Laimonas Kelbauskas, Shashanka Ashili, Jeff Houkal, Dean Smith, Aida Mohammadreza, Kristen Lee, Jessica Forrester, Ashok Kumar, Cody Youngbull, Yanqing Tian, Mark Holl, Roger Johnson, Deirdre Meldrum, Yasser Anis, Thomas Paulson
Intercellular heterogeneity is a key factor in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis, and drug resistance. However, cell-to-cell variability studies at the single-cell level have been hampered by the lack of enabling experimental techniques. We present a measurement platform that features the capability to quantify oxygen consumption rates of individual, non-interacting and interacting cells under normoxic and hypoxic conditions. It is based on real-time concentration measurements of metabolites of interest by means of extracellular optical sensors in cell-isolating microwells of subnanoliter volume. We present the results of a series of measurements of oxygen consumption rates (OCRs) of individual non-interacting and interacting human epithelial cells. We measured the effects of cell-to-cell interactions by using the system's capability to isolate two and three cells in a single well. The major advantages of the approach are: 1. ratiometric, intensity-based characterization of the metabolic phenotype at the single-cell level, 2. minimal invasiveness due to the distant positioning of sensors, and 3. ability to study the effects of cell-cell interactions on cellular respiration rates.
Shashanka Ashili, Laimonas Kelbauskas, Jeff Houkal, Dean Smith, Yanqing Tian, Cody Youngbull, Haixin Zhu, Yasser Anis, Michael Hupp, Kristen Lee, Ashok Kumar, Juan Vela, Andrew Shabilla, Roger Johnson, Mark Holl, Deirdre Meldrum
We have developed a fully automated platform for multiparameter characterization of physiological response of
individual and small numbers of interacting cells. The platform allows for minimally invasive monitoring of cell
phenotypes while administering a variety of physiological insults and stimuli by means of precisely controlled
microfluidic subsystems. It features the capability to integrate a variety of sensitive intra- and extra-cellular fluorescent
probes for monitoring minute intra- and extra-cellular physiological changes. The platform allows for performance of
other, post- measurement analyses of individual cells such as transcriptomics.
Our method is based on the measurement of extracellular metabolite concentrations in hermetically sealed ~200-pL
microchambers, each containing a single cell or a small number of cells. The major components of the system are a) a
confocal laser scan head to excite and detect with single photon sensitivity the emitted photons from sensors; b) a
microfluidic cassette to confine and incubate individual cells, providing for dynamic application of external stimuli, and
c) an integration module consisting of software and hardware for automated cassette manipulation, environmental
control and data collection. The custom-built confocal scan head allows for fluorescence intensity detection with high
sensitivity and spatial confinement of the excitation light to individual pixels of the sensor area, thus minimizing any
phototoxic effects. The platform is designed to permit incorporation of multiple optical sensors for simultaneous
detection of various metabolites of interest. The modular detector structure allows for several imaging modalities,
including high resolution intracellular probe imaging and extracellular sensor readout. The integrated system allows for
simulation of physiologically relevant microenvironmental stimuli and simultaneous measurement of the elicited
phenotypes. We present details of system design, system characterization and metabolic response analysis of individual
eukaryotic cells.
Laimonas Kelbauskas, Shashanka Ashili, Jeff Houkal, Dean Smith, Aida Mohammadreza, Kristen Lee, Ashok Kumar, Yasser Anis, Tom Paulson, Cody Youngbull, Yanqing Tian, Roger Johnson, Mark Holl, Deirdre Meldrum
Non-genetic intercellular heterogeneity has been increasingly recognized as one of the key factors in a variety
of core cellular processes including proliferation, stimulus response, carcinogenesis and drug resistance. Many diseases,
including cancer, originate in a single or a few cells. Early detection and characterization of these abnormal cells can
provide new insights into the pathogenesis and serve as a tool for better disease diagnosis and treatment. We report on a
novel technology for multiparameter physiological phenotype characterization at the single-cell level. It is based on real-time
measurements of concentrations of several metabolites by means of extracellular optical sensors in microchambers
of sub-nL volume containing single cells. In its current configuration, the measurement platform features the capability
to detect oxygen consumption rate and pH changes under normoxic and hypoxic conditions at the single-cell level. We
have conceived, designed and developed a semi-automated method for single-cell manipulation and loading into
microwells utilizing custom, high-precision fluid handling at the nanoliter scale.
We present the results of a series of measurements of oxygen consumption rates (OCRs) of single human
metaplastic esophageal epithelial cells. In addition, to assess the effects of cell-to-cell interactions, we have measured
OCRs of two and three cells placed in a single well. The major advantages of the approach are a) multiplexed
characterization of cell phenotype at the single-cell level, b) minimal invasiveness due to the distant positioning of
sensors, and c) flexibility in terms of accommodating measurements of other metabolites or biomolecules of interest.
A digital light modulation microscope (DLMM) using a digital micro-mirror device (DMD, Texas Instruments) has been developed to enable detection of O2 concentration in micro-bioreactors using O2-quenching porphyrin phosphorescent dyes. The emission intensity and phosphorescence lifetime of such dyes are both a function of O2 concentration. While emission intensity can vary in these dye systems as a function of concentration and illumination intensity, phosphorescence lifetime is primarily sensitive to only O2 concentration. In contrast to conventional phosphorescence lifetime imaging, the DLMM eliminates the need for a pulsed light source, scanning mirrors, or a high-speed camera for time-gated imaging. This technique can selectively address structured light illumination to each sensor location, which is a beneficial feature for analysis of large micro-sensor arrays within lab-on-a-chip devices. The mirrors on the DMD perform as electronically addressable optical switches, each having a ~15 μs switching time, shorter than the phosphorescence lifetimes of potential O2 sensing dyes (~25-100 μs). The structured light pattern of the DMD and the switching rate of the mirrors are controlled by a PC. An arc lamp illuminates the DMD uniformly and then projects to the specimen through a filter cube for the selected phosphorescent sensor compound. The emitted light returns to the filter cube and is detected by a photo multiplier tube (PMT). An oscilloscope is used to record the emission signal waveform from the PMT. To demonstrate O2 sensing with lab-on-a-chip devices, an array of 150-μm-diameter micro-wells coated with phosphorescent porphyrin were observed using the DLMM. The goal of this platform is to measure the O2 consumption of individual cells trapped in the microwells.
Monitoring of biologically active agents such as bacteria, viruses, proteins and small molecules in environmental samples poses complex analytical problems. The particulate nature of the analytes and potential interferents is of particular concern for microfluidic systems in which the channels may not be much larger than the particles themselves. For this reason, sample preconditioning upstream of a chemical analytical device will usually be required. However, the small dimensions of microfluidic devices also allow unique methods of sample purification, concentration, and detection. In our laboratory we have developed a series of microfluidic chemical analytical devices for such purposes. These devices rely on the low Reynolds number flow conditions. In such conditions field flow fractionation based on sedimentation, diffusion and electrophoresis perpendicular to the flow direction can be profitably harnessed to precondition samples. The H-filter is one such device in which a simple 4-port device that allows two fluids to be brought into adjacent flow, and then separated downstream into two (or more) flow streams after exchange of material under the influence of one or more fields. It can be fabricated using anodically bonded silicon and Pyrex channels, or using polymeric devices formed using `soft lithography' techniques. We have tested the ability of this device to be used for purification of bacteria and their spores from complex samples containing silica and other interferent particles. We will present results of our tests of this device, as well as initial attempts to integrate the H-filter into a sample preconditioning system that includes on-chip pumps.
The purpose of this study was to develop a miniaturized CO- oximeter for hemoglobin derivative measurement using microfabrication technology. A microcuvette (volume equals 507 nl) was fabricated for analysis of percent oxyhemoglobin (O2Hb%) in whole blood. A cuvette of 50 micrometer pathlength produced optimal absorbance sensitivity to changes in O2Hb%. The pressure differential for a nominal blood flow rate of approximately 1 microliter/second was 4.1 kPa (16.6 in water, 0.6 psi). Entrained bubbles were easily discharged at these pressures. Spectral measurements were made using an ocean optics miniaturized spectrophotometer (500 - 700 nm). A fiber optic probe with one receiving and six emitting fibers (200 micrometer core and 0.22 NA) was used for spectral measurement. Heparinized fresh blood from a healthy volunteer was tonometered with N2, CO2, and O2 mixtures to produce six samples with O2Hb% from 22 - 97%. Chemometrics was used for data analysis. The second derivatives of spectra were taken to eliminate baseline changes caused by RBC light scattering. Indirect calibration by principal component regression was applied to the second derivative. Four factor cross validation showed a correlation coefficient of 0.9994 between measured O2Hb% of lysed blood using an OSM3 CO- oximeter (Radiometer America, Ohio) and whole blood using the microfabricated cuvette. The linear relationship is: O2Hb%micro-cuvette equals 0.8411% plus 0.9882 multiplied by O2Hb%OSM3. We conclude that O2Hb% measurement on unlysed whole blood using a silicon microfabricated cuvette is practical and that results are similar to traditional CO- oximetry.
A mechanical interlock system is reported that achieves the manual alignment of two components accurate to within plus or minus 10 micrometers in three spatial coordinates. In addition, the system allows rapid component interchange. The system is based on a novel two stage application of the principles of the kinematic location of instrument components. A macro scale kinematic mount allows manual handling while a micro scale mount delivers the accuracy required. Silicon microfabrication methods are used to create features in the micron size range accurately and repeatable for the micro scale mount. Such a system could be used for fluidic, pneumatic, electrical, optical, or mechanical interconnects. We use it for the alignment of optics to flow channel in an optical flow cytometer which is part of a microfluidic total chemical analysis system we are developing.
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