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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.
IEEE Transactions on Automation Science and Engineering 02/2008; · 1.46 Impact Factor
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ABSTRACT: Advancements in methods and algorithms for the measurement of oxygen consumption rates of single cells is presented. In this system a low density of randomly seeded eukaryotic cells are sealed in an array of microwells etched in glass (zero to three cells per microwell). The decrease in oxygen concentration inside each microwell in the array is measured yielding the oxygen consumption rates of the cells trapped in the array. While fundamentally simple in concept, the system requires advanced algorithms for data collection and image processing. The data collection technique enabling the oxygen sensors in each microwell has been modified to increase speed and sensor precision. Utilizing internal triggering and an integrate-on-chip mode rather than external triggering and an off-chip accumulation mode improves sensor precision by 45% and increases collection speed by a factor of seven. Furthermore, an optimized sensor locator algorithm has reduced the time to process image data for a single oxygen measurement point five-fold. A new measurement technique involving custom image-processing algorithms has also been developed revealing the microwell volumes to be 0.54 nL on average with a 6% maximum spread from the mean. To demonstrate the utility of the system, we present an experiment that successfully measured the oxygen consumption rates of 1, 2 or 3 cells in nine individual microwells simultaneously.
Automation Science and Engineering, 2007. CASE 2007. IEEE International Conference on; 10/2007
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ABSTRACT: The analysis of cell function comprises an examination of gene expression, protein synthesis, and metabolic activity. In order to measure these parameters in single cells a means for signal transduction and amplification is required. Fluorescent molecules have been demonstrated to provide a powerful tool for this detection need when performing living cell analysis. The development of an image analysis tool is the first step in automating multi-parameter cell function analysis where the objective is to ascertain cell membrane integrity and by extension, to obtain an estimate of cell health. A live/dead fluorescent stain was used to make this distinction. Two image analysis algorithms were implemented from the literature and one new method was developed. Three methods were tried: threshold segmentation, matched filtering, and an original method named morphological subtraction. The threshold technique produced the greatest overall accuracy in reducing spurious counts, closely followed by the morphological subtraction and then the matched filter. However, the original morphological subtraction method may be more appropriate in single cell studies because it overestimates live cells, aiding in the identification of unsuitable data.
Automation Science and Engineering, 2007. CASE 2007. IEEE International Conference on; 10/2007
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ABSTRACT: 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 O<sub>2</sub> 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
IEEE Sensors Journal 07/2007; · 1.52 Impact Factor
