Multisite analysis of metabolic transients in single living cells by multichannel microfluorometry

Summary A multichannel microspectrofluorometer allows the continuous monitoring of NAD(P)-linked dehydrogenases throughout the intact cell simultaneously in correlation with intracellular topography (morphological mode) or the spectral properties of NAD(P)H emission (spectral mode). Changes in NAD(P)H levels corresponding in absolute amounts to 10^–15–10^–16 mol can be followed with a signal-to-noise ratio over 100 to 1, following imposition of metabolic transients (e. g. with glycolytic intermediates). With glucose-6-phosphate, the level of fluorescence changes in individual cell regions imaged over separate channels may differ considerably. With glucose-1-phosphate an asynchronous response is observed, with initially a few only of the channels on which the cell is imaged, joining in the response which later on spreads throughout. The natural fluorescence of the cell shows on the spectral mode a multichannel distribution practically superposable to the emission curve of NAD(P)H crystals. Since spectral scan may be completed in 32 msec, changes in the spectral curve upon imposition of a metabolic transient can be observed down to such time resolution. Using the multichannel approach a new perspective is gained of the living cell, which suggests a certain individualization of various intracellular regions (e. g. metabolic asynchronicities, local residual glycolysis despite aerobic inhibition).

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Zusammenfassung Ein Vielkanalmikrofluorimeter ermöglicht die kontinuierliche Messung NAD(P)-gebundener Dehydrogenasen innerhalb der intakten Zelle, gleichzeitig in Wechselbeziehung mit der intrazellulären Topographie (morphologische Form) oder der spektralen Eigenschaften der NAD(P)H-Emission (spektrale Form). Änderungen der NAD(P)H-Gehalte in absoluten Mengen von 10^–15 bis 10^–16 Mol infolge Überlagerung von Stoff Wechselschwankungen können mit einem Signal-Untergrund-Verhältnis von über 1001 registriert werden (z. B. glykolytische Zwischenformen). Bei Glucose-6-phosphat kann das Ausmaß der Fluoreszenzänderung, die über getrennte Kanäle beobachtet wird, in den einzelnen Zellregionen erhebliche Unterschiede aufweisen. Bei Glucose-1-phosphat wird nur mit einigen Kanälen, bei denen die Zelle betrachtet wird, anfangs ein asynchrones Verhalten beobachtet; sie stimmt dann in ihrem Verhalten überein, das sich später durchgehend ausbreitet.Die natürliche Fluoreszenz der Zelle zeigt in der spektralen Form eine Vielkanalverteilung, die praktisch der Emissionskurve der NAD(P)-Kristalle überlagert werden kann. Da die spektrale Aufzeichnung in 32 msec beendet werden kann, lassen sich Änderungen in der Spektralkurve infolge Überlagerung einer Stoffwechselschwankung bis zu einer solchen Zeitauflösung messen. Bei Verwendung der Vielkanalmethode wird eine neue Perspektive der lebenden Zelle gewonnen, die eine gewisse Individualisierung verschiedener intrazellulärer Regionen vermuten läßt (z. B. Stoffwechselasynchronitäten, lokale Restglykolyse trotz aerober Hemmungen).

     

Fast quantification of water in single living cells by near-infrared microscopy

We have set up a near-infrared microscope using a tuneable diode laser in the range from 1530 to 1570 nm. This spectral range is close to the peak of the water overtone absorption. We used this new microscope to study liver cells, hepatocytes, showing that quantitative information of the intracellular water concentration in living cells can be extracted.     

SERS-based plasmonic nanobiosensing in single living cells

In this paper, we describe the development and application of a pH-sensitive plasmonics-active fiber-optic nanoprobe suitable for intracellular bioanalysis in single living human cells using surface-enhanced Raman scattering (SERS) detection. The effectiveness and usefulness of SERS-based fiber-optic nanoprobes are illustrated by measurements of intracellular pH in HMEC-15/hTERT immortalized “normal” human mammary epithelial cells and PC-3 human prostate cancer cells. The results indicate that fiber-optic nanoprobe insertion and interrogation provide a sensitive and selective means to monitor cellular microenvironments at the single cell level.     

Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy

Our recently developed in-cell NMR procedure now enables one to observe protein conformations inside living cells. Optimization of the technique demonstrates that distinguishing the signals produced by a single protein species depends critically on protein overexpression levels and the correlation time in the cytoplasm. Less relevant is the selective incorporation of (15)N. Poorly expressed proteins, insoluble proteins, and proteins that cannot tumble freely due to associations within the cell cannot yet be observed. We show in-cell NMR spectra of bacterial NmerA and human calmodulin and discuss limitations of the technique as well as prospects for future applications.     

Accurately measuring respiratory activity of single living cells by scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) is a powerful tool to examine the respiratory activity of living cells. However, in SECM measurements of cell respiratory activity, the signal recorded usually also includes the signal corresponding to the cell topography. Therefore, measurements of cell respiratory activity using conventional SECM techniques are not accurate. In the present work, we develop a method for accurate measurement of the respiratory activity of single living cells using SECM. First, cells are immobilized on a glass substrate modified with collagen. Then, a Pt ultramicroelectrode tip of SECM held at −0.50 V is scanned along the central line across a living cell and a SECM scan curve, i.e., the relationship of the tip current versus the displacement (the first scan curve) is recorded with a negative peak. The peak current i_p on this first scan curve is composed of i_p1, which corresponds to the cell respiratory activity and i_p2, which corresponds to the cell topography. In order to isolate the i_p2 component, the cell is killed by exposing it to 1.0 × 10⁻³ mol/L KCN for 10 min. The tip is then scanned again with the same trace over the dead cell, and a second SECM scan curve is recorded. Noting that the topography of the dead cell is the same as that of the living cell, this second scan curve with a negative peak corresponds now only to the cell topography. Thus, i_p2 is obtained from the second SECM scan curve. Finally, i_p1 corresponding to the respiratory activity of the living cell can be accurately calculated using i_p1 = i_p − i_p2. This method can be used to monitor real-time change in the respiratory activity of single cells after exposing them to KBr, NaN₃ and KCN.     

Slowed diffusion of single nanoparticles in the extracellular microenvironment of living cells revealed by darkfield microscopy

We obtained vertical distribution of diffusion coefficients of single gold nanoparticles (AuNPs) in the extracellular solution space of living cells with optical sectioning darkfield microscopy. It was identified that before reaching the plasma membrane surface during their cellular uptake process, AuNPs must diffuse through a viscous pericellular “buffer zone” several microns thick where their motion is retarded significantly. The pericellular layer exists in two different cell types and is unrelated to the surface chemistry of AuNPs. Further studies on its properties and manipulation may help the development of nanoparticle probes and carriers.     

O-GlcNAcylation mapping of single living cells by in situ quantitative SERS imaging

O-GlcNAcylation is involved in many biological processes including cancerization. Nevertheless, its in situ quantification in single living cells is still a bottleneck. Here we develop a quantitative SERS imaging strategy for mapping the O-GlcNAcylation distribution of single living cells. O-GlcNAcylated compounds (OGCs) can be quantified through their in situ azide labeling and then a click reaction competing with azide and Raman reporter labeled 15 nm-gold nanoparticles (AuNPs) for linking to dibenzocyclooctyne labeled 40 nm-AuNPs to produce OGC-negatively correlated SERS signals. The calibration curve obtained in vitro can be conveniently used for detecting OGCs in different areas of single living cells due to the negligible effect of cell medium on the click linkage and Raman signal. This method has been successfully applied in mapping O-GlcNAcylation distribution in different cell lines and monitoring O-GlcNAcylation variation during cell cycling, which demonstrate its great practicability and expansibility in glycosylation related analysis.

A quantitative SERS imaging strategy is developed for O-GlcNAcylation mapping of single living cells through a competitive click reaction.     

Multiplexed detection of ions and mRNA expression in single living cells

In order to push forward into new areas of medical and biological research, new techniques must be developed that will enable a complex investigation into cellular processes. This involves investigating not only the different expression levels inside of a cell but also the ability to analyze how those expression levels are connected to one another. In order to accomplish this level of exploration, different types of analytes must be investigated simultaneously inside of single cells, thereby allowing their expression levels to be directly compared. To accomplish this, we have developed a method of detecting and monitoring mRNA expression levels and ion concentrations simultaneously inside of the same single cell. We have utilized this technique in studying the effects of an anti-cancer agent on human breast carcinoma cells. Using this approach, we are able to shed light onto the complex connections between genes and ions inside the cell that is not possible with any other existing technique.     

High-speed separation system of randomly suspended single living cells by laser trap and dielectrophoresis

We developed a new system for random separation of a single microorganism, such as a living cell and a microbe, in the microfluidic device under the microscope by integrating the laser-trapping force and dielectrophoretic (DEP) force. An arbitrarily selected single microbe could be isolated in a microchannel, despite the presence of a large number of microbes in solution. Once the target microbe is trapped at the focal point of the laser, we can easily realize exclusion of excess microbes around the target by controlling the electric field, while keeping the target trapped by the laser at the focal point. To realize an efficient separation system, we proposed a new separation cell and produced it by microfabrication. Flow speed in the microchannel is adjusted and balanced to realize high-speed and high-purity extraction of the target. Some preliminary experiments are conducted to show the effectiveness. The target is trapped by the laser, transported, and is taken out from the extraction port. Total separation time is less than 20 s. Our method is extremely useful in the pure cultivation of the cell and will be a promising method for biologists in screening useful microbes.     

The potential of autofluorescence for the detection of single living cells for label-free cell sorting in microfluidic systems

A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and microfluidic devices have the potential to facilitate high-throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three-port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF-based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high-throughput live cell detection and differentiation without the need for cell-specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single-cell analysis.     

Imaging the cell wall of living single yeast cells using surface-enhanced Raman spectroscopy

The surface of a living yeast cell (Saccharomyces cerevisiae strain W303-1A) has been labeled with silver (Ag) nanoparticles that can form nanoaggregates which have been shown to have surface-enhanced Raman scattering (SERS) activity. The cell wall of a single living yeast cell has been imaged by use of a Raman microspectroscope. The SERS spectra measured from different Ag nanoaggregates were found to be different. This can be explained on the basis of detailed spectral interpretation. The SERS spectral response originates from mannoproteins which cover the outermost regions of the yeast cell wall. Analysis of SERS spectra from the cell wall and the extracted mannoproteins from the yeast has been performed for the clarification of variation in SERS spectra.     

Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system

We evaluated the intracellular NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione–ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 μM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 μM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system.     

Probing of multidrug ABC membrane transporters of single living cells using single plasmonic nanoparticle optical probes

Currently, molecular mechanisms of multidrug ABC (ATP-binding cassette) membrane transporters remain elusive. In this study, we synthesized and characterized purified spherically shaped silver nanoparticles (Ag NPs) (11.8 ± 2.6 nm in diameter), which were stable (non-aggregation) in PBS buffer and inside single living cells. We used the size-dependent localized surface plasmon resonance (LSPR) spectra of single Ag NPs to determine their sizes and to probe the size-dependent transport kinetics of the ABC (BmrA, BmrA-EGFP) transporters in single living cells (Bacillus subtilis) in real time at nanometer resolution using dark-field optical microscopy and spectroscopy (DFOMS). The results show that the smaller NPs stayed longer inside the cells than larger NPs, suggesting size-dependent efflux kinetics of the membrane transporter. Notably, accumulation and efflux kinetics of intracellular NPs for single living cells depended upon the cellular expression level of BmrA, NP concentrations, and a pump inhibitor (25 μM, orthovanadate), suggesting that NPs are substrates of BmrA transporters and that passive diffusion driven by concentration gradients is the primary mechanism by which the NPs enter the cells. The accumulation and efflux kinetics of intracellular NPs for given cells are similar to those observed using a substrate (Hoechst dye) of BmrA, demonstrating that NPs are suitable probes for study of multidrug membrane transporters of single living cells in real-time. Unlike fluorescent probes, single Ag NPs exibit size-dependent LSPR spectra and superior photostability, enabling them to probe the size-dependent efflux kinetics of membrane transporters of single living cells in real-time for better understanding of multidrug resistance.     

Spatial desynchronization of glycolytic waves as revealed by Karhunen-Loeve analysis

The dynamics of glycolytic waves in a yeast extract have been investigated in an open spatial reactor. At low protein contents in the extract, we find a transition from inwardly moving target patterns at the beginning of the experiment to outwardly moving spiral- or circular-shaped waves at later stages. These two phases are separated by a transition phase of more complex spatiotemporal dynamics. We have analyzed the pattern dynamics in these three intervals at different spatial scales by means of a Karhunen-Loeve (KL) decomposition. During the initial phase of the experiment, the observed patterns are sufficiently described by the two dominant KL modes independently of the spatial scale. However, during the last stage of the experiment, at least 6 KL modes are needed to account for the observed patterns at spatial scales larger than 3 mm, while for smaller scales, 2 KL modes are still sufficient. This indicates that in the course of the experiment, the local glycolytic oscillators become desynchronized at spatial scales larger than 3 mm. Possible reasons for the desynchronization of the glycolytic waves are discussed.     

Nanoelectrodes for intracellular measurements of reactive oxygen and nitrogen species in single living cells

Reactive oxygen and nitrogen species (ROS and RNS) play important roles in various physiological processes (e.g. phagocytosis) and pathological conditions (e.g. cancer). The primary ROS/RNS, viz., hydrogen peroxide, peroxynitrite ion, nitric oxide, and nitrite ion, can be oxidized at different electrode potentials and therefore detected and quantified by electroanalytical techniques. Nanometer-sized electrochemical probes are especially suitable for measuring ROS/RNS in single cells and cellular organelles. In this article, we survey recent advances in the localized measurements of ROS/RNS inside single cells and discuss several methodological issues, including optimization of nanoelectrode geometry, precise positioning of an electrochemical probe inside a cell, and interpretation of electroanalytical data.     

DNA analysis by application of Pt nanoparticle electrochemical amplification with single label response

This study demonstrates a highly sensitive sensing scheme for the detection of low concentrations of DNA, in principle down to the single biomolecule level. The previously developed technique of electrochemical current amplification for detection of single nanoparticle (NP) collisions at an ultramicroelectrode (UME) has been employed to determine DNA. The Pt NP/Au UME/hydrazine oxidation reaction was employed, and individual NP collision events were monitored. The Pt NP was modified with a 20-base oligonucleotide with a C6 spacer thiol (detection probe), and the Au UME was modified with a 16-base oligonucleotide with a C6 spacer thiol (capture probe). The presence of a target oligonucleotide (31 base) that hybridized with both capture and detection probes brought a Pt NP on the electrode surface, where the resulting electrochemical oxidation of hydrazine resulted in a current response.     

Evaluation of critical parameters for measurement of pH by flow injection analysis determination of pH in soil extracts

A systematic evaluation of the parameters affecting the measurement of pH in a flow injection system incorporating a tubular PVC-based pH-sensitive membrane electrode is presented. In order to obtain reliable results, it is necessary to have a dispersion coefficient D^max ? 1.008 at peak maximum and to adjust the buffering capacity of the carrier stream to be less than that of the sample solution. A suitable design of system to meet these criteria is discussed. The applicability of this approach is demonstrated for the determination of pH in a series of soil extracts; the results are compared with those obtained by conventional glass electrode measurements.