Time-resolved fluorescence-decay measurement and analysis on single cells by flow cytometry

A novel method is described for the measurement and analysis of fluorescence decays of individual cells and particles in flow. It combines the rapid measurement capabilities of a flow cytometer and the robust measurement and analysis procedures of time-domain fluorescence-lifetime spectroscopy. For excitation we use a cw laser that is pulse modulated by an electro-optic modulator. The characteristics and the repetition rate of the excitation pulses can be easily adjusted to accommodate fluorescence decays with a wide range of lifetimes.     

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.     

Electroporation of Ishikawa cells: analysis by flow cytometry

Electroporation facilitates loading of cells with molecules and substances that are normally membrane impermeable. Flow cytometry is used in this study to examine the effects of the application of electroporation‐level monopolar electric field pulses of varying electrical field strength on Ishikawa endometrial adenocarcinoma cells. Analysis of the fluorescence versus forward scatter plots corroborates the well‐recognised threshold and cell size dependence characteristics of electroporation, but also shows the progression of cell lysis and generation of particulate material. Two 500 µs monopolar rectangular pulses ranging from 1.0 × 10⁵ to 2.5 × 10⁵ V/m were used to electroporate the cells. Electroporation yields (fraction of viable cells exhibiting significant propidium iodide uptake) ranged from 0 to 97%, with viability ranging between 78 and 34% over the electric field strength range tested. The higher electric field strength pulses not only reduced cell viability, but also generated a substantial amount of sub‐cellular sized particulate material indicating cells have been physically disrupted enough to create these particles.Inspec keywords: biomembranes, fluorescence, bioelectric phenomena, cancer, cellular effects of radiationOther keywords: sub‐cellular sized particulate material, electric field strength pulses, monopolar rectangular pulses, electric field strength, propidium iodide uptake, electroporation, cell lysis, cell size dependence characteristics, Ishikawa endometrial adenocarcinoma cells, electrical field strength, electroporation‐level monopolar electric field pulses, flow cytometry, cell viability     

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.     

Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry

We report a method to increase the resolution of single pair fluorescence resonance energy transfer (spFRET) measurements in aqueous solutions. Solution-based spFRET measurements of fluorescently labeled biological molecules (proteins, RNA, DNA) are often used to obtain histograms of molecular conformation without resorting to sample immobilization. However, for solution-phase spFRET studies, the number of photons detected from a single molecule as it diffuses through an open confocal volume element are quite limited. An "average" transit may yield on the order of 40 photons. Shot noise on the number of detected photons substantially limits the resolution of the measurement. The method reported here uses a hydrodynamically focused sample stream to ensure molecules traverse the full width of an excitation laser beam. This substantially increases the average number of photons detected per molecular transit (approximately 85 photons/molecule), which increases measurement precision. In addition, this method minimizes another source of heterogeneity present in diffusive measures of spFRET: the distribution of paths taken through the excitation laser beam. We demonstrate here using a FRET labeled protein sample (a FynSH3 domain) that superior resolution (a factor of approximately 2) can be obtained via molecular cytometry compared to spFRET measurements based upon diffusion through an open confocal volume element.     

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%.     

Monitoring drug efflux from sensitive and multidrug-resistant single cancer cells with microvoltammetry.

Multidrug resistance (MDR) is the eventual cross-resistance of certain cancer cells to a series of chemically unrelated drugs. It is attributed to a number of possible biophysical processes, one of them being increased drug efflux from resistant cells which leads to a decreased intracellular drug accumulation and retention. In this work, a carbon fiber microdisk electrode was used to monitor directly doxorubicin efflux from single preloaded cancer cells. Electrochemical cleaning, adsorptive preconcentration, and an electrocatalytic effect due to ambient oxygen made it possible to detect eventually very low drug concentrations (down to 1 nM) at good temporal resolution (down to 30 s/measurement) very close (< or = 1 micron) to single cancer cells for the first time. The results from a sensitive (AUXB1) and a drug-resistant (CHRC5) version of Chinese hamster ovarian cancer cells show that resistant cells exhibit a much higher initial efflux rate and shorter efflux time constant when both cell lines are preloaded up to the same intracellular drug concentration. These observations are consistent with results obtained from populations of the same cells by conventional techniques, proving that microvoltammetry can be used to monitor doxorubicin efflux at the single-cell level. Compared with existing methodologies, however, whose data represent only average cell behavior at typically low temporal resolution, the technique described here can provide information on the microheterogeneity of cancer cell populations in terms of drug efflux at high temporal resolution. The actual driving force of efflux is obtained since concentrations are measured directly at individual cells. This approach may lead to important new information on the mechanisms and prospective treatments of MDR.     

Oxygen microsensor and its application to single cells and mouse pancreatic islets.

An oxygen microsensor with a < 3-micron tip diameter was developed for monitoring oxygen levels at single cells and mouse pancreatic islets. The sensor was fabricated by electrochemically recessing an etched Pt wire inside a pulled glass micropipet and then coating with cellulose acetate. This fabrication process was found to be simpler than previous oxygen electrode designs of comparable size. The microsensors had a average sensitivity of 0.59 +/- 0.29 pA/mmHg (mean +/- SD, n = 42), signals that were minimally perturbed by convection, and response times of < 1 s. The electrode was used to measure the oxygen gradient around and inside single mouse islets. The measurements demonstrate that oxygen levels within even the largest islets at maximal glucose stimulation are 67 +/- 1.6 mmHg (mean +/- SD, n = 5), indicating that islets have adequate oxygen supplies by diffusion under tissue culture conditions to support insulin secretion. The electrode was also used to record the dynamics of oxygen level at single islets as a function of glucose concentration. As glucose level was changed from 3 to 10 mM, oxygen level decreased by 15.8 +/- 2.3 mmHg (mean +/- SEM, n = 6) and oscillations with a period of 3.3 +/- 0.6 min (mean +/- SEM, n = 6) appeared in the oxygen level. In islets bathed in quiescent solutions containing 10 mM glucose, similar oscillations could be observed. In addition, in the quiet solutions it was possible to detect faster oscillations with a period of 12.1 +/- 1.7 s (mean +/- SEM, n = 6) superimposed on the slower oscillations. Oxygen consumption could also be observed at single insulinoma cells using the electrode. Individual cells also showed oscillations in oxygen consumption with a period of a few seconds. The results demonstrate that the electrode can be used for dynamic oxygen level recordings in biological microenvironments.     

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.     

Raman microscopic analysis of single microbial cells

We demonstrate the utility of the Raman confocal microscope to generate a spectral profile from a single microbial cell and the use of this approach to differentiate bacterial species. In general, profiles from different bacterial taxa shared similar peaks, but the relative abundances of these components varied between different species. The use of multivariate methods subsequently allowed taxa discrimination. Further investigations revealed that the single-cell spectra could be used to differentiate between growth phases of a single species, but these differences did not obscure the overall interspecies discrimination. Finally, we tested the efficacy of the method as a means to identify cells responsible for the uptake of a specific substrate. A single strain was grown in media containing incrementally varying ratios of (13)C(6) to (12)C(6) glucose, and it was found that (13)C incorporation shifted characteristic peaks to lower wavenumbers. These findings suggest that Raman microscopy has significant potential for studies requiring the taxonomic identity and functioning of single microbial cells to be determined.     

Chemical force microscopy of single live cells

Traditionally, cell surface properties have been difficult to study at the subcellular level, especially on hydrated, live cells. Here, we demonstrate the ability of chemical force microscopy to map the hydrophobicity of single live cells with nanoscale resolution. After validating the technique on reference surfaces with known chemistry, we probe the local hydrophobic character of two medically important microorganisms, Aspergillus fumigatus and Mycobacterium bovis, in relation with function. Applicable to a wide variety of cells, the chemically sensitive imaging method presented here provides new opportunities for studying the nanoscale surface properties of live cells and for understanding their roles in mediating cellular events.