Sarah C. McQuaide

A New Approach for Measuring Single-Cell Oxygen Consumption Rates

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.

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.

50.4: Three-dimensional Virtual Retinal Display System using a Deformable Membrane Mirror

A deformable membrane mirror is being used to generate multiple focal planes within a scanned light display, or virtual retinal display (VRD). The MEMS deformable mirror changes the focal plane of the display by varying the wavefront of the optical beam entering the eye. Dynamically changing the focal plane will allow the viewer to see 3-D objects using the natural accommodative response of the eye, unlike current stereographics that rely on retinal disparity. The display system can successfully generate a focal range from infinity to −3 diopters, verified through measurements on human subjects made with a laser autorefractor.

Interface of an Array of Five Capillaries with an Array of One-Nanoliter Wells for High-Resolution Electrophoretic Analysis as an Approach to High-Throughput Chemical Cytometry

We report a system that allows the simultaneous aspiration of one or more cells into each of five capillaries for electrophoresis analysis. A glass wafer was etched to create an array of 1-nL wells. The glass was treated with poly(2-hydroxyethyl methacrylate) to control cell adherence. A suspension of formalin-fixed cells was placed on the surface, and cells were allowed to settle. The concentration of cells and the settling time were chosen so that there was, on average, one cell per well. Next, an array of five capillaries was placed so that the tip of each capillary was in contact with a single well. A pulse of vacuum was applied to the distal end of the capillaries to aspirate the content of each well into a capillary. Next, the tips of the capillaries were placed in running buffer and potential was applied. The cells lysed upon contact with the running buffer, and fluorescent components were detected at the distal end of the capillaries by laser-induced fluorescence. The electrophoretic separation efficiency was outstanding, generating over 750,000 theoretical plates (1,800,000 plates/m). In this example, AtT-20 cells were used that had been treated with TMR-G(M1). The cells were allowed to metabolize this substrate into a series of products before the cells were fixed. The number of cells found in each well was estimated visually under the microscope and was described by a Poisson distribution with mean of 0.98 cell/well. This system provides an approach to high-throughput chemical cytometry.

Phosphorescence lifetime based oxygen micro-sensing using a digital micromirror device

A digital light modulation microscope (DLMM) that utilizes a digital micromirror device (DMD) on an epifluorescence microscope has been developed to modulate excitation light in spatial and temporal domains for phosphorescence lifetime detection. Local O2 concentration can be inferred through the detected lifetime around an O2-quenching phosphorescent porphyrin microsensor. Combined with microsensor arrays, the DLMM can sequentially address light to each microsensor element to construct a discrete lifetime image or O2 distribution. In contrast to conventional phosphorescence lifetime imaging, the new method eliminates the need for a pulsed light source and a time-gated camera. To demonstrate O2 sensing with lab-on-a-chip devices, an array of 150-mum-diameter micro-wells coated with phosphorescent porphyrin were observed. The locations of the sensor elements were automatically identified though image analysis. The goal of this platform is to measure the O2 consumption of individual cells trapped in the microwells.

Algorithm Advancements for the Measurement of Single Cell Oxygen Consumption Rates

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.

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

Single Cell Methods for Methane Oxidation Analysis

Respiration is widely used for evaluation of the metabolic capabilities or physiological state of the microbial culture. This chapter describes novel approaches for characterization of respiration at a single cell level: (1) flow cytometry-based redox sensing (FCRS) of actively metabolizing microbes; (2) respiration response imaging (RRI) for real-time detection of substrate stimulated redox responses of individual cells; (3) respiration detection system: microobservation chamber (RDS: MC), a single cell analysis system for carrying out the physiological and genomic profiling of cells capable of respiring C(1) compounds. The techniques are suitable for description of physiological heterogeneity among cells in a single microbial population and could be used to characterize distribution of methylotrophic ability among microbial cells in the natural environmental samples.

Forming self-assembled cell arrays and measuring the oxygen consumption rate of a single live cell

We report a method for forming arrays of live single cells on a chip using polymer micro-traps made of SU8. We have studied the toxicity of the microfabricated structures and the associated environment for two cell lines. We also report a method for measuring the oxygen consumption rate of a single cell using optical interrogation of molecular oxygen sensors placed in micromachined micro-wells by temporarily sealing the cells in the micro-traps. The new techniques presented here add to the collection of tools available for performing “single-cell” biology. A single-cell self-assembly yield of 61% was achieved with oxygen draw down rates of 0.83, 0.82, and 0.71 fmol/minute on three isolated live A549 cells.

Automated Classification of Macrophage Membrane Integrity using a Fluorescent Live/Dead Stain

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.