxindole Syk inhibitor OXSI-2 on various aspects of nigericin-induced inflammasome signaling. Our results indicate that OXSI-2 inhibits inflammasome assembly, caspase-1 activation, IL-1ß processing and release, mitochondrial ROS generation, and pyroptotic cell death. Using a novel live cell potassium sensor we show that Syk inhibition with OXSI-2 has no effect on potassium efflux kinetics and that blockade of potassium efflux with extracellular potassium alters Syk phosphorylation. The effects of OXSI-2 identified in this study provide context for the role of Syk in inflammasome signaling and demonstrate its importance in oxidative signaling upstream of inflammasome activation and downstream of ion flux.

1,8-Naphthalimide Derivative Dyes with Large Stokes Shifts for Targeting Live-Cell Mitochondria.

An ideal fluorescent dye for staining cell organelles should have multiple properties including specificity, stability, biocompatibility, and a large Stokes shift. Tunable photophysical properties enable 1,8-naphthalimide to serve as an excellent fluorophore in biomedical applications. Many naphthalimide derivatives have been developed into drugs, sensors, and other dyes. In this study, a series of 1,8-naphthalimide derivatives targeting live cell mitochondria were synthesized. Among these probes, Mt-4 was characterized as the best one, with highly specific mitochondrial localization, low cytotoxicity, and a large Stokes shift. More importantly, Mt-4 stood out as a potential mitochondrial dye for living-cell experiments involving induced mitochondrial stress arising from the treatments because Mt-4 shows enhanced fluorescence in mitochondrial stress situations.

Premethylation of foreign DNA improves integrative transformation efficiency in Synechocystis sp. strain PCC 6803.

Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5' untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803.

Measuring gene expression in single bacterial cells: recent advances in methods and micro-devices.

Populations of bacterial cells that grow under the same conditions and/or environments are often considered to be uniform and thus can be described by ensemble average values of their physiologic, phenotypic, genotypic or other parameters. However, recent evidence suggests that cell-to-cell differences at the gene expression level could be an order of magnitude greater than previously thought even for isogenic bacterial populations. Such gene expression or transcriptional-level heterogeneity determines not only the fate of individual bacterial cells in a population but could also affect the ultimate fate of the population itself. Although techniques for single-cell gene expression measurement in eukaryotic cells have been successfully implemented for a decade or so, they have only recently become available for single bacterial cells. This is due to the difficulty of efficient lysis of most bacterial cells, as well as short half-life time (low stability) of bacterial mRNA. In this article, we review the recent progress and challenges associated with analyzing gene expression levels in single bacterial cells using various semi-quantitative and quantitative methods. In addition, a review of the recent progress in applying microfluidic devices to isolate single bacterial cells for gene expression analysis is also included.

A highly selective mitochondria-targeting fluorescent K(+) sensor.

Regulation of intracellular potassium (K(+) ) concentration plays a key role in metabolic processes. So far, only a few intracellular K(+) sensors have been developed. The highly selective fluorescent K(+) sensor KS6 for monitoring K(+) ion dynamics in mitochondria was produced by coupling triphenylphosphonium, borondipyrromethene (BODIPY), and triazacryptand (TAC). KS6 shows a good response to K(+) in the range 30-500 mM, a large dynamic range (Fmax /F0 ≈130), high brightness (ϕf =14.4 % at 150 mM of K(+) ), and insensitivity to both pH in the range 5.5-9.0 and other metal ions under physiological conditions. Colocalization tests of KS6 with MitoTracker Green confirmed its predominant localization in the mitochondria of HeLa and U87MG cells. K(+) efflux/influx in the mitochondria was observed upon stimulation with ionophores, nigericin, or ionomycin. KS6 is thus a highly selective semiquantitative K(+) sensor suitable for the study of mitochondrial potassium flux in live cells.

A convenient, optimized pipeline for isolation, fluorescence microscopy and molecular analysis of live single cells.

BACKGROUND: Heterogeneity within cell populations is relevant to the onset and progression of disease, as well as development and maintenance of homeostasis. Analysis and understanding of the roles of heterogeneity in biological systems require methods and technologies that are capable of single cell resolution. Single cell gene expression analysis by RT-qPCR is an established technique for identifying transcriptomic heterogeneity in cellular populations, but it generally requires specialized equipment or tedious manipulations for cell isolation. RESULTS: We describe the optimization of a simple, inexpensive and rapid pipeline which includes isolation and culture of live single cells as well as fluorescence microscopy and gene expression analysis of the same single cells by RT-qPCR. We characterize the efficiency of single cell isolation and demonstrate our method by identifying single GFP-expressing cells from a mixed population of GFP-positive and negative cells by correlating fluorescence microscopy and RT-qPCR. CONCLUSIONS: Single cell gene expression analysis by RT-qPCR is a convenient means for investigating cellular heterogeneity, but is most useful when correlating observations with additional measurements. We demonstrate a convenient and simple pipeline for multiplexing single cell RT-qPCR with fluorescence microscopy which is adaptable to other molecular analyses.

Engineering cyanobacteria for photosynthetic production of 3-hydroxybutyrate directly from CO2.

(S)- and (R)-3-hydroxybutyrate (3HB) are precursors to synthesize the biodegradable plastics polyhydroxyalkanoates (PHAs) and many fine chemicals. To date, however, their production has been restricted to petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economical feasibility. With the ability to fix CO2 photosynthetically, cyanobacteria have attracted increasing interest as a biosynthesis platform to produce fuels and chemicals from alternative renewable resources. To this end, synthesis metabolic pathways have been constructed and optimized in cyanobacterium Synechocystis sp. PCC 6803 to photosynthetically produce (S)- and (R)-3HB directly from CO2. Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing pathway by deleting slr1829 and slr1830 (encoding PHB polymerase) from the Synechocystis genome further promoted the 3HB production. Up to 533.4mg/L 3HB has been produced after photosynthetic cultivation of the engineered cyanobacterium Synechocystis TABd for 21 days. Further analysis indicated that the phosphate consumption during the photoautrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. For the first time, the study has demonstrated the feasibility of photosynthetic production of (S)- and (R)-3HB directly from sunlight and CO2.

Monitoring the single-cell stress response of the diatom Thalassiosira pseudonana by quantitative real-time reverse transcription-PCR.

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.

A fluorescent colorimetric pH sensor and the influences of matrices on sensing performances.

A fluorescent colorimetric pH sensor was developed by a polymerization of a monomeric fluorescein based green emitter (SM1) with a monomeric 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran derived red emitter (SM2) in poly(2-hydroxyethyl methacrylate)-co-polyacrylamide (PHEMA-co-PAM) matrices. Polymerized SM1 (PSM1) in the polymer matrices showed bright emissions at basic conditions and weak emissions at acidic conditions. Polymerized SM2 (PSM2) in the polymer matrices exhibited a vastly different response when compared to PSM1. The emissions of PSM2 are stronger under acidic conditions than those under basic conditions. When SM1 and SM2 were polymerized in the same polymer matrix, a dual emission sensor acting as a ratiometric pH sensor (PSM1,2) was successfully developed. Because the PSM1 and PSM2 exhibited different pH responses and separated emission windows, the changes in the emission colors were clearly observed in their dual color sensor of PSM1,2, which changed emission colors dramatically from green at pH 7 to red at pH 4, which was detected visually and/or by using a color camera under an excitation of 488 nm. In addition to the development of the dual color ratiometric pH sensor, we also studied the effects of different matrix compositions, crosslinkers, and charges on the reporting capabilities of the sensors (sensitivity and pKa).

Integrated metagenomic and metatranscriptomic analyses of microbial communities in the meso- and bathypelagic realm of north pacific ocean.

Although emerging evidence indicates that deep-sea water contains an untapped reservoir of high metabolic and genetic diversity, this realm has not been studied well compared with surface sea water. The study provided the first integrated meta-genomic and -transcriptomic analysis of the microbial communities in deep-sea water of North Pacific Ocean. DNA/RNA amplifications and simultaneous metagenomic and metatranscriptomic analyses were employed to discover information concerning deep-sea microbial communities from four different deep-sea sites ranging from the mesopelagic to pelagic ocean. Within the prokaryotic community, bacteria is absolutely dominant (~90%) over archaea in both metagenomic and metatranscriptomic data pools. The emergence of archaeal phyla Crenarchaeota, Euryarchaeota, Thaumarchaeota, bacterial phyla Actinobacteria, Firmicutes, sub-phyla Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria, and the decrease of bacterial phyla Bacteroidetes and Alphaproteobacteria are the main composition changes of prokaryotic communities in the deep-sea water, when compared with the reference Global Ocean Sampling Expedition (GOS) surface water. Photosynthetic Cyanobacteria exist in all four metagenomic libraries and two metatranscriptomic libraries. In Eukaryota community, decreased abundance of fungi and algae in deep sea was observed. RNA/DNA ratio was employed as an index to show metabolic activity strength of microbes in deep sea. Functional analysis indicated that deep-sea microbes are leading a defensive lifestyle.

Micro-patterning and characterization of PHEMA-co-PAM-based optical chemical sensors for lab-on-a-chip applications.

We report a novel method for wafer level, high throughput optical chemical sensor patterning, with precise control of the sensor volume and capability of producing arbitrary microscale patterns. Monomeric oxygen (O(2)) and pH optical probes were polymerized with 2-hydroxyethyl methacrylate (HEMA) and acrylamide (AM) to form spin-coatable and further crosslinkable polymers. A micro-patterning method based on micro-fabrication techniques (photolithography, wet chemical process and reactive ion etch) was developed to miniaturize the sensor film onto glass substrates in arbitrary sizes and shapes. The sensitivity of fabricated micro-patterns was characterized under various oxygen concentrations and pH values. The process for spatially integration of two sensors (Oxygen and pH) on the same substrate surface was also developed, and preliminary fabrication and characterization results were presented. To the best of our knowledge, it is the first time that poly (2-hydroxylethyl methacrylate)-co-poly (acrylamide) (PHEMA-co-PAM)-based sensors had been patterned and integrated at the wafer level with micron scale precision control using microfabrication techniques. The developed methods can provide a feasible way to miniaturize and integrate the optical chemical sensor system and can be applied to any lab-on-a-chip system, especially the biological micro-systems requiring optical sensing of single or multiple analytes.

High Throughput Micropatterning of Optical Oxygen Sensor for Single Cell Analysis.

In this paper, we present our results from process development and characterization of optical oxygen sensors that are patterned by traditional UV lithography. An oxygen sensitive luminescent probe, platinum octaethylporphyrin (PtOEP), was encapsulated in commercially purchased photoresist (AZ5214) to form uniform thin sensor films on fused silica substrates. Plasticizer ethoxylated trimethylolpropane triacrylate (SR454) was added to the dye-photoresist sensor mixtures to improve the oxygen sensitivity. The optimum sensor mixture composition that can be patterned with maximum sensitivity was identified. The microfabrication process conditions, cell adherence and oxygen sensitivity results from patterned structures were characterized in detail. Down to 3 µm features have been fabricated on fused silica substrates using the developed techniques. The result implies the developed methods can provide a feasible way to miniaturize the optical sensor system for single cell analysis with precise control of sensor volume and response.

Stability of DNA origami nanoarrays in cell lysate.

Scaffolded DNA origami, a method to create self-assembled nanostructures with spatially addressable features, has recently been used to develop water-soluble molecular chips for label-free RNA detection, platforms for deterministic protein positioning, and single molecule reaction observatories. These applications highlight the possibility of exploiting the unique properties and biocompatibility of DNA nanostructures in live, cellular systems. Herein, we assembled several DNA origami nanostructures of differing shape, size and probes, and investigated their interaction with lysate obtained from various normal and cancerous cell lines. We separated and analyzed the origami-lysate mixtures using agarose gel electrophoresis and recovered the DNA structures for functional assay and subsequent microscopic examination. Our results demonstrate that DNA origami nanostructures are stable in cell lysate and can be easily separated from lysate mixtures, in contrast to natural, single- and double-stranded DNA. Atomic force microscope (AFM) and transmission electron microscope (TEM) images show that the DNA origami structures are fully intact after separation from cell lysates and hybridize to their targets, verifying the superior structural integrity and functionality of self-assembled DNA origami nanostructures relative to conventional oligonucleotides. The stability and functionality of DNA origami structures in cell lysate validate their use for biological applications, for example, as programmable molecular rafts or disease detection platforms.

A new highly selective fluorescent K+ sensor.

We describe the synthesis, properties, and application of a new fluorescent potassium chemosensor, KS2, for K(+) sensing and imaging in live cells. By virtue of a strong electron-withdrawing group, 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF), with a triazacryptand ligand, the new sensor can respond to K(+) up to 1.6 M. This is the first highly selective intracellular sensor suitable for sensing K(+) over a broad and high concentration range. Confocal fluorescence microscopy has established the utility of KS2 for live-cell K(+) detection. The application of KS2 combined with other sensors will be of great benefit for investigating cellular metabolism, detecting and diagnosing diseases including cancer, and monitoring responses to therapy.

Quantitative characterization of preneoplastic progression using single-cell computed tomography and three-dimensional karyometry.

The development of morphological biosignatures to precisely characterize preneoplastic progression necessitates high-resolution three-dimensional (3D) cell imagery and robust image processing algorithms. We report on the quantitative characterization of nuclear structure alterations associated with preneoplastic progression in human esophageal epithelial cells using single-cell optical tomography and fully automated 3D karyometry. We stained cultured cells with hematoxylin and generated 3D images of individual cells by mathematically reconstructing 500 projection images acquired using optical absorption tomographic imaging. For 3D karyometry, we developed novel, fully automated algorithms to robustly segment the cellular, nuclear, and subnuclear components in the acquired cell images, and computed 41 quantitative morphological descriptors from these segmented volumes. In addition, we developed algorithms to quantify the spatial distribution and texture of the nuclear DNA. We applied our methods to normal, metaplastic, and dysplastic human esophageal epithelial cell lines, analyzing 100 cells per line. The 3D karyometric descriptors elucidated quantitative differences in morphology and enabled robust discrimination between cell lines on the basis of extracted morphological features. The morphometric hallmarks of cancer progression such as increased nuclear size, elevated nuclear content, and anomalous chromatin texture and distribution correlated with this preneoplastic progression model, pointing to the clinical use of our method for early cancer detection.

Diaphragm pico-liter pump for single-cell manipulation.

A pico-liter pump is developed and integrated into a robotic manipulation system that automatically selects and transfers individual living cells of interest to analysis locations. The pump is a displacement type pump comprising one cylindrical chamber connected to a capillary micropipette. The top of the chamber is a thin diaphragm which, when deflected, causes the volume of the fluid-filled cylindrical chamber to change thereby causing fluid in the chamber to flow in and out of the micropipette. This enables aspirating and dispensing individual living cells. The diaphragm is deflected by a piezoelectric actuator that pushes against its center. The pump aspirates and dispenses volumes of fluid between 500 pL and 250 nL at flow rates up to 250 nL/s. The piezo-driven diaphragm arrangement provides exquisite control of the flow rate in and out of the capillary orifice. This feature, in turn, allows reduced perturbation of live cells by controlling and minimizing the applied shear stresses.