No abstract

A novel system that has enabled the measurement of single-cell oxygen consumption rates is presented. The experimental apparatus includes a temperature controlled environmental chamber, an array of microwells etched in glass, and a lid actuator used to seal cells in the microwells. Each microwell contains an oxygen sensitive platinum phosphor sensor used to monitor the cellular metabolic rates. Custom automation software controls the digital image data collection for oxygen sensor measurements, which are analyzed using an image-processing program to yield the oxygen concentration within each microwell versus time. Two proof-of-concept experiments produced oxygen consumption rate measurements for A549 human epithelial lung cancer cells of 5.39 and 5.27 fmol/min/cell, closely matching published oxygen consumption rates for bulk A549 populations.

The development of a cellular isolation system (CIS) that enables the monitoring of singlecell 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-porphyrinembedded 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.

The lux operon derived from Photorhabdus luminescens incorporated into bacterial genomes, elicits the production of biological chemiluminescence typically centered on 490 nm. The light-producing bacteria are widely used for in vivo bioluminescence imaging. However, in living samples, a common difficulty is the presence of blue-green absorbers such as hemoglobin. Here we report a characterization of fluorescence by unbound excitation from luminescence, a phenomenon that exploits radiating luminescence to excite nearby fluorophores by epifluorescence. We show that photons from bioluminescent bacteria radiate over mesoscopic distances and induce a red-shifted fluorescent emission from appropriate fluorophores in a manner distinct from bioluminescence resonance energy transfer. Our results characterizing fluorescence by unbound excitation from luminescence, both in vitro and in vivo, demonstrate how the resulting blue-to-red wavelength shift is both necessary and sufficient to yield contrast enhancement revealing mesoscopic proximity of luminescent and fluorescent probes in the context of living biological tissues.ight-based optical methods are emerging at the cutting edge of molecular imaging technologies. However, many challenges remain including significant practical limitations, and especially the poor propagation of blue-green light in living tissues (1, 2). Peak photon absorption by mammalian tissues is mainly determined by the presence of oxyheamoglobin, deoxyheamoglobin, and melanin, which dramatically reduce light propagation, deteriorating efficient photon detection (3, 4). Numerous studies have aimed at the development of red-shifted optical probes that emit in the range 700-900 nm where absorption of photons is minimal, reducing signal loss and allowing for deep tissue imaging in vivo. However, although there has been considerable success with efforts to red-shift fluorescent chemical and genetically engineered probes (4, 5), there has been rather less progress toward the goal of red-shifting chemiluminescent and bioluminescent probes (6, 7). For example, methods like random mutagenesis of Renilla reniformis luciferase have achieved red-emission shifts of only 547 nm (4, 8), a peak luminescence wavelength far from the optimal target range of 700-900 nm. As an alternative approach, some studies have proposed red shift of blue bioluminescence using bioluminescence resonance energy transfer (BRET). For example, So et al. (9) demonstrated "self-illuminating quantum dots," where blue luminescence from a mutant of Renilla reniformis luciferase was red-shifted by covalently coupling to carboxylate-functionalized quantum dots (QDs), providing a new emission maximum at 655 nm. Likewise, a modified Photinus pyralis luciferase was closely bound to the commercially available Alexa Fluor 750, resulting in light emission of 780 nm (10). Similarly, far-red bioluminescent protein was constructed from Cypridina luciferase conjugated to an indocyanine dye, giving a red shift to 675 nm (11). In another study, BRET3 used a...

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.Respiration rates of an aerobic bacterial population can be used as a gauge of the metabolic state. For metabolic modes not involving primary oxygenases (e.g., methane monooxygenase) (9), changes in oxygen uptake reflect alterations in respiratory chain activity due to a phenotypic response or genetic manipulation (11). Given the tight coupling between energy metabolism and a cell's metabolic network (14), changes in respiration rates can reflect shifts in overall metabolism and how a specific metabolic state adapts to change (11).A number of methods are available to detect oxygen concentrations, such as the use of Clark electrodes, electrochemical cells, electrochemical microscopy, and paramagnetic cells (7,15). One of the most commonly performed techniques is the use of a Clark electrode. However, the caveats of this method are low sensitivities, signal drift, probe fragility, electrode consumption of oxygen, and the ability to only measure the immediate microenvironment (15,17,22). In addition, high-throughput analysis requires a number of individual devices, increasing the cost and decreasing reproducibility. One method that has seen a rapid increase in use recently is the application of optical sensors, such as phosphorescent dyes (4, 12), which impart greater signal-to-noise ratios, signal independence of the dye concentration and photobleaching, rapid response characteristics, and functionality while imbedded in a variety of materials (15, 23). Additionally, optical methods are amenable to high-throughput screening using high-density well formats (1), but existing systems tend to be custom designed and not broadly available.Recently, commercially available polystyrene beads doped with a platinum(Pt)-porphyrin dye and inexpensive off-theshelf components have become available for O 2 measurements. We examined this system to demonstrate its utility in measuring respiration rates of Methylobacterium extorquens AM1 cultures. M. extorquens AM1 has the ability to grow on C 1 substrates, e.g., methanol, as a sole source of carbon and energy and is an inexpensive renewable biofeedstock, which can reduce production costs of value-added products (5, 13). A broad range of biochemical and genetic tools along with a metabolic flux balance model has allowed a comprehensive mapping of central metabolism during C 1 and multicarbon growth (19,20).In addition to the wild type, two mutant strains were analyzed. The first mutant (20) was null for NADH-ubiquinone oxidoreductase subunit B (NADH-UOR; NADH-UOR subunit B::Tet r ), which couples NADH oxidation to the respiratory chain during multicarb...