Tim J. Strovas

Cell-to-cell heterogeneity in growth rate and gene expression in Methylobacterium extorquens AM1.

Cell-to-cell heterogeneity in gene expression and growth parameters was assessed in the facultative methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized methylotrophy promoter (P(mxaF)) and gfp(uv) (encoding a variant of green fluorescent protein [GFPuv]) was used to assess single-cell gene expression. Using a flowthrough culture system and laser scanning microscopy, data on fluorescence and cell size were obtained over time through several growth cycles for cells grown on succinate or methanol. Cells were grown continuously with no discernible lag between divisions, and high cell-to-cell variability was observed for cell size at division (2.5-fold range), division time, and growth rate. When individual cells were followed over multiple division cycles, no direct correlation was observed between the growth rate before a division and the subsequent growth rate or between the cell size at division and the subsequent growth rate. The cell-to-cell variability for GFPuv fluorescence from the P(mxaF) promoter was less, with a range on the order of 1.5-fold. Fluorescence and growth rate were also followed during a carbon shift experiment, in which cells growing on succinate were shifted to methanol. Variability of the response was observed, and the growth rate at the time of the shift from succinate to methanol was a predictor of the response. Higher growth rates at the time of the substrate shift resulted in greater decreases in growth rates immediately after the shift, but full induction of P(mxaF)-gfp(uv) was achieved faster. These results demonstrate that in M. extorquens, physiological heterogeneity at the single-cell level plays an important role in determining the population response to the metabolic shift examined.

A cellular isolation system for real-time single-cell oxygen consumption monitoring

The development of a cellular isolation system (CIS) that enables the monitoring of single-cell oxygen consumption rates in real time is presented. The CIS was developed through a multidisciplinary effort within the Microscale Life Sciences Center (MLSC) at the University of Washington. The system comprises arrays of microwells containing Pt-porphyrin-embedded polystyrene microspheres as the reporter chemistry, a lid actuator system and a gated intensified imaging camera, all mounted on a temperature-stabilized confocal microscope platform. Oxygen consumption determination experiments were performed on RAW264.7 mouse macrophage cells as proof of principle. Repeatable and consistent measurements indicate that the oxygen measurements did not adversely affect the physiological state of the cells measured. The observation of physiological rates in real time allows studies of cell-to-cell heterogeneity in oxygen consumption rate to be performed. Such studies have implications in understanding the role of mitochondrial function in the progression of inflammatory-based diseases, and in diagnosing and treating such diseases.

Population heterogeneity in Methylobacterium extorquens AM1

Heterogeneity of cells within exponentially growing populations was addressed in a bacterium, the facultative methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized methanol-inducible promoter (P(mxaF)) and gfp(uv) was used with flow cytometry to analyse the distribution of gene expression in populations grown on either succinate or methanol, correlated with forward scatter as a measure of cell size. These cell populations were found to consist of three major subpopulations defined by cells that were actively growing and dividing, newly divided, and non-dividing. Through the use of flow cytometry, it was demonstrated that a significant percentage of the total population did not respond to carbon shift. In addition, these experiments demonstrated that a small subset of the total population was significantly brighter than the rest of the population and dominated fluorimetry data. These results were corroborated with a continuous flow-through system and laser scanning microscopy, confirming that subpopulations, not discernible in the population average, dominate population response. These results demonstrate that the combination of flow cytometry and microscopic single-cell analysis can be effectively used to determine the dynamics of subpopulations in population response. In addition, they support the concept that physiological diversity in isogenic populations can poise some proportion of the population to respond appropriately to changing conditions.

Utilization of micelles formed from poly(ethylene glycol)-block- poly(ε-caprolactone) block copolymers as nanocarriers to enable hydrophobic red two-photon absorbing emitters for cells imaging

A hydrophobic two-photon absorbing (2PA) red emitter (R) was successfully incorporated into micelles formed from two block copolymers, poly(epsilon-caprolactone)-block-poly(ethylene glycol)s, for imaging and toxicity studies. In micelles, the chromophore R exhibits a 2PA cross-section of 400 GM (1 GM = 1 x 10(-50) cm(4) s photon(-1) molecule(-1)) at 820 nm, which is among the highest values reported for red 2PA emitters. The micelles with a cationic amino moiety-containing poly(ethylene glycol) corona showed an enhancement of cell internalization and delivered the dye into the cytoplasmic regions of the mouse macrophage RAW 264.7 cells. In comparison, the dye in micelles with neutral poly(ethylene glycol) as corona could not be delivered into the cells. Cytotoxicity of the micelle-R constructs was studied using a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. More than 90% of the cells were viable after they were stained with the dye-containing micelles at different concentrations (dye concentrations of 2-6 muM and polymer concentrations of 0.05-0.15 mg/mL) for 16 h. This is the first reported application of a hydrophobic 2,1,3-benzothiadiazole-containing 2PA red emitter delivered into the cytoplasm of cells for bioimaging and toxicity assessment.

Respiration response imaging for real time detection of microbial function at the single cell level

ABSTRACT The ability to detect specific functions of uncultured microbial cells in complex natural communities remains one of the most difficult tasks of environmental microbiology. Here we present respiration response imaging (RRI) as a novel fluorescence microscopy-based approach for the identification of microbial function, such as the ability to use C1 substrates, at a single-cell level. We demonstrate that RRI could be used for the investigation of heterogeneity of a single microbial population or for functional profiling of microbial cells from complex environmental communities, such as freshwater lake sediment.

Measurement of Respiration Rates of Methylobacterium extorquens AM1 Cultures by Use of a Phosphorescence-Based Sensor

ABSTRACT Respiration rates of bacterial cultures can be a powerful tool in gauging the effects of genetic manipulation and environmental changes affecting overall metabolism. We present an optical method for measuring respiration rates using a robust phosphorescence lifetime-based sensor and off-the-shelf technology. This method was tested with the facultative methylotroph Methylobacterium extorquens AM1 to demonstrate subtle mutant phenotypes.

A Living Cell Array (LCA) for Multiparameter Cell Metabolism Studies

A proof-of-platform-concept architecture for the analysis of living cell arrays is presented. Initial experiments conducted show a conceptual design for single-cell multiparameter measurements in real time. As a first parameter, extensive development and experimentation have been conducted on a sensor that will measure single-cell oxygen consumption. Wafer-level processing for chip fabrication and sensor deposition has been achieved, making possible the future goal of an automated system using disposable chips. The oxygen sensor has been tested for long-term adherence to a substrate in aqueous environments, biocompatibility with cell types of interest, and sensitivity to small changes in local oxygen concentration. A test device was made to verify the use of a gold foil barrier as an oxygen seal and shows good isolation of sealed areas

Two-Photon Lithography of Platinum-Porphyrin Oxygen Sensors

Oxygen sensing structures were generated by two-photon microfabrication. By copolymerizing metalloporphyrins with a two-photon (2P) photo-initiated polymer, oxygen sensors were patterned into complex 3-D shapes. The sensors were generated on the interior walls of small bore capillaries to allow for controlled concentrations of oxygenated water and cell-rich media to be pumped through their local environment. Phosphorescence lifetime of the patterns were acquired at known levels of O2 as a standard for measuring the respiration rate of a tiny population of bacterial cells. In addition, we report that the inclusion of the Pt-Porphyrin significantly reduces the 2P polymerization threshold. Fabricating near the inferred polymerization threshold, 3-D structures as small as 50 nm were observed in both the Pt-Porphyrin enhanced and the pure photopolymerizable monomers

Single-cell information extraction and viability analysis using automated microscopy

We present the use of an automated microscope routine for long-term single cell viability analysis. Murine macrophage cells were monitored for more than 10 hours at physiological conditions. The information of each cell was extracted from time-lapse raw fluorescence and bright-field images to study single cell dynamic behaviors. Two methods were applied to analyze single cell viability: the popular method using live/dead fluorescent dye, and a new morphology-based, dye-free method that estimates viability with optical appearance. Both methods yielded similar death event estimation, indicating the new morphology-based method can be an alternative when using live/dead fluorescent dye is difficult or not allowed

High-throughput, long-term imaging of Salmonella infecting macrophages in a micro-environmental system

Real-time single cell analysis is necessary to understand dynamic cellular functions in time and space. Such analyses require the simultaneous measurement of multiple variables in real-time, due to heterogeneity in cellular populations. We report the application of using a micro-environmental chamber on an automatic laser scanning confocal microscope to observe murine macrophage cells in incubation conditions for more than 18 hours. The motorized stage of the microscope was programmed to scan through pre-defined monitoring locations to increase the observation throughput. The acquired images were post-processed to extract the information of each cell. In contrast to current single-cell technologies, such as fluorescence-activated cell sorter (FACS) based systems, the reported architecture records the history of the physiological responses of individual cells.Copyright