cRGD functionalized 2,1,3-benzothiadiazole (BTD)-containing two-photon absorbing red-emitter-conjugated amphiphilic poly(ethylene glycol)-block-poly(-caprolactone) for targeted bioimaging.

A two-photon absorbing (2PA) red emitter group was chemically conjugated onto amphiphilic poly(ethylene glycol)--poly(-caprolactone) (PEG--PCL) copolymers, and further grafted with cyclo (Arg-Gly-Asp) (cRGD) peptide to form micelle 1. Micelle 1 with cRGD targeting groups were used for targeted bioimaging. For comparison, micelle 2 without the cRGD targeting groups were also prepared and investigated.

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.

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.

Simultaneous Multiparameter Cellular Energy Metabolism Profiling of Small Populations of Cells.

Functional and genomic heterogeneity of individual cells are central players in a broad spectrum of normal and disease states. Our knowledge about the role of cellular heterogeneity in tissue and organism function remains limited due to analytical challenges one encounters when performing single cell studies in the context of cell-cell interactions. Information based on bulk samples represents ensemble averages over populations of cells, while data generated from isolated single cells do not account for intercellular interactions.

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.

Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells.

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image.

Multifunctional PHPMA-Derived Polymer for Ratiometric pH Sensing, Fluorescence Imaging, and Magnetic Resonance Imaging.

In this paper, we report synthesis and characterization of a novel multimodality (MRI/fluorescence) probe for pH sensing and imaging. A multifunctional polymer was derived from poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) and integrated with a naphthalimide-based-ratiometric fluorescence probe and a gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex (Gd-DOTA complex). The polymer was characterized using UV-vis absorption spectrophotometry, fluorescence spectrofluorophotometry, magnetic resonance imaging (MRI), and confocal microscopy for optical and MRI-based pH sensing and cellular imaging.

A versatile method for dynamically controlled patterning of small populations of epithelial cells on substrates via non-contact piezoelectric inkjet printing.

Intercellular interactions play a central role at the tissue and whole organism level modulating key cellular functions in normal and disease states. Studies of cell-cell communications are challenging due to ensemble averaging effects brought about by intrinsic heterogeneity in cellular function which requires such studies to be conducted with small populations of cells. Most of the current methods for producing and studying such small cell populations are complex to implement and require skilled personnel limiting their widespread utility in biomedical research labs.

A platform for high-throughput bioenergy production phenotype characterization in single cells.

Driven by an increasing number of studies demonstrating its relevance to a broad variety of disease states, the bioenergy production phenotype has been widely characterized at the bulk sample level. Its cell-to-cell variability, a key player associated with cancer cell survival and recurrence, however, remains poorly understood due to ensemble averaging of the current approaches. We present a technology platform for performing oxygen consumption and extracellular acidification measurements of several hundreds to 1,000 individual cells per assay, while offering simultaneous analysis of cellular communication effects on the energy production phenotype.

Platform for combined analysis of functional and biomolecular phenotypes of the same cell.

Functional and molecular cell-to-cell variability is pivotal at the cellular, tissue and whole-organism levels. Yet, the ultimate goal of directly correlating the function of the individual cell with its biomolecular profile remains elusive. We present a platform for integrated analysis of functional and transcriptional phenotypes in the same single cells.

Identification of stable reference genes for lipopolysaccharide-stimulated macrophage gene expression studies.

Gene expression studies which utilize lipopolysaccharide (LPS)-stimulated macrophages to model immune signaling are widely used for elucidating the mechanisms of inflammation-related disease. When expression levels of target genes are quantified using Real-Time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), they are analyzed in comparison to reference genes, which should have stable expression. Judicious selection of reference genes is, therefore, critical to interpretation of qRT-PCR results.

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.

Transcriptional regulation by normal epithelium of premalignant to malignant progression in Barrett's esophagus.

In carcinogenesis, intercellular interactions within and between cell types are critical but remain poorly understood. We present a study on intercellular interactions between normal and premalignant epithelial cells and their functional relevance in the context of premalignant to malignant progression in Barrett's esophagus. Using whole transcriptome profiling we found that in the presence of normal epithelial cells, dysplastic cells but not normal cells, exhibit marked down-regulation of a number of key signaling pathways, including the transforming growth factor beta (TGFβ) and epithelial growth factor (EGF).

Ratiometric fluorescent pH-sensitive polymers for high-throughput monitoring of extracellular pH.

Extracellular pH has a strong effect on cell metabolism and growth. Precisely detecting extracellular pH with high throughput is critical for cell metabolism research and fermentation applications. In this research, a series of ratiometric fluorescent pH sensitive polymers are developed and the ps-pH-neutral is characterized as the best one for exculsive detection of extracellular pH.

A simple non-perturbing cell migration assay insensitive to proliferation effects.

Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region.

Vorinostat differentially alters 3D nuclear structure of cancer and non-cancerous esophageal cells.

The histone deacetylase (HDAC) inhibitor vorinostat has received significant attention in recent years as an 'epigenetic' drug used to treat solid tumors. However, its mechanisms of action are not entirely understood, particularly with regard to its interaction with the aberrations in 3D nuclear structure that accompany neoplastic progression. We investigated the impact of vorinostat on human esophageal epithelial cell lines derived from normal, metaplastic (pre-cancerous), and malignant tissue.

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.

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.

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.

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).

Parallel RNA extraction using magnetic beads and a droplet array.

Nucleic acid extraction is a necessary step for most genomic/transcriptomic analyses, but it often requires complicated mechanisms to be integrated into a lab-on-a-chip device. Here, we present a simple, effective configuration for rapidly obtaining purified RNA from low concentration cell medium. This Total RNA Extraction Droplet Array (TREDA) utilizes an array of surface-adhering droplets to facilitate the transportation of magnetic purification beads seamlessly through individual buffer solutions without solid structures.

A minimally invasive method for retrieving single adherent cells of different types from cultures.

The field of single-cell analysis has gained a significant momentum over the last decade. Separation and isolation of individual cells is an indispensable step in almost all currently available single-cell analysis technologies. However, stress levels introduced by such manipulations remain largely unstudied.

A polymer-based ratiometric intracellular glucose sensor.

The glucose metabolism level reflects cell proliferative status. A polymeric glucose ratiometric sensor comprising poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) and poly[2-(methacryloyloxy)ethyl]trimethylammonium chloride (PMAETMA) was synthesized. Cellular internalization and glucose response of the polymer within HeLa cells were investigated.

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.

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.