Metabolic cytometry: capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons

Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.     

Probing single molecules in single living cells

Single-molecule detection in single living cells has been achieved by using confocal fluorescence microscopy and externally tagged probe molecules. The intracellular background fluorescence is substantially higher than that in aqueous buffer, but this background is continuous and stable and does not significantly interfere with the measurement of single-molecule photon bursts. As a result, single-molecule data have been obtained on three types of fluorescent probes at spatially resolved locations (e.g., cytoplasm and nucleus) inside human HeLa cells. First, the iron transport protein transferrin labeled with tetramethylrhodamine undergoes rapid receptor-mediated endocytosis, and single transferrin molecules are detected inside living cells. Second, the cationic dye rhodamine 6G (R6G) enters cultured cells by a potential-driven process, and single R6G molecules are observed as intense photon bursts when they move in and out of the intracellular laser beam. Third, we report results on synthetic oligonucleotides that are tagged with a fluorescent dye and are taken up by living cells via a passive, nonendocytic pathway.     

Microfluidic lysis of human blood for leukocyte analysis using single cell impedance cytometry

This paper demonstrates an integrated microfluidic system that performs a full blood count using impedance analysis. A microfluidic network design for red blood cell (RBC) lysis is presented, and the diffusive mixing processes are analyzed using experimental and simulated results. Healthy and clinical bloods analyzed with this system, and the data shows good correlation against data obtained from commercial hematology machines. The data from the microfluidic system was compared against hospital data for 18 clinical samples, giving R(2) (coefficient of determination) values of 0.99 for lymphocytes, 0.89 for monocytes, and 0.99 for granulocytes in terms of relative counts and 0.94 for lymphocytes, 0.91 for monocytes, and 0.95 for granulocytes in terms of absolute counts. This demonstrates the potential clinical utility of this new system for a point-of-care purpose.     

Asymmetry between sister cells in a cancer cell line revealed by chemical cytometry

We introduce instrumentation and methodology for two-channel chemical cytometry of sister cells-two cells born from division of the same mother cell. The method is based on capillary electrophoresis with laser-induced fluorescence detection and allows simultaneously probing multiple intracellular components in sister cells. To test the new technology, we compared the expression patterns of green fluorescent protein (GFP) between the sisters in cultured cancer cells stably transfected with a GFP-expressing construct. We found that all sister cells had detectable asymmetry in the GFP expression patterns with a confidence level of higher than 95%. To our best knowledge, this is the first reported observation of asymmetric patterns of protein expression in sister cells in a cancer cell line. The proposed technology can reliably detect minor differences in chemical contents between sister cells, which makes it a potentially indispensable tool in studying the molecular mechanisms of developmental processes. It will be especially valuable in quantitative studies of cells with complex proliferation kinetics (e.g., stem cells).     

Optical tweezers for single cells

Optical tweezers (OT) have emerged as an essential tool for manipulating single biological cells and performing sophisticated biophysical/biomechanical characterizations. Distinct advantages of using tweezers for these characterizations include non-contact force for cell manipulation, force resolution as accurate as 100aN and amiability to liquid medium environments. Their wide range of applications, such as transporting foreign materials into single cells, delivering cells to specific locations and sorting cells in microfluidic systems, are reviewed in this article. Recent developments of OT for nanomechanical characterization of various biological cells are discussed in terms of both their theoretical and experimental advancements. The future trends of employing OT in single cells, especially in stem cell delivery, tissue engineering and regenerative medicine, are prospected. More importantly, current limitations and future challenges of OT for these new paradigms are also highlighted in this review.     

Nanowire substrate-based laser scanning cytometry for quantitation of circulating tumor cells

We report on the development of a nanowire substrate-enabled laser scanning imaging cytometry for rare cell analysis in order to achieve quantitative, automated, and functional evaluation of circulating tumor cells. Immuno-functionalized nanowire arrays have been demonstrated as a superior material to capture rare cells from heterogeneous cell populations. The laser scanning cytometry method enables large-area, automated quantitation of captured cells and rapid evaluation of functional cellular parameters (e.g., size, shape, and signaling protein) at the single-cell level. This integrated platform was first tested for capture and quantitation of human lung carcinoma cells from a mixture of tumor cells and leukocytes. We further applied it to the analysis of rare tumor cells spiked in fresh human whole blood (several cells per mL) that emulate metastatic cancer patient blood and demonstrated the potential of this technology for analyzing circulating tumor cells in the clinical settings. Using a high-content image analysis algorithm, cellular morphometric parameters and fluorescence intensities can be rapidly quantitated in an automated, unbiased, and standardized manner. Together, this approach enables informative characterization of captured cells in situ and potentially allows for subclassification of circulating tumor cells, a key step toward the identification of true metastasis-initiating cells. Thus, this nanoenabled platform holds great potential for studying the biology of rare tumor cells and for differential diagnosis of cancer progression and metastasis.     

SOFC单电池测试方法

固体氧化物燃料电池(SOFC)性能的测试结果能揭示材料和制备方法及其性能之间的复杂关系,确定燃料电池内部损耗的各种来源,并指导有关材料和制备技术的研发。在SOFC进入商业化发展的前期,测试方法的标准化有助于建立基础研究和开发研究间的有效和可靠联系,实现各研究机构所得测试结果的可比性,从而推动基础研究成果转化为现实的生产力。本文综述了国际上有关单电池标准测试系统的建立和步骤的制定以及测试结果报告的标准化,指出了在我国建立完善的SOFC发电技术标准体系的重要性和迫切性。     

Flow cytometry of Escherichia coli on microfluidic devices.

Flow cytometry of the bacterium Escherichia coli was demonstrated on a microfabricated fluidic device (microchip). The channels were coated with poly(dimethylacrylamide) to prevent cell adhesion, and the cells were transported electrophoretically by applying potentials to the fluid reservoirs. The cells were electrophoretically focused at the channel cross and detected by coincident light scattering and fluorescence. The E. coli were labeled with a membrane-permeable nucleic acid stain (Syto15), a membrane-impermeable nucleic acid stain (propidium iodide), or a fluorescein-labeled antibody and counted at rates from 30 to 85 Hz. The observed labeling efficiencies for the dyes and antibody were greater than 94%.     

Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry coupled with single-cell capillary sieving electrophoresis

Study of cell cycle-dependent protein expression is important in oncology, stem cell research, and developmental biology. In this paper, we report the first protein fingerprint from a single cell with known phase in the cell cycle. To determine that phase, we treated HT-29 colon cancer cells with Hoescht 33342, a vital nuclear stain. A microscope was used to measure the fluorescence intensity from one treated cell; in this form of image cytometry, the fluorescence intensity is proportional to the cell's DNA content, which varies in a predictable fashion during the cell cycle. To generate the protein fingerprint, the cell was aspirated into the separation capillary and lysed. Proteins were fluorescently labeled with 3-(2-furoylquinoline-2-carboxaldehyde, separated by capillary sieving electrophoresis, and detected by laser-induced fluorescence. This form of electrophoresis is the capillary version of SDS-PAGE. The single-cell electropherogram partially resolved approximately 25 components in a 30-min separation, and the dynamic range of the detector exceeded 5000. There was a large cell-to-cell variation in protein expression, averaging 40% relative standard deviation across the electropherogram. The dominant source of variation was the phase of the cell in the cell cycle; on average, approximately 60% of the cell-to-cell variance in protein expression was associated with the cell cycle. Cells in the G1 and G2/M phases of the cell cycle had 27 and 21% relative standard deviations in protein expression, respectively. Cells in the G2/M phase generated signals that were twice the amplitude of the signals generated by G1 phase cells, as expected for cells that are soon to divide into two daughter cells. When electropherograms were normalized to total protein content, the expression of only one component was dependent on cell cycle at the 99% confidence limit. That protein is tentatively identified as cytokeratin 18 in a companion paper.     

Profiling signaling peptides in single mammalian cells using mass spectrometry

The peptide content of individual mammalian cells is profiled using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Both enzymatic and nonenzymatic procedures, including a glycerol cell stabilization method, are reported for the isolation of individual mammalian cells in a manner compatible with MALDI MS measurements. Guided microdeposition of MALDI matrix allows samples to be created with suitable analyte-to-matrix ratios. More than 15 peptides are observed in individual rat intermediate pituitary cells. The combination of accurate mass data, expected cleavages by proteolytic enzymes, and postsource decay sequencing allows identification of 14 of these peptides as pro-opiomelanocortin prohormone-derived molecules. These protocols permit the classification of individual mammalian cells by peptide profile, the elucidation of cell-specific prohormone processing, and the discovery of new signaling peptides on a cell-to-cell basis in a wide variety of mammalian cell types.     

Three-dimensional super-localization and tracking of single gold nanoparticles in cells

We introduce a precise three-dimensional (3D) localization method of spherical gold nanoparticle probes using model-based correlation coefficient mapping. To accomplish this, a stack of sample images at different z-positions are acquired, and a 3D intensity profile of the probe serving as the model is used to map out the positions of nanoparticles in the sample. By using this model-based correlation imaging method, precise localization can be achieved in imaging techniques with complicated point spread functions (PSF) such as differential interference contrast (DIC) microscopy. We demonstrated the localization precision of 4-7 nm laterally and 16 nm axially for 40-nm gold nanospheres at an imaging rate of 10 frames per second. The 3D superlocalization method was applied to tracking gold nanospheres during live endocytosis events.     

Optical and acoustic detection of laser-generated microbubbles in single cells

Acoustically monitored laser-induced optical breakdown (LIOB) has potential as an important tool to diagnose and treat living cells. Laser-induced intracellular microbubbles are readily detectable using high-frequency ultrasound, and LIOB can be controlled to operate within two distinct regimes. In the nondestructive regime, a single, short-lived bubble can be generated within a cell, without affecting its immediate viability. In the destructive regime, the induced photodisruption quickly can kill a targeted cell. To generate and monitor this range of bioeffects in real time, we have developed a system integrating an ultrafast laser source with optical and acoustic microscopy. Experiments were performed on monolayers of Chinese hamster ovary (CHO) cells. A 793 nm, 100 fs laser pulsed at 3.8 kHz was tightly focused within each cell to produce the photodisruption, and a 50 MHz ultrasonic transducer monitored the resultant bubble via continuous pulse-echo recordings. Photodisruption was also observed using bright field microscopy, and cell viability was assessed following laser exposure with a trypan blue assay. By controlling laser pulse fluence and exposure duration, either nondestructive or destructive LIOB could be produced. The intracellular position of the laser focus was also varied to demonstrate that cell viability was affected by the specific location of material breakdown.     

Hydrodynamic tweezers: 1. Noncontact trapping of single cells using steady streaming microeddies

A key need for dynamic single-cell measurements is the ability to gently position cells for repeated measurements without perturbing their behavior. We describe a new method that uses a gentle secondary flow to trap and suspend single cells, including motile cells, at predictable locations in 3-D. Trapped cells can be more dense or less dense than the surrounding medium. The cells are suspended without surface contact in one of four steady streaming eddies created by audible-frequency fluid oscillation (< or =1000 Hz) in a microchannel containing a single fixed cylinder (radius = 125 microm). Comparison of measured trap locations to computations of the eddy flow show that each trap is located near the eddy center, and the location is controlled via the oscillation frequency. We use the motile phytoplankton cell (Prorocentrum micans) to experimentally measure the trapping force, which is controlled via the oscillation amplitude. Trapping forces up to 30 pN are generated while exerting moderate shear stresses (shear stresses < or = 1.5 N/m2) on the trapped cell. The magnitude of this trapping force is comparable to that of optical tweezers or dielectrophoretic traps, without requiring an external field outside the physiological range for cells (the shear stresses are comparable to those found in arterial blood flow). The unique combination of predictable 3-D positioning, insensitivity to cell and medium properties, strong adjustable trapping forces, and a gentle fluid environment makes hydrodynamic tweezers a promising new option for noncontact trapping of single cells in suspension.     

Tracking individual kinesin motors in living cells using single quantum-dot imaging

We report a simple method using semiconductor quantum dots (QDs) to track the motion of intracellular proteins with a high sensitivity. We characterized the in vivo motion of individual QD-tagged kinesin motors in living HeLa cells. Single-molecule measurements provided important parameters of the motor, such as its velocity and processivity, as well as an estimate of the force necessary to carry a QD. Our measurements demonstrate the importance of single-molecule experiments in the investigation of intracellular transport as well as the potential of single quantum-dot imaging for the study of important processes such as cellular trafficking, cell polarization, and division.     

Use of single wall carbon nanohorns in polymeric electrolyte fuel cells

Single wall carbon nanohorns (SWNH), produced by AC arc discharge in air, were used as Pt and PtRu supports in polymer electrolyte membrane fuel cells (PEMFC). These electrocatalysts were compared with equivalent electrocatalysts supported on commercial carbon back. The SWNH were characterized by differential thermal analysis (DTA), TEM, SEM, and XRD. The produced SWNH were 84.5 wt% pure, containing 3 wt% of amorphous carbon and 12.5 wt% of graphitic carbon. SWNH were used as electrocatalyst supports and tested in the electrodes of two types of polymer electrolyte fuel cells: H₂-fed PEMFC and direct methanol fuel cells (DMFC). The electrocatalyst nanoparticles anchored on both carbon supports were ca. 2.5 nm in diameter obtained by employing ethylene glycol as the reducing agent. The use of SWNH showed catalytic activities 60% higher than using carbon black as the electrocatalyst support in both types of fuel cells.     

Electroanalysis of metabolic flux from single cells in simple picoliter-volume microsystems

A picoliter-volume electrochemical analytical chamber has been developed for detecting the metabolic flux resulting from the stress responses of a single plant cell. Electrochemical cells, with volumes as small as 100 pL, were fabricated by controlled electrochemical dissolution of a gold wire sealed in glass (the back-etching of the metal realizing an ultralow-volume titer chamber). In the first instance, the electrode contained within the chamber was characterized by the microinjection of standard aliquots of either ascorbic acid or hydrogen peroxide. In all cases, experimental currents obtained correlated well with theoretical calculations. Subsequently, single plant cells were micromanipulated into the chambers and were exposed to amounts of the detergent SDS (which permeabilized the cell membrane and released the intracellular contents). The flux of metabolite released from a single cell was estimated by using electrochemical-linked assays based upon the enzymes catalase, ascorbate oxidase, and horseradish peroxidase (in each case), in the presence of a mediator. In so doing, we investigated the activity of the cellular protection mechanisms through the determination of peroxides, while the individual cell was "stressed". The technique was found to provide a reliable and reproducible method for making single-cell measurements, using fabrication procedures that are both simple and do not require photolithographic methods.