Chemical Analysis of Single Cells - Analytical ... - ACS Publications

Review pubs.acs.org/ac

Chemical Analysis of Single Cells Raphael̈ Trouillon,†,§ Melissa K. Passarelli,†,§ Jun Wang,†,§ Michael E. Kurczy,‡,§ and Andrew G. Ewing*,†,‡ †

University of Gothenburg, Department of Chemistry and Molecular Biology, 41296 Gothenburg, Sweden Chalmers University, Department of Chemistry and Biological Engineering, 41296 Gothenburg, Sweden

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CONTENTS

Single Cell Fluorescent Imaging Fluorescent Indicators for Atomic and Molecular Species Protein Probes Nanoparticle Probes Lifetime Fluorescence Super-Resolution Fluorescence Microscopy Electrochemistry Single Microelectrodes Exocytosis Oxidative Stress Arrays of Microelectrodes and Microwells Exocytosis Enzymatic Activity and Respiration Scanning Electrochemical Microscopy Electrochemical Detection in Lab-on-a-Chip Systems Mass Spectrometric Analysis of Single Cells Mass Spectrometric Analysis of Single Cells with Electrospray Ionization Nanoelectrospray Ionization (nano-ESI) Desorption Electrospray Ionization Mass Spectrometry (DESI) Capillary Electrophoresis Electrospray Ionization Mass Spectrometry (CE-ESI-MS) Laser Ablation Electrospray Ionization Mass Spectrometry (LA-ESI-MS) Mass Spectrometric Analysis of Single Cells Based on Laser Desorption Laser Desorption/Ionization Mass Spectrometry (LDI-MS) Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) Mass Spectrometric Analysis of Single Cells Based on Secondary Ion Mass Spectrometry (SIMS) Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) Dynamic SIMS (NanoSIMS) Mass Spectrometric Analysis of Single Cells Based on Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Laser Ablation Inductively Coupled Plasma Mass Spectroscopy (LA-ICP-MS) Mass Cytometry (Flow Cell-Nebulizer-ICP-MS) Lab-on-a-Chip for Single Cell Manipulation and Analysis Single Cell Trapping and Analysis on a Chip

Microwell or Microhurdle-Based Single Cell Trapping and Analysis Electrokinetic or Hydrodynamic Flow-Based Single Cell Trapping and Analysis Optical Tweezer-Based Single Cell Trapping and Analysis Droplet-Based Single Cell Trapping and Analysis Chemical Patterning-Based Single Cell Trapping and Analysis Other Techniques for Single Cell Analysis Author Information Corresponding Author Author Contributions Notes Biographies Acknowledgments References

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n this Review, we provide an overview of methods developed for chemical analysis of single cells over the last two years. Many biological systems contain an ensemble of cells with heterogeneous chemistry; therefore, it is important to analyze them on an individual basis in order to elucidate the role each cell plays in the function of these systems. In clinical diagnostics, the development of extremely sensitive measurements, down to single cells, may provide the best ability for diagnoses. Single cell analysis has, in fact, been present for quite some time. Investigators in life sciences consider the cell as the unit of life and so the pursuit to quantify, image, and modulate the cell has been ongoing for decades. We have made an attempt to be comprehensive, but this Review focuses on the last two years. There are many great works in related areas, and it was difficult to draw a specific line between quantitative chemical analysis of cells and qualitative analysis. For example, we have again not covered a great deal of the studies that are truly at the single cell level concerning biological applications of fluorescence imaging with agents like FURA II, FM1-43, and green fluorescent protein (GFP). There are simply too many applications as these tools become more highly used in biology. We have chosen to aim mostly at new techniques or variations of techniques in the areas of quantitative fluorescence, mass spectrometry and mass spectrometry imaging, and electrochemical analysis. We have chosen to not cover other

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Published: November 14, 2012 © 2012 American Chemical Society

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mitchodrial targeting ability of triphenylphosphonium salt; a disulfide group acts as the cysteine sensor, and 6-(benzo[d]thiazol-2′-yl)-2-(N,N-dimethylamino)naphthalene (BTDAN) provides the ratiometric signal. This label shows good specificity for mitochondrial thiols and low cytotoxicity. Researchers at Hunan University have developed a ratiometric dye for the detection of cysteine in living cells.13 In addition to the advantages of ratiometric labeling, this label is highly specific for cysteine. This is evidenced by their results from challenging the indicator with similar thiols. The ratio variation registered for cysteine (300 equiv.) was a 115-fold increase while homocysteine (300 equiv.) and glutathione (300 equiv.), common interfering agents, produced 4.6- and 1.6-fold increases, respectively. The authors suggest that the reason for the selectivity is the kinetic considerations of a ring closing reaction, which they propose as the sensing mechanism. On the basis of Baldwin’s rules, the ring closure between the dye molecule and cysteine is more favorable than either homocysteine or glutathione. The authors admit, however, that the mechanism still needs further investigation. Cardoso et al. have used synchrotron radiation X-ray fluorescence (SR-XRF) to map the elemental distribution of embroid bodies (EB) composed of neural-induced and naive pluripotent stem cells.14 In addition to elemental identification, the advantages of SR-XRF include that analysis can be carried out at ambient pressure and the spatial resolution is on the micrometer scale. Although the exact mechanism remains ambiguous, this work demonstrates that EBs composed of neural-induced pluripotent stem cells from both humans and mice display elemental polarization over the cellular aggregate. Protein Probes. A fluorescence method to detect microbial function at the single cell level has been developed by researchers at the University of Washington called respiration response imaging (RRI).15 The motivation behind this work is the need to isolate bacteria cells from diverse populations based on metabolism. This imaging technique relies on RedoxSensor green (RSG), as an indicator of bacterial reductase activity in conjunction with substrate stimulation. Bacteria that respire using a given substrate will increase respiration in the presence of that substrate. The increase in reductase activity will then be reflected in the RSG signal. The authors show that this straightforward method is effective for identifying metabolically viable cells. Hirata and co-workers have used two-photon fluorescence and confocal microscopy to track the invasion of individual glioblastoma cells that were implanted into the brains of rats.16 These tumor cells were transfected with fluorescence resonance energy transfer (FRET)-based enzyme activity probes that showed clear differences in the activities of Rho-family GTPases in cells associated with blood vessels and cells that were not (Figure 1A). The sensor is based on a GFP-YFP FRET pair biosensor which is activated by protein folding that follows GTP binding.17 Due to some instability in transfection, which results from using both a donor and acceptor derived from the A. victoria jelly, the researchers chose to substitute the donor for teal fluorescent protein and the acceptor for a YFP variant. This modification allowed for the successful transfection of the sensor into the glioblastoma cells. In addition to the advancement of the label strategy, this work also highlights the heterogeneity of tumor forming cancer cells. Motivated by the inherent difficulties of investigations of protein−DNA interactions in vitro, Sharavan et al. have presented an in vivo FRET-based bioprobe called FREP.18 In vivo FRET assays, however, suffer from a relativity high instance

areas that do not have as many new developments and are not as central as these three mainstream areas of analytical chemistry. In the area of microfluidic devices, there have been so many new developments in the last two years that we made a special section for this and this includes a lot of separation chemistry as well. There is clearly a great deal of cross-fertilization in an area such as chemical analysis of single cells where many methods are hybrid methods and there is a great deal of collaboration with biologists. In fact, as the technology advances, it is assumed that its use in life science will expand to be nearly routine. We sincerely hope we have paid attention to all the work done in this field and would be happy to hear from anyone whose work we omitted.

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SINGLE CELL FLUORESCENT IMAGING Fluorescence microscopy has been ubiquitous in biological laboratories for decades and plays a prominent role in the chemical analysis of single cells. Fluorescence imaging offers high contrast chemical specific images. If the target is not intrinsically fluorescent, it must be labeled either chemically or genetically. The design of unique labels has been an active area of research over the past two years as evidenced by the reviews that have been published in this short time.1−4 In addition, work in subdiffraction limit imaging has been reviewed extensively,5,6 and reviews on general fluorescence imaging topics7−9 have also been recently published. Our goal here is to give some illustrative examples of fluorescence microscopy as it has been applied to single cell chemical analysis over the past two years Fluorescent Indicators for Atomic and Molecular Species. A chemosensor designed for Fe3+ detection based on the properties of rhodamine has been synthesized and characterized by Chereddy et al.10 By altering the number and the nature of the coordination sites, this work culminated in a Bis-rhodamine indicator which exists in a nonfluorescent spirocyclic form until it complexes with Fe3+. The specificity for Fe3+ was demonstrated by challenging the indicator against Cu2+ and by measuring Fe3+ in the presence of 17 other cations. The specificity was achieved by tailoring the length of the linker between the two rhodamine molecules, thus defining the size of the chelating cavity of the probe. This fluorescent probe was also utilized to detect Fe3+ in living fibroblast cells. A fluorescent indicator for the detection of Al3+ in living cells has been synthesized and characterized by chemists and microbiologists from the University of Burdwan.11 Emission spectra of the molecule, HL (2-((naphthalen-6-yl)methylthio)ethanol), show a maximum at 449 nm with a low quantum yield. The emission is credited to an exciplex that forms between the two lone electrons on the sulfur atom in the methylthioethanol and the excited naphthalene. The low quantum yield of the signal is attributed to quenching done by the alcohol. The molecule senses Al3+ when it complexes to the alcohol, thus disrupting quenching and increasing signal. The indicator was tested against 11 common cations and was shown to be specific for Al3+; however, signal was reduced when Al3+ was measured in the presence of Cu2+ and Fe3+. The probe was used to measure aluminum-treated yeast cells showing the permeability of the ligand and indicating biocompatibility. A new two-photon probe for biological detection of mitochondrial thiols has been reported by Lim et al.12 Twophoton excitation affords advantages such as deeper penetration depth and localized excitation. In addition, this label is also ratiometric, which serves to normalize environmental effects making quantification less ambiguous. The label utilizes the 523

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ratiometric probe for detection of hydroxyl radicals using a lactide-co-glycolide (PLGA) nanoparticle platform formed by use of micelles containing lysine-coumarin 3-carboxylic acid conjugates (lysine-C3C).23 Coumarin acts as the reporter dye for hydroxyl radicals while neutral red, which is encapsulated in the nanoparticle, acts as a reference. The probe showed specificity for OH• when compared to several other reactive oxygen species. The biocompatibility of this coumarin−neutral red (CONER) nanoprobe was utilized to detect OH• in viable MCF-7 cell in response to hydrogen peroxide induced oxidative stress. Using semiconductor polymer-based nanoparticles (Pdots) as a FRET donor and fluorescein as an acceptor (Figure 1B), Chan et al. have designed a ratiometric pH sensor.24 Most of the absorption of the fluorescein molecules is due to FRET and the efficiency of the transfer from the Pdot is quite stable over pH 5− 8. The pH sensitivity of the probe is based on the increased quantum efficiency of the deprotonated forms of fluorescein. Following endocytosis of the Pdots, the pH of acidic endocytotic organelles were measured with values in good agreement with literature values. Pdots have several advantages; they are extremely bright, and because no dye is encapsulated in the particle, there is no danger of leaching. A potentially useful fluorescent nanoparticle, which utilizes upconversion luminescence (UCL), has been presented by Liu et al. from Fudan University.25 Low-energy photons are converted to high-energy photons by UCL processes without the need for pulsed excitation. This work exploits triplet−triplet annihilation (TTA) in which energy transfer between a sensitizer and an annihilator (both molecules are in a triplet state) results in a blueshifted emission from the annihilator molecule. The sensitizer and annihilator molecules were incorporated into a silica nanoparticle platform for this transducer and showed quantum yields as high as 4.5% in water. The probe was also transported into and detected in living HeLa cells. Lifetime Fluorescence. Fluorescence lifetime image microscopy (FLIM) using the phasor approach has recently been used to map free and bound nicotinamide adenine dinucleotide (NADH) in differentiated and undifferentiated myoblast cells. NADH is a metabolic coenzmye that is intrinsically fluorescent. Additionally, the fluorescence lifetime of NADH is reduced when it is bound to a substrate, allowing the determination of the bound and unbound state. Typical analysis of FLIM images requires each pixel to be fitted to a signal decay. In cell experiments, low signals make fitting multiple decays problematic. The phasor approach transforms the time delays in each pixel to a phase vector or phasor. The phasor for each pixel is plotted, and molecular species can be identified from their position on the plot. Wright et al. found using this treatment on FLIM images that differentiated cell differed from undifferentiated cells in that concentration of free NADH was reduced in the nucleus (Figure 1C), indicating the expected increase in nuclear activity.26 Localized measurements of viscosity at the nucleus, the cytosol, and the plasma membrane in living HEK 293 T cells have been reported using FLIM and a hybrid genetic-chemical rotor tag.27 To make localized measurements, structure-specific proteins were first genetically labeled with E. coli dihydrofolate reductase (eDHFR). The eDHFR has a strong noncovalent interaction with the drug trimethoprim (TMP), and this interaction can be exploited to tag the eDHFR labeled protein with a TMP−dye conjugate. The viscosity indicator used was the cyamine dye CY3. In addition to radiative decay, the excited CY3 molecule can also return to the ground state via a torsion motion.

Figure 1. Fluorescence for single cell analysis. (A) Intensity modulated FRET images showing the activity of Cdc42 and RhoA in glioblastoma cells, which have been inoculated into rat brains. The arrows indicate cells advancing toward blood vessels, and the arrow points indicate cells growing in the parenchyma. Adapted with permission from ref 16. Copyright 2012 The Company of Biologists Ltd. (B) A cartoon representation of a Pdot pH sensor shown with brightfield and fluorescent images of an individual HeLa which has internalized the sensors. Adapted from ref 24. Copyright 2011 American Chemical Society. (C) Confocal and FLIM images of undifferentiated and differentiated myoblast cells. The FLIM images, processed using the phasor method, show the spatial distribution of bound (red) and unbound (blue) NADH. Adapted with permission from ref 26. Copyright 2012 Elsevier Inc. (D) An x−y projection of a 3D STORM image of Alexa647 labeled clathrin coated pits (magenta) loaded with Alexa568 labeled transferin (green). The inset shows an expanded view of the indicated clathrin coated pit in the x−y and x−z planes. Adapted with permission from ref 28. Copyright 2011 Nature Publishing Group.

of false positives and false negatives, in part because the acceptor−donor distance is critical. The unique solution based on works by Fields and Song19 and Waldo et al.,20 described in this article, utilizes an E. coli cell containing expression vectors for a transcription factor and the reporter, which can be designed for optimal fluoresce transfer. The expression vector for the reporter contains genes for the FRET pair (CFP and YFP) separated by the target DNA sequence. If the transcription factor interacts with the reporter vector, expression of the FRET pair is impeded yielding a ratio of CFP to CFP−YFP reporter molecules. Likewise, no interaction results in complete FRET pair expression. The probe is not a true sensor as the signal is not generated by the actual binding event, but its indirect nature is key to obtaining reliable whole cell fluorescence signal. Nanoparticle Probes. Si et al. have devised a free calcium sensor using a PEBBLE (photonic explorers for bioanalysis with biologically localized embedding) motif.21 The sensor uses a rhodamine-based Ca2+ indicator embedded in polyacrylamide nanoparticles. PEBBLE methods decrease several disadvantages implicit in using free dyes such as nonspecific binding and cytotoxicity. The authors also exploit the ratiometric capability of the PEBBLE platform by conjugating a reference dye (Hylite 647) to the particle. The sensor was successfully employed to measure intracellular Ca2+ in 9L gliosarcoma cells down to nanomolar concentrations. Utilizing a similar strategy presented by King et al.,22 the Warner group at Louisiana State University reported a 524

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Figure 2. Electrochemical analysis of single cells. (A) Microelectrode investigation of exocytosis at PC12 cells has revealed three different spike populations (top) which may arise from different vesicle/membrane interactions (bottom). Adapted with permission from ref 41. Copyright 2011 Elsevier. (B) Simultaneous TIRF imaging (top) and amperometric recording (bottom) of a single amperometric event at a BON enterochromaffin cell. Adapted with permission from ref 55. Copyright 2011 John Wiley & Sons, Inc. (C) Electrochemical imaging of the spatial heterogeneity of exocytosis at the surface of a single PC12 cells with a ring microelectrode array (inset: electron micrograph and electrogenerated chemiluminescence imaging of an 8electrode array; the bars indicate 5 μm; Adapted from ref 57. Copyright 2012 American Chemical Society.). (D) SECM imaging of hippocampal neurons obtained from SECM compared to a fluorescence picture, and (E) SECM imaging of differentiated PC12 cells coupled to amperometric detection of exocytosis. Adapted with permission from ref 69. Copyright 2012 National Academy of Sciences.

incorporating a weak lens in the emission pathway, an asymmetry in the point-spread function was induced and the degree of the asymmetry indicated the z-position of the molecule. C. cresentus expressing FtsZ proteins labeled with the photoswitchable fluorophore Dendra2 were imaged both before and during division. Before division, the Z-ring appeared as a sphere with a radius of approximately 150 nm, and during division, the Z-ring was imaged in an expanded conformation as appearing as a ≈ 650 nm disk with ≈150 nm aperture. This work demonstrates the possibly of using super-resolution 3D fluorescence imaging to look at dynamic subcellular structures.

This rotation is impeded in a viscous environment, and therefore, the lifetime of fluorescence can be used to measure viscosity. The ability to make localized measurements is complemented by relatively rapid acquisition time when compared to FCS or FRAP experiments. Super-Resolution Fluorescence Microscopy. Developments in super-resolution fluorescence microscopy have produced spectacular 3-dimensional images of subcellular structures. Using stochastic optical reconstruction microscopy (STORM) and employing astigmatism, researchers at Harvard University have produced 3D images (Figure 1D) of clathrin coated pits loaded with transferin in live epithelial cells.28 STORM utilizes photoswitchable fluorophores densely distributed within a cellular structure such as RecA filaments.29 The fluorophores are initially switched off globally with a strong laser pulse; next, a second pulse of an activating wavelength switches a fraction of the molecules into a fluorescent state. The diffuse distribution of the active fluorophores allows individual molecules to be localized and yields high spatial accuracy. After a number of iterations, a subdiffraction limit image of the whole structure emerges. The brightness and the switching kinetics of the labels are critical to minimize motion blurring. This paper evaluated several fluorophores and found that Alexa647 performed very well but pointed out the need to develop faster labels for better characterization of ultrastructural dynamics. Similar work using astigmatism and super-resolution fluorescence imaging was carried out in the Moerner lab to create 3D images of the Z-ring in living Caulobacter crescentus cells.30 The Z-ring is the result of the polymerization of the bacterial tubulin like protein FtsZ and is important for cell division. By

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ELECTROCHEMISTRY Electrochemistry is a powerful alternative to most of the methods used in biology. Electrochemical sensors are cheap and easy to manufacture with benchtop techniques but also highly suitable for miniaturization and microfabrication. Furthermore, this technology allows for the real-time, quantitative analysis of species released in minute amounts in biological systems. This capability has in particular established the strong efficacy of electrochemical methods in neuroscience, and electrodes have been routinely used to detect neurotransmitters since the seminal work of Ralph N. Adams in the 1970s.31 The biological applications of electrochemical methods are nevertheless not limited to this purpose, and several reviews focusing totally or partially on electrochemical detection in single cells have been published over the last 2 years.32−37 Single Microelectrodes. Because of their small size, microelectrodes are ideally suited for the chemical study of 525

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experiments provided similar information, but with improved time resolution. The actin cytoskeleton has also been found to regulate granule secretion in platelets.44 Actin depolymerization, induced by exposure to latrunculin A and cytochalasin D, increased the rate of serotonin release, and cytochalasin D also enhanced the serotonin quantal secretion rate. These results suggest mechanical control of the cytoskeleton over exocytosis. The impact of cell exposure to drugs or nanoparticles on their exocytotic ability has also been studied. Hondebrink et al. have investigated the effect of 3,4-methylenedioxymethampetamine (MDMA, ecstasy) as well as its metabolite 3,4-methylenedioxyamphetamine on intracellular Ca2+ levels and levels of released dopamine in PC12 cells.45 A 15 min incubation with a highly concentrated (1 mM) solution of these drugs was found to inhibit depolarization-induced exocytosis and to increase intracellular [Ca2+], without affecting the vesicular content. This report indicates that MDMA can inhibit exocytosis through indirect inhibition of Ca2+ influx, possibly via the activation of the protein kinase C. The effect of morphine on the physiology of sickle cell disease has also been studied, as the serious pains induced by this condition motivate the use of opioids.46 By monitoring serotonin exocytosis at mast cells obtained from transgenic mouse models expressing sickle cell human hemoglobin or normal human hemoglobin, it was found that the sickle hemoglobin decreased the exoytotic frequency, as well as the amount of serotonin released per event. The chronic inflammation state present in the sickle cell disease models appears to modify both the serotonin loading of the granules as well as the membrane dynamics controlling the release of the vesicular content. Furthermore, incubation with morphine was found to reverse this phenomenon and to compensate for the lower exocytotic efficiency induced by this pathology. The increasing presence of nanoparticles into manufactured goods has motivated the study of the effect of noble metal nanoparticles on exocytosis. It was found that small (15−20 nm) Ag nanoparticles are more likely to be translocated into mouse chromaffin cells and alter the exocytosis by increasing the release kinetics.47 Similarly, poly(ethylene glycol)-coated Au nanoparticles can also significantly modify the amount released. The effect of the ζ-potential of Au and Ag nanoparticles, controlled during the synthesis by surfactant exchange, on exocytosis was studied in mouse peritoneal mast cells.48 Positively and negatively charged Au and Ag nanoparticles were prepared. Positively charged Au nanoparticles and negatively charged Ag nanoparticles were found to decrease the amount of serotonin released during exocytosis. All the nanoparticles, apart from the negative Ag ones, were also found to decrease the release frequency. These results indicate that the interaction between the cellular machinery and the nanoparticles can be very complex and that both intragranular and extragranular mechanisms are involved. Oxidative Stress. Electrochemical methods are an increasingly popular method to study oxidative stress. Most of the reactive oxygen and nitrogen species released during this stress can be detected at the surface of a platinized carbon fiber microelectrode. This technique has been used to investigate the effect of an artificial superoxide dismutase (SOD mimics) on the different fluxes of species released during oxidative stress from a single interferon-γ stimulated macrophage.49 It was found that this SOD mimics did not change the initial amounts of nitric oxide and hydrogen peroxide released by the cells but significantly decreased the formation of the highly cytotoxic peroxynitrite. This result supports the development of SOD

single cells. Work to measure exocytosis and oxidative stress in two general categories is presented here. Exocytosis. Microelectrodes are powerful tools for the study of exocytosis. This phenomenon is the basis of neuronal communication, and electrochemical methods offer the ability to detect and quantify, in real time, individual exocytotic events. Because of the limited magnitude of the recorded currents, limiting the noise is of paramount importance to achieve reliable measurements. It has been recently shown that the theoretical noise limit in amperometric detection of exocytosis is thermal in origin, generated at the electrode/electrolyte interface, and proportional to the real part of the electrode admittance.38 Interestingly, this article suggests that cell adhesion does not significantly increase the noise and that printed microdevices (gold, indium tin oxide (ITO), or diamond like carbon) might show a lower noise level than carbon fiber microelectrodes of the same surface area, in particular by offering a better control over the fabrication process. Furthermore, the analysis of the recorded current traces is critical for understanding the mechanisms of exocytosis. Watson et al. have recently proposed a new classification of exocytotic spikes.39 Some of the characteristics of these peaks were analyzed and were found to be different for each class, showing that these different types of spike may correspond to different biological functions. Adams et al. have used a gold-nanoparticle-network microelectrode to detect dopamine released from PC12 cells.40 The surfaces of carbon fiber microelectrodes were modified with a sol−gel silicate network seeded with Au nanoparticles. The exocytotic peaks recorded with this device were found to show faster kinetics, when compared to the ones obtained with unmodified microelectrodes. Furthermore, these new surfaces showed a higher resistance to fouling. Microelectrodes have been used in several studies to investigate the fundamental mechanisms of exocytosis in neuroendocrine cells or platelets. Exocytosis was stimulated in PC12 cells, and a precise analysis and processing of the peak parameters obtained, involving a cluster analysis and Gaussian fitting, revealed the presence of 3 subpopulations.41 These clusters were attributed to three different exocytotic modes (Figure 2A): apparent full distention (accounting for 70% of the released catecholamine), where the vesicle is fully integrated into the membrane, and the transient fusion events kiss-and-run and kiss-and-stay (respectively, accounting for 20% and 10% of the released catecholamine). The exocytotic release from small dense core vesicles of the carotid glomus chemosensory cells was triggered with whole cell dialysis, thus increasing the intracellular [Ca2+] and initiating vesicle fusion, and this was compared to events recorded at chromaffin cells.42 It was found that the prevalence of flickering events was 9-fold in the carotid glomus cells, and for a comparable amount of catecholamines released, the peak kinetics were faster than for the chromaffin cells. These results suggest a higher matrix release efficiency in the carotid glomus cells than in chromaffin cells, as well as the facilitated triggering of kiss-and-run exocytosis after a limited elevation of the intracellular [Ca2+] in these cells. Serotonin release from platelet granules was observed electrochemically upon thrombin stimulation.43 Fast scan cyclic voltammetry was used at the single cell level to clearly identify the released chemical as serotonin. Amperometry was then preferred for the dynamic study of exocytosis, owing to its improved temporal resolution. The results obtained from these single cell experiments were compared to the dynamics of serotonin release observed in platelet suspensions, showing that single cell 526

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insulator have been tested.56 It has been found that a spiral-like aperture offers the best trade-off between noise and cell deposition. The exocytotic spikes obtained from the base of the cell with the ITO electrodes have been compared to those obtained from the apex of the cell and are found to be significantly different. Lin et al. described the fabrication of a microelectrode array from small carbon rings, featuring from 8 to 15 electrodes.57 These electrodes were produced from acetylene pyrolysis, and the whole fabrication process can be performed with benchtop equipment. Electrochemical imaging of the surface of a single PC12 cell was performed, with 8 electrodes, showing the possibility to identify sites of higher exocytotic activity (Figure 2C). Kim et al. have recently described the design of a CMOS-based electrode array.58 This chip features 100 Pt electrodes and was designed by lift-off photolithography. Simultaneous recordings of exocytotic events from several individual chromaffin cells were performed, thus demonstrating the efficacy of this chip as a highthroughput, massively parallel platform for the study of secretion from populations of cells. Enzymatic Activity and Respiration. Matsue et al. reported a microwell-based microfabricated device for the electrochemical detection of secreted alkaline phosphatase (SEAP) from single transfected HeLa cells.59 This device consists of 256 microwells on top of the corresponding individually addressable ring−disk electrodes that were modified with anti-SEAP. SEAP from pSEAP2 transfected HeLa cells was successfully captured and analyzed with this chip. An array of six microwells was used to study the photosynthetic activity of a single cyanobacterium.60 The microwell containing the cell was closed with a urethane cover, thus allowing accumulation of the released oxygen. The levels of oxygen were determined with two Pt electrodes printed at the bottom of the microwell, showing that a single cyanobacterium releases an average of 10−17 mole of oxygen per second. Similarly, the same group used an array of Pt microelectrodes printed at the bottom of a microwell to observe the respiration of a single mouse embryo.61 Embryos at different development stages were studied (two cells, morula and blastocyst), and the rate of oxygen consumption was found to increase with the development of the embryo. Electrochemical desorption of a self-assembled monolayer of alkanethiol was used as a dynamic method for cell manipulation.62 An array of ITO microelectrode was coated with a monolayer of alkanethiol, providing a substrate for fibroblast adhesion. Electrochemical desorption of the alkanethiol could be triggered at each electrode, thus selectively detaching the cells adhering to its surface. Scanning Electrochemical Microscopy. In SECM, an electrode is scanned over the sample, thus allowing simultaneous topological and chemical mappings of the surface.63 This method has been applied to the study of cell metabolism, respiration, and the measurement of the permeability of a cell monolayer to chemicals. Bergner et al. used platinum micro- and nanoelectrodes to image fixed kidney epithelial cell monolayers.64 The system was used in constant height mode, with ferrocene−methanol or Ru(NH3)63+ as redox reporters. The topology of the monolayer can be resolved with 1.5 μm and 550 nm electrodes, and single cells can be observed. The same technique was used to observe the passive diffusion of these redox mediators across a monolayer of kidney epithelial cells grown on a porous membrane.65 This

mimics as therapeutic agents in pathologies induced by high levels of oxidative stress. Similarly, these platinized microsensors have been miniaturized to the nanoscale to achieve intracellular detection of oxidative stress.50 These nanosensors were introduced into a macrophage, thus rupturing and depolarizing the membrane and triggering an oxidative response. This response was monitored both intracellularly and extracellularly, showing that the amount of reactive oxygen and nitrogen species is much more important outside of the cell. This indicates that the cell has developed mechanisms allowing for the fast inactivation of these species, thus preventing self-damage. Zheng et al. reported the development of a hybrid optical/ electrochemical nanoprobe, prepared from a pulled optical fiber sputter coated with a thin layer of Au, modified with Prussian blue.51 The simultaneous detection of hydrogen peroxide at the Prussian blue modified Au ring electrode and the monitoring of the redox state, or the level of reduced thiols, with fluorescent dyes using the optical core can be carried out with this device. These highly spatially resolved measurements in single cells have been carried out, indicating that the levels of oxidative stress in phorbol 12-myristate 13-acetate stimulated mammary epithelial cells and human breast cancer cells are correlated with the level of tumor malignancy and that the center of the cell is more active than its periphery. Takami et al. built an ion selective nanoprobe for the intracellular measurement of K+ levels.52 The small size (diameter ∼100 nm) allows insertion of the device into a single HeLa cell, and a gradient of [K+] of 100 mM was measured across the membrane. The time response of the device was ∼0.1 s. Arrays of Microelectrodes and Microwells. The design of microelectrode arrays, allowing for massively parallel analysis of single cells, is an increasing trend in the electrochemical study of cellular release. We report here work done to study exocytosis and enzymatic activity in cells. Exocytosis. Several recent reports were focused at the fabrication and use of microelectrode arrays specifically designed to study exocytosis. The possibility of combining electrochemical detection with microscopy has spurred the use of transparent substrates. Zhao et al. reported a microwell-based ITO microelectrode (50 μm diameter) for the study of exocytosis at single SH-SY5Y cells.53 In this study, they observed that cells kept in long-term culture have higher release frequency of norepinephrine than the spherical cells recently seeded. Similarly, a device featuring several microwells, with embedded ITO electrodes, has been used to study exocytosis at bovine chromaffin cells.54 The SU-8 photoresist insulating the electrodes was coated with poly(ethylene glycol) to prevent cell adhesion. This ensured a seeding efficiency of ∼75% in the microwells. Good detection of amperometric spikes was obtained upon K+ stimulation. The transparency of ITO was used to simultaneously perform amperometric detection and total internal reflection fluorescence microscopy (TIRFM) during exocytosis.55 This device featured four ITO band electrodes, patterned on a glass substrate. The surface was coated with collagen to promote cell adhesion. BC21 cells, stable clones of enterochromaffin BON cells expressing a fluorescent marker of secretory granule, were grown on the surface of the chip, and cells were individually stimulated with an ionomycin solution. The dynamics of exocytosis could be recorded, for a single cell, electrochemically at the ITO electrodes as well as optically using TIRFM, thus enabling correlation of the opening of the fusion pore with the flux of neurotransmitter (Figure 2B). Different geometries for the SU-8 527

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Figure 3. The trade-off between chemical information and spatial resolution is illustrated for imaging methodologies, (A) MALDI (Adapted from ref 107. Copyright 2012 American Chemical Society.), (B) TOF-SIMS (Adapted from refs 113 and 115. Copyright 2012 and 2011, respectively, American Chemical Society.), and (C) nanoSIMS (Adapted with permission from ref 123. Copyright 2012 Nature Publishing Group.). Mass spectrometric profiles of single cells were obtained with (D) LDI on special nanofabricated surfaces (Adapted from ref 97. Copyright 2012 American Chemical Society.), (E) nano-ESI via live video mass spectrometry (Adapted with permission from ref 85. Copyright 2012 the Japan Society for Analytical Chemistry.), and (F) mass cytometry ICP-MS (Adapted with permission from ref 141. Copyright 2011 American Association for the Advancement of Science.).

in microwells covered with a slide coated with anti-SEAP. The amount of immobilized SEAP was then detected in substrate generation/tip collection mode via the enzymatic hydrolysis of paminophenyl phosphate at SEAP. Alternating current SECM is a powerful technique for measurement of local impedance at the surface of substrates. This method was recently combined with fast scan cyclic voltammetry to achieve simultaneous mapping of the topology (with the alternating current wave) and oxygen consumption (with the voltammetry) at PC12 cells.68 In particular, it was demonstrated that the presence of the fast frequency component, of low amplitude, on the potential waveform does not jeopardize the quality of the voltammetric measurements. Pyrolytic carbon nanoelectrodes (6.5−100 nm radius), built by the pyrolysis of butane into a quartz capillary, have been used to image different cell types (boar sperm cell, differentiated PC12

imaging technique has been used to show that the hydrophilic mediator clearly diffuses through the layer using a paracellular pathway, whereas the lipophilic mediators are transported transcellularly. The influence of the size of the electrode during the imaging in substrate generation/tip collection mode at a single Saccharomyces cerevisiae yeast cell has been investigated.66 Lipophilic menadione and hydrophilic ferricyanide were then used as coupled mediators to observe the activity of the intracellular NAD(P)H-oxidizing enzymes. Electrode diameters down to 199 nm were used to image the cell, and no disturbances in the concentration profile of the mediator were observed. However, downscaling the electrode did not significantly improve the resolution of the system because of the natural diffusion of the mediators. The same group described a 2-step method to detect SEAP in transfected HeLa cells.67 First, the cells were incubated 528

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Table 1. List of All the Techniques Reviewed in the Mass Spectrometry Section of This Worka MS method nano-ESI

general sample description

LC-ESI LAESI-MS

plant plant mammalian mammalian mammalian mammalian mammalian gastropod/mollusk algae plant plant

LAESI-MS LDI

mammalian plant algae

DESI CE-ESI

LDI MALDI

SIMS (TOF-SIMS)

a

Pelargonium zonale Tulipa gesneriana (tulip) Homo sapiens (human) Mus musculus (mouse) Rattus norvegicus (rat) Mus musculus (mouse) Mus musculus (mouse) Aplysia californica (sea slug) Chara australis Arabidopsis thaliana Citrus aurantium Allium cepa (onion) Citrus aurantium (Bitter orange) Homo sapiens Allium cepa (onion) Chlamydomonas reinhardtii

HepG2 C2C12 PC12 L929

mammalian reptile gastropod/mollusk gastropod/mollusk mammalian mammalian mammalian protozoan mammalian mammalian

Homo sapiens (human) Rattus norvegicus (rat) Tetrahymena pyriformis Homo sapiens (human) Mus musculus (mouse)

HepG2 PC12

mammalian mammalian

Homo sapiens (human) Homo sapiens (human)

HeLa HeLa

mammalian mammalian

Homo sapiens (human) Rattus norvegicus (rat) Mus musculus (mouse) Rattus norvegicus (rat)

HeLa-M

mammalian cyanobacteria

phytoplankton plant plant mammalian

LA-ICP-MS mass cytometry

cell line

Euglena gracilis Saccharomyces cerevisiae Saccharomyces cerevisiae Periplaneta americana (American cockroach) Homo sapiens Axolotl Mexicanum (salamander) Aplysia californica (sea slug) Aplysia californica (sea slug) Homo sapiens (human)

yeast yeast insect

mammalian SIMS (nanoSIMS)

detailed sample description (common name)

mammalian Archaea and bacteria mammalian mammalian mammalian bacteria mammalian mammalian

HeLa

HeLa NIH/3T3

NIH/3T3 NRK (clone 52E) MDCK II

Mus musculus (mouse) UCYN-A Richelia intracellularis Calothrix rhizosoleniaes Phaeocystis globosa Alyssum lesbiacum Hedera helix Homo sapiens (human) Rattus norvegicus (rat) Homo sapiens (human)

H295R GH3.TRE MDA-MB-231

Mus musculus (mouse) human kidney cells Homo sapiens (human)

Clone 15 cell line HepG2 GFAJ 3T3

Mus musculus (mouse) Homo sapiens (human)

analyte

reference

imaging/ profiling

metabolites metabolites drug metabolites lipids

82 83 85 86

P P P P

lipids metabolites/amino acids metabolites proteins metabolites

87 89, 90 91 92 93

P P P

metabolites metobolites, lipids (βcarotene)

94 96

I P

metobolites metobolites neuropeptides

97 100 101, 102

P P P

proteins lipids neuropeptide peptides metabolites and intact lipids elements lipid fragments lipid fragments lipid fragment fragments and small molecules BrdU lipid fragments and small molecules lipid fragments lipid fragments

103 104 105 106 107

P P P P I

109 110 111 112 113

I I I I I

114 115

I I

116 117

I I

amino acids elements elements elements elements

118 119 123 124 125

elements elements elements elements

126 127 128 129

I I I I I I I I I I

elements elements elements

130 131 132 133 134 135 138 140

I I I I I I I P

elements elements elements elements

P

The sample information, type of analyte detected, and type of analysis (i.e., imaging (I) or profiling (P)) are detailed and listed. 529

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Review

MASS SPECTROMETRIC ANALYSIS OF SINGLE CELLS Mass spectrometry (MS) is a well-established detection scheme for analyzing biological materials at the single cell level. The high sensitivity, broad detection range, and high chemical specificity of mass spectrometry make it well-suited for single cell analyses. A number of ablation and ionization methods can be coupled to a mass spectrometer, including electrospray ionization (ESI), laser ablation/laser desorption ionization (LA/LDI), secondary ionization (SIMS), and inductively coupled plasma (ICP). With these ionization methods, a wide range of chemicals are detectable, including proteins, peptides, lipids, metabolites,74,75 and elements. Reviews published concerning the mass spectrometric analyses of single cells discuss its main challenges, recent technological developments, and applications.76−80 Here, we briefly review the methods which have been utilized in research articles published in the past two years. The subsections are organized based on the desorption/ionization mechanism for the approach. An emphasis was placed on the growing field of imaging mass spectrometry (IMS) and its impact on single cell analyses. The majority of the mass spectrometric single cell analyses techniques fall under two categories: imaging and profiling (Figure 3). In the profiling modality, only one mass spectrum is obtained per cell. As a general rule, ESI, LDI, and ICP ionization methods are typically employed to obtain single cell profiles; however, there are some exceptions to this rule (see Table 1). These ionization methods are limited by their effective working spatial resolution, which is greater or only slightly smaller than the size of a single cell. Spatial information can still be obtained with these techniques by profiling adjacent cells; this method is known as cell-by-cell imaging. In the imaging modality, multiple mass spectra are obtained for a given cell. Imaging provides information on the spatial distribution of chemicals within or across the surface of a single cell. This requires an ionization probe that is significantly smaller than the size of an individual cell. Techniques employing focused ion beams (e.g., SIMS) have been used to image single cells for over two decades, but the earliest work was mostly elemental determination. Recently, several technological and methodological advances, oversampling, improved laser optics (i.e., smart beam technology), and improved matrix application techniques, have reduced the spatial resolution of laser-based ionization approaches. Imaging mass spectrometry is an exciting new bioanalytic platform.81 However, as shown in Figure 3A,B,C, there is an inherent trade-off between molecular information and lateral resolution observed in IMS. Mass Spectrometric Analysis of Single Cells with Electrospray Ionization. Electrospray ionization continues to be a dominant ionization approach for small volume analyses, and it's new approaches and results are summarized here. Nanoelectrospray Ionization (nano-ESI). Nanoelectrospray tips have been coupled with a variety of mass spectrometers for single cell analysis. The nanoelectrospay tips are commercially available; however, the protocol for interfacing sprayers with commercial mass spectrometers has not yet been standardized. Tejedor et al. have used the technique to compare the metabolomics profiling obtained between individual cells in the same plant.82 They removed the contents of cells in the stem, leaf, and petal of a Pelargonium zonale and compared their metabolic profiles using multivariable analyses (MVA).

cells, A431 cells, cardiac myocytes, and hair cells) with a voltage switching mode SECM technique (Figure 2D).69 The topology of the sample was first obtained, using feedback from the hindered diffusion during reduction of Ru(NH3)63+. When the sensor approached the surface of the cell, the potential was switched to carry out detection of the analyte of interest. For instance, the distribution of epithelial growth factor receptor, tagged with an alkaline phosphatase-tagged antibody, was mapped via the hydrolysis of p-aminophenyl phosphate. Similarly, exocytosis could be observed at single PC12 cells (Figure 2E). Finally, a high correlation was found between the topography obtained electrochemically and fluorescence imaging. Zhang et al. used time-lapse SECM to observe oxidative stress at single bladder tumor cells and the effect of cisplatin on this event.70 Obtaining successive constant height SECM images and maintaining the electrode at −0.8 v vs Ag|AgCl, they were able to examine the x−y profile of a current arising from the reduction of the produced hydrogen peroxide (indicating the level of oxidative stress) and the feedback current from the reduction of dissolved oxygen (describing the topology). These pictures show that the cell switches from an active oxidative state to a resting state and back to activation again, over a period of ∼6 h. Induction of apoptosis with cisplatin enhanced the frequency of this cycle and increased the level of oxidative stress. Electrochemical Detection in Lab-on-a-Chip Systems. Electrochemistry provides an important detection mode for analysis of single cell chemistry with lab-on-a-chip devices. This is because it offers high sensitivity and selectivity for intracellular content analytes. Han et al. reported an integrated microfluidic system that performs a count of white blood cells using electrochemical impedance analysis.71 This approach contains on-chip hydrodynamic red blood cell lysis and subsequent microfluidic impedance cytometry. Counting of three leukocyte subpopulations was obtained from the microfluidic impedance system against control data acquired using the central lab blood analysis system. The correlation results between this impedance cytometry data, and control data showed this chip can be used for samples with low subpopulation counts. Wu et al. reported a microfluidic device for the detection of neurotransmitters released from single PC12 cells.72 This microchip combined collagen/carbon nanotube modified platinum electrodes for single cell trapping and a silicon dioxide nanochannel for neurotransmitter preconcentration, electrophoresis separation, and amperometric detection. The total analysis time from cell sampling to neurotransmitter detection was about 15 min. Dopamine and norepinephrine released from single cells have been successfully separated by nanochannel electrophoresis and detected by amperometry. A microfluidic device, combined with electrophoretic manipulation and electrochemical detection, was used to quantify the amount of oxygen photosynthetically generated by a single cyanobacteria Microcystis cell.73 In this approach, each cell is individually guided to the main chamber containing the electrochemical setup by electrophoresis. The cells are then exposed to light irradiation, and the amount of oxygen produced is measured via its reduction current. The average oxygen generation rate has been found to be ∼10−18 mole per second. 530

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A similar experiment was performed by Gholipour et al.83 In this experiment, a quartz capillary tip filled with an oil mixture, known as a modified cell pressure probe, is used to extract sap from plant cells. The capillary tips are used for precise subfemtoliter solution management and can be loaded with an internal calibrant for mass calibration or quantitation. In the reported work, the probe was coupled to an Orbitrap mass spectrometer via ESI. Metabolite profiling for tulip bulbs, Tulipa gesneriana, stored at room temperature, 25 ± 0.5 °C, and in cool temperatures, 15 ± 0.5 °C, was examined. In video mass spectrometry, a form of live cell mass spectrometry,84 a nanoelectrospray tip is utilized as a pipet to extract the contents of subcellular regions before direct injection into a mass spectrometer. The experimental setup provides a platform to directly detect drug metabolites in a live single cell in real time. This technique is a viable platform for pharmacological studies since it allows for the detection of drugs and their respective metabolites in various regions of a single cell. Date et al. used this technique to track Tamoxifen, a drug using in breast cancer treatments at the single cell level.85 In this study, nonmetabolized Tamoxifen was present in both the cell’s cytoplasm and a vacuole and its metabolites were only found in the cytoplasm. Ellis et al. used inkjet technology to deposit living cells suspended in a bioink onto a microarray.86 The cells were extracted from the array via a robot-controlled liquid microextraction coupled to a chip-based nanoelectospray mass spectrometer. In this study, lipid profiles were characterized for three different cell lines: C2C12 (skeletal muscle, mouse), PC12 (adrenal pheochromocytoma, rat), and L929 (fibroblast, mouse). Principal component analysis (PCA) was used to simplify the data set. The highly automated approach resulted in accurate and reproducible analyses. The report shows that the technique is a viable platform for single cell-based drug screening assays. Desorption Electrospray Ionization Mass Spectrometry (DESI). Ferreira et al., in the Cooks Laboratory, used DESI to examine changes in the lipid profiles of cells during embryonic development.87 In this technique, an electrospray tip is used to directly spray the surface of the sample and ions that are desorbed from the surface are collected into the mass spectrometer. One of the great advantages of this system is its compatibility with ambient conditions. Lipid profiles obtained for unfertilized oocytes (large single cells), two- and four-cell embryos, and blastocysts have been compared. Comparing embryos grown under in vitro and in vivo conditions, they found lower variation in the lipid profile obtained from the in vitro cultured samples owing to the nutrient restriction in the culture medium. Although capable of imaging analysis, the limited lateral resolution of these techniques generally prevents single cell imaging. However, its sensitivity and atmospheric capabilities allow profiles of whole single cells. Capillary Electrophoresis Electrospray Ionization Mass Spectrometry (CE-ESI-MS). For single cell analysis, capillary electrophoresis (CE) has commonly been employed with ESI to separate cellular compounds before MS detection. Brown et al. have summarized the contribution of CE in single cell analysis in a book chapter recently published.88 Briefly, CE provides an additional degree on analyses to deconvolute the complex chemical nature of a single cell. This technique is also compatible with the extremely small sample volumes (nanoliter) associated with single cells, and quantification is carried out by removing competitive ionization effects. Recently, CE-ESI-MS was successfully employed to characterize metabolites in various

neurons isolated from the Aplysia californica central nervous system89,90 and in various cellular compartments of the alga, Chara australis.91 Nemes et al. in the Sweedler Lab have linked the chemical composition of an individual cell with its physiological function.89 Neurons with similar functions were found to be chemically similar. For instance, the neurons responsible for gut motility (B1 and B2) were chemically indistinguishable but significantly different from the metabolomics profile of the metacerebral cell (MCC) neurons, a neuron linked to modified feeding behaviors. Some metabolites, such as glycine betaine and proline betaine, were ubiquitous to all neurons. The large degree of variation in the cells chemical composition points to the significance of single cell measurements. The same team has also compared the metabolomics profiles of these neurons with respect to sample preparation procedure.90 Significant variations in metabolomics species were found when the neurons were analyzed freshly after extraction compared to those in culture. Oikawa et al. have utilized CE-MS to study the effects of environmental conditions on the distribution of metabolites within a single cell.91 The algae Chara australis was used as a model system. Fluctuations in metabolite levels in the vacuole and the cytoplasm of a single cell under different light conditions were examined. Approximately 125 metabolites within a single vacuole and cytoplasm were detected with CE-MS. With the ability to detect hundreds of metabolites in a single organelle, Oikawa describes a platform for elucidating cellular compartmentalization. Oikawa found that metabolites are spatially regulated within the cell, transferring metabolites between the organelles in response to stress. In addition, metabolites labeled with stable isotopes were injected via microinjection into the cell, and their distribution was tracked using this technique. Koroleva et al. used nano-LC-ESI-MS with MS/MS to analyze protein from glucosinolate-rich S-cells in Arabidopsis thaliana at the single cell level.92 The S-cells were extracted with a microcapillary and enzymatically digested in buffer before being injected into a nano-LC-ESI-MS. With this technique, they were able to successfully identify nine unique proteins in a single cell sample taken from the flower. Altogether, 56 proteins were identified from a total of 15 single cell samples. These proofof-concept experiments show that nano-LC-MS/MS is a viable platform for single cell proteomic analysis. Laser Ablation Electrospray Ionization Mass Spectrometry (LA-ESI-MS). LA-ESI-MS employs an infrared laser that ablates the sample. The desorbed ions are mixed with charged droplets from the ESI source and are ionized. Like DESI, this technique is compatible with ambient conditions. In the Vertes laboratory, this technique has been used to obtain in situ cell-by-cell profiles of plant tissue.93,94 A variety of metabolites and lipids have been detected with this method. MVA was used to identify potential biomarkers by extraction of the metabolites responsible for the majority of the variance detected from cell to cell. With this method, the detection of cyanidin, the purple pigmentation in the epidermal cells of an onion (Allium cepa), was correlated to the tissue physiology. The flavonoid, quercetin, was also found to be localized to the pigmented cells, whereas sucrose was distributed uniformly throughout all the studied cells with slightly higher intensities in the nonpigmented cells. The metabolite alliin, a precursor to the chemical compound responsible for the smell of onion, was concentrated in only 2 cells and absent in the other cells. Mass Spectrometric Analysis of Single Cells Based on Laser Desorption. Approaches in mass spectrometry with laser 531

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desorption have again been widely used in chemical analysis of single cells. Here, we discuss results in the last two years with direct laser desorption and MALDI, Laser Desorption/Ionization Mass Spectrometry (LDI-MS). In LDI-MS, a laser is employed to desorb and ionize the analyte without fragmentation. This method works well for plants and algae because they contain pigments that readily absorb UV light. However, without an organic matrix, the method is not highly compatible with most mammalian cells. A number of substrates have been designed specifically to overcome this issue and assist in the ionization of labile analytes via laser ablation without the assistance of a matrix. These specialized surfaces are designed to absorb laser energy, which causes localized heating and leads to the vaporization and ionization of the analyte. The latest developments and applications associated with this technology have been recently reviewed.95 Urban et al. in the Zenobi laboratory employed a multimodal approach, including optical microscopy, fluorescence imaging, Raman imaging, and LDI profiling to characterize algal cells, specifically Euglena gracilis.96 In this report, they correlated structures visible with optical microscopy, such as proplastids with β-carotene detected in the Raman band (1515 cm−1) and chloroplasts with chlorophyll pigment detected with fluorescence (emission wavelength, 618 nm). In addition, a variety of phospholipid species were detected in the mass spectrometry analysis. Since proplastids, detected by their β-carotene content, are only present in the early stages of the cell cycle and develop into chloroplasts (detected by their chlorophyll content), one can determine the cell cycle stage of an individual cell by measuring the relative amount of β-carotene and chlorophyll. This work describes a platform that allows for the visualization of cell development and the ability to correlate the cell stage with the relative phospholipids content. Walker et al. have developed a functionalized surface for laser ablation of labile molecules without an organic matrix.97 Nanopost arrays (NAPA) are highly ordered columns of silicon geometrically designed to significantly reduce the ionization threshold allowing matrix-free LDI-MS to be carried out. Nanofabrication techniques have been used to control the height (H), diameter (D), and periodicity (P) of the columns in the array. The aspect ratio (H/D), determined to be the most important parameter controlling strong ion yield resonance, was optimized. NAPA-LDI-MS is capable of ultratrace analysis (e.g., LOD ∼800 zmol of verapamil). In a proof-of-concept experiment, they demonstrated the ability to detect multiple metabolites from a single yeast cell deposited on the NAPA chip. Altogether, 24 metabolites, representing 4% of the yeast metabolome, were identified in the single cell spectrum. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). Matrix-assisted laser desorption ionization (MALDI) mass spectrometry is carried out with an organic matrix to assist in the ionization of laser ablated material. It is the leading IMS method for tissue analyses and is emerging as a potential platform for single cell analyses.98,99 The main factors that limit the spatial resolution of MALDI are matrix crystal size and laser beam size. As previously mentioned, several technological and methodological advances have been employed to overcome these hurdles and push the spatial resolution of MALDI down near the single cell level. Urban et al. monitored ATP metabolism in single yeast cells using high-density microarrays.100 The microarrays consist of a high density of single cell aliquot wells for high throughput single cell MS profiling. Yeast cells were incubated in media in which

C2-ethanol was the only exogenous available source of carbon. In a time-based study, biosynthetic activity of the cells was determined by measuring the incorporation of the heavy isotope carbon (13C) into ATP. The results revealed a discrepancy between single cell and multiple cell analysis, which highlighted the need for single cell analysis. In addition, large cell-to-cell variations in the ATP isotope distributions were measured. In this proof-of-concept experiment, the ability to interface heavy isotopic labeling MAMS-MALDI-MS to measure biosynthetic activity in single yeast cells was demonstrated. Neupert et al. have correlated neuropeptides with specific neurons in order to elucidate the precise functions of individual cells.101,102 Peptide profiles were obtained for various cells in the ganglia of cockroaches (Periplaneta Americana) and compared. In this study, MALDI-MS was used to profile the neuropeptides. In addition, immunochemical labeling and optical microscopy were used to image the distribution of specific peptides in the neural network. One article used the combined techniques to examine expression patterns in the neural network of the antennal lobe of the cockroach.101 They demonstrated that the sensitivity of their method was sufficient to detect neuropeptides in individual cells, known to be at extremely low levels. In a second paper, they examined peptidergic cells of the pars intercerebralis.102 Immunocytochemistry was used to visualize target cells, and MALDI-MS was used to profile the peptides and metabolites. In addition, the paper demonstrated the durability of the paraformaldehyde-fixed immunolabeled neurons as a proper sample storage procedure. Lagarrigue et al. employed a variety of methods and techniques to push the resolution of MALDI imaging down to the single cell level.103 They examined spectral quality with respect to laser profiles and found higher spectral quality using the smartbeam II laser profile compared to the Gaussian profile employed in oversampling methods. A commercial matrix application device (e.g., Imageprep) was employed to ensure optimal extraction with minimal crystal sizes. As a proof-of-concept experiment, they characterized spermatogenesis, the development of germ cells in testicular tissue at the single cell level. Overall, they managed to image proteins at 20 μm spatial resolution and achieved single cell resolution if not imaging across single cells. They use MALDI imaging as a platform to search for highly sensitive and highly selective biomarkers for clinical diagnostics. Roy et al. also achieved cellular resolution using MALDI-MS imaging on retinal tissue from the salamander.104 In this experiment, the heterogeneous distribution of polyunsaturated fatty acids, containing lipids in the various layers of the retina, was examined. One mass spectrum was obtained for each cell in the tissue; however, the spectral quality was low, and the intact lipids detected were close to the noise threshold. Zimmerman et al. in the Sweedler lab have characterized neuropeptides in neurons extracted and cultured from the sea slug, Aplysia californica.105 A technique was employed to stretch the sample in order to compensate for the limited spatial resolution associated with MALDI imaging. The neurons were cultured on a special substrate consisting of glass beads embedded in parafilm. In situ tandem MS was employed to identify Aplysia pedal peptide, PLDSVYGTHGMSGFA, at m/z 1540. Fan et al., also in the Sweedler lab, used functionalized particle-embedded monolithic capillaries to collect the neuropeptides released from individual cultured neurons during stimulation, using the same model system. MALDI was used to analyze the extract from the capillary. The columns were functionalized with two particles pyrrolidone and ethylenedi532

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freeze-drying ex situ causes the cilia to collapse and cover the plasma membrane. Lanekoff et al. also used a freeze-fracture device to study phospholipids in the cell membrane of PC12 cells, a model system commonly used in exocytosis studies.111 The cells were incubated with various concentrations of deuterated phosphocholines and phosphoethanolamines. Electrochemical-based analyses have shown significant changes in the rate of exocytosis when the lipid composition is changed. They demonstrated that their experimental setup was able to determine the relative concentration of phospholipids in the cell membrane and suggested only a small change in lipid is observed following incubation. Piwowar et al. examined the efficacy of the sample preparation procedure known as frozen hydration.112 In this method, the cells are washed, freeze-plunged, and introduced into the high vacuum analysis chamber without dehydration and without a freezefracturing device. HeLa cells were used as the model system. The cells were washed with 150 mM ammonium formate, which removes the phosphate-rich salt media and coats the cells with an osmotically favorable, pH balanced solution that sublimates off in the vacuum. The excess solution was wicked away with a KimWipe, and the cells were dried under nitrogen gas. Cell viability was retained throughout these procedures. The cells were then plunge frozen in liquid ethane, and the sample temperature was closely monitored during its transfer into the analysis chamber. With this method, Piwowar et al. were able to image cells with their membranes intact. In SIMS analyses, the method for measuring sputter rates, the rate at which an ion beam removes material during the sputtering process, for flat homogeneous samples is well established. However, determining the sputtering rate through a nonflat heterogeneous environment, such as a cell, remains a challenge. Robinson et al. in the Castner lab have tackled this challenge in a recent paper.113 In this experiment, NIH/3T3 fibroblast cells were used as a model system. Atomic force microscopy (AFM) was utilized to measure the height of a cell and account for topographical features. The AFM information was used to determine the sputter rate and adjust the depth-scale for more accurate 3D renderings of the cell. In this article, they detected a lipid-rich subcellular region, which was possibly the nuclear membrane, and reported these results in a three-dimensional corrected rendering. Using TOF-SIMS and AFM analysis of the same dried cell, the sputter rate was found to be approximately 10 nm per 1.25 × 1013 ions C602+/cm2. The article also introduced an add-on to their free MATLAB-based software (complements of the NIH) that will correct the depth scale in a 3D data set. Brison et al. examined HeLa cells treated with bromodeoxyuridine (BrdU), a nucleus marker that is incorporated into DNA during cell division.114 It was used to facilitate identification and localization of subcellular features during depth profiling. A number of sample preparation procedures were also examined. The efficacy of ammonium acetate washing, fixing with phosphate-buffered paraformaldehyde, trehalose in 0.3% glycerol water solution, freeze-drying using liquid nitrogen, and freezedrying using liquid ethane and frozen hydrated were examined. They found that washing with ammonium acetate was the most effective method for removing salts. In addition, C60 was used to remove surface contamination prior to imaging with the Bi3+ source. Molecular depth profiling of the BrdU marker was combined with AFM to determine the erosion rate through a HeLa cell, which was found to be 1.3 × 1015 C60+ ions per cm2.

amine. The binding efficiencies and column properties were optimized. In this study, a single bag cell neuron was stimulated and its secretions were collected for 30 min. The peptides α-bag cell peptide, acidic peptide, and egg-laying hormone were detected.106 Schober et al. imaged a single HeLa cell with 7 μm pixel size using an AP-MALDI mass spectrometer.107 The cell was stained with the fluorescent probe (DIOC6) and imaged with both optical microscopy (wavelength 501 nm) and mass spectrometry imaging (m/z 445). The cell was completely consumed by the laser beam, and the resulting mass spectrum contained a complex mixture of m/z ratios, indicating molecules associated with multiple cellular components (i.e., cell walls, cytoplasm, and nucleus). A number of metabolites and lipids were detected, including adenine, guanine, cholesterol, phosphatidylcholines, sphingomyelins, diglycerides, and triglycerides. The high mass resolution of an Orbitrap mass spectrometer provides confident chemical assignments without tandem MS analyses. Despite the ability to obtain multiple pixels per cell, subcellular components were not resolved. Mass Spectrometric Analysis of Single Cells Based on Secondary Ion Mass Spectrometry (SIMS). A great deal of the single cell work with mass spectrometry has been carried out with SIMS owing to its high spatial resolution. Here, we discuss work with both static and dynamic SIMS aimed at discovery in single cell analysis. Time-of-Flight Secondary Ion Mass Spectrometry (TOFSIMS). For single cell analyses, time-of-flight secondary ion mass spectrometry utilizes an ion beam to desorb secondary ions from the surface of a single cell. This technique is capable of detecting biological material under 1000 Da (i.e., lipids,108 metabolites, small fragments, and elements) and can easily achieve subcellular resolution. The ion beam is easily controlled via electronic rastering for acquisition of images. A unique feature of clusterTOF SIMS is its ability to carry out molecular depth profiling. As a result, it is the only IMS technique capable of 3D molecular imaging of single cells. The major challenges associated with this technique often pertain to sample preparation and protocols for 3D analyses. The majority of the reports published with regards to TOF-SIMS on single cells, in the last two years, have been focused on ameliorating these issues. Sample preparation is a crucial step in biological mass spectrometric analyses, especially when the analysis method requires the interfacing of biological samples into a vacuum environment. Several spring-loaded freeze-fracturing devices have been manufactured specifically to analyze cells in high vacuum. Chang et al. claim to have improved the success rate of sample preparation using a freeze-fracturing device that utilized powerful magnets.109 In a proof-of-concept experiment, HepG2 cells in a solution of quantum dots were freeze-fractured with the device and imaged. The report claims that the cells were evenly separated due to the uniform force applied during the fracture by the magnetics. The device is currently not incorporated into the instrument’s analysis chamber. Lanekoff et al. examined the importance of sample preparation techniques when analyzing single cells in TOF-SIMS.110 Tetrahymena pyriformis was used as a model system for this study. Two sample preparation procedures were examined, ex situ freeze-drying and freeze-fracturing, and the ability to detect lipid fragments with each procedure was examined. They attempted to examine phospholipid changes during cell division but were unable to find changes in the lipid fragments in the junction between the dividing cells. The results suggest that 533

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Lechene et al. have recently showcased the potential of multiisotope imaging mass spectrometry (MIMS) in cell biology.123 MIMS combines stable isotope labels with the high resolution imaging capabilities of the NanoSIMS methodology. In this study, 15N-thymidine was incorporated into the DNA of stem cells. After a chase period, to allow for cell division, the retention of the tracer was measured. The full retention of the tracer in some cells is direct evidence of biased DNA segregation in stem cells and supports the “immortal strand hypothesis”. However, after examining a statistically significant number of cells, they were unable to locate any stem cells in which the 15N-thymidine labeled DNA was retained at its original level. The dilution of the tracer, 50% every cell cycle, suggests that random DNA segregation is the likely mechanism dictating stem cell division. The paper also included a proof-of-concept experiment in which human cells were infused with radiolabeled thymidine. The lag time between infusion and the appearance of labeled white blood cells in the blood was about 4 weeks, consistent with the approximate time the cells have been released from the bone marrow. The experiment shows that MIMS is a potentially powerful platform for studying human metabolism and cell tracking. The Zehr Laboratory used NanoSIMS imaging to study the symbiotic relationship between cyanobacterium UCYN-A, a nitrogen-fixing prokaryote, and the photosynthetic eukaryotes, prymnesiophyte.124 The transfer of the isotopically enriched nitrogen (15N) from the cyanobacterium to its eukaryotic partner in exchange for carbon (13C) was monitored with nanoSIMS. This symbiotic relationship has led to the genetic reduction of the cyanobacteria UCYN-A, which lacks various functions that typical cyanobacteria possess and are typically required for survival. In a separate study, cyanobacteria, particularly, Richelia intracellularis and Calothrix rhizosoleniae, in a symbiotic relationship with various diatoms (Hemiaulus, Rhizosolenia, and Chaetoceros) were found to have upregulated nitrogen fixation rates compared to independent cyanobacteria.125 Overall, these model systems for symbiosis are a potential platform for investigating the endosymbiotic theory, the theory which suggests that eukaryotic cells evolved from symbiotic relationships among multiple microorganisms. Sheik et al. used nanoSIMS to examine changes in host cells due to viral infection.126 The phytoplankton, Phaeocystis globosa, sequesters inorganic carbon in intercellular chitinous, star-like structures. The effect of viruses on the carbon assimilation of the phytoplankton provides a model system to show how viral infections alter host physiology at the single cell level. Phytoplankton play an important role in the ecosystem; their survival is crucial to the health of the oceans. Therefore, understanding the viruses that threaten the viability of this phytoplankton is important. They found that noninfected phytoplankton produced more star-like structures and particles compared to viral infected phytoplankton. Smart et al. examined the subcellular distribution of elements, in particular nickel, in the leaf of an Alyssum lesbiacum plant.127 The images show an enriched distribution of calcium in the cell walls, nickel in the vacuoles, and magnesium, potassium, and sodium in the cytoplasm. This proof of concept paper highlights the importance of sample preparation procedures in the preservation of the original molecular distribution. In this case, high pressure freezing followed by freeze substitution was effective in preserving the distribution of soluble elements. NanoSIMS provides a platform to study the adverse effects of pollution at the subcellular level. NanoSIMS was used to visualize

An alternative approach to examining the inside of a cell was described by Szakal et al.115 In this study, the high resolution of focus ion beam (FIB) was used to cut cells in a controlled manner before TOF-SIMS analysis. The FIB cut, performed at 90 degrees off normal, removed topographical features, reduced bombardment related deposition, and was independent of the samples heterogeneities. They also introduced an alternative multivariate analysis approach, which differentiates between ion specific images. Specifically, the k-means clustering method was used to classify peaks in a hierarchical system based on their commonalities. For the specific aim of single cell mass spectrometric imaging, TOF-SIMS analysis combined with FIB-milling and a new novel MVA method provided a plausible and comprehensive approach for 3D analysis. Fletcher et al. also examined the importance of sample preparation procedures when analyzing single cells in TOFSIMS; however, their analysis focused on 3D imaging analysis.116 The work was performed on the J105 3D Chemical Imager, with a buncher-TOF mass spectrometer and a C60 primary ion beam. Two sample preparation procedures were examined: (1) chemically fixed and freeze-dried cells and (2) nonfixed, frozen-hydrated cells freeze-fractured exposed with a freezedried device called a Mousetrap. They found reduced chemical redistribution in the cells prepared with the frozen hydrated method. In addition, better spectral quality, cleaner spectra, and improved chemical contrast were also found for the frozen hydrated cells. Finally, the paper presented 3D reconstruction of the data using MVA techniques. Barnes et al. demonstrated the potential of TOF-SIMS as a tool in tissue engineering.117 In a proof of concept paper, TOFSIMS and MVA were used to resolve different cell types cocultured together based on their different chemical signatures. In this article, primary rat esophageal epithelial cells were cultured with NIH 3T3 mouse fibroblasts on tissue. Principal component analysis and partial least-squares discriminant analysis were used to extract peaks that contributed to the variance associated with each cell type’s chemical signature. Overall, this method was capable of successfully distinguishing different cell types visually and chemically. This ability will be useful for characterizing seeded cells on biocompatible substrates for tissue engineering applications. A proof-of-concept experiment was performed to showcase the ability of TOF-SIMS to localize nonluminescent, unlabeled particles buried in cells, a task that has been proven to be difficult for a number of experimental techniques. Hagenhoff et al. examined the cells ability to internalize silica nanoparticles of various sizes, mapped the distribution of nanoparticles inside cells, and characterized the cells lipid membrane composition.118 The TOF-SIMS analysis shows that epithelial-like NRK cells (normal rat kidney, clone 52E) were able to uptake silica nanoparticles with a variety of sizes: 2, 500, and 150 nm. The particles were mainly localized in the cellular cytoplasm. This was reaffirmed by Kollmer et al. in a paper where gold NPs were examined using a different cell line.119 Dynamic SIMS (NanoSIMS). NanoSIMS, a dynamic SIMS methodology, employs a focused atomic primary ion capable of obtaining images with spatial resolution below 50 nm. Typically, only atomic or diatomic species are detected with this method. Its role in detecting metabolic activities in single cells120 and in plants121,122 has recently been reviewed. In the past two years, NanoSIMS technology has had some exciting applications in cell biology. 534

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confirmed the chemical nature of dense body features observed in the TEM analysis as protein accumulations. Audinot et al. examined nanoparticle toxicity in Daphnia magna, and the distribution of CuO nanoparticles in individual HepG2 cells.134 Subcellular features detected in a TEM image were correlated to regions of high copper signal. The relatively low 31P signal from the cell infused with copper nanoparticles suggests cell death and links nanoparticle toxicity with cell viability. Wolfe-Simon and co-workers used NanoSIMS to image arsenic (As) incorporated into the bacterium strain GFAJ-1 of the Halomonadaceae, isolated from Mono Lake, California.135 Bacteria incubated in arsenic-doped media and normal media were imaged. The ratio of 75As−:12C− was compared to 31 P−:12C− for cells incubated in arsenic and phosphate. Arsenic is clearly present in the cell doped with arsenic. However, the authors hypothesized that the AsO43− replaced the PO43− backbone in the organism’s DNA. The authors also believe that the bacterium, which is able to sustain life in harsh environments (i.e., hypersaline and alkaline water), possess the mechanisms necessary to cope with the inherent instability of AsO43−. The paper has been highly criticized; concerns that the experiments did not provide enough evidence to support the claims made by the authors have been expressed.136 Mass Spectrometric Analysis of Single Cells Based on Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The trends and developments achieved by inductively coupled plasma mass spectrometry were recently reviewed.137 Here, we present recent advances in this area related to chemical analysis of single cells. Laser Ablation Inductively Coupled Plasma Mass Spectroscopy (LA-ICP-MS). The Jakubowski group used laser ablation inductively coupled plasma mass spectrometry for cellular imaging. Giesen et al. described a new method in which iodine is utilized to stain cell nuclei for LA-ICP-MS imaging analyses.138 Fibroblast cells were incubated with potassium iodine for 60 s before fixation and LA-ICP-MS imaging. The iodine was detected in the cytoplasm but was concentrated in the cell nucleus. The same method was used to stain the nuclei of cells in tissue sections. Iodine was used in tissue-based analyses as an internal standard for measuring tissue thickness and was used to assist the relative quantification of possible biomarkers within the tissue. Drescher et al. used the technique to image nanoparticles in individual 3T3 fibroblast cells.139 They found an accumulation of silver and gold nanoparticles in the perinuclear region. This accumulation increased with increasing incubation times. This proof-of-concept experiment shows that LA-ICP-MS is a potential platform for studying for nanoparticle toxicity. Mass Cytometry (Flow Cell-Nebulizer-ICP-MS). Single cell mass cytometry is a technique developed by Garry Nolan’s lab at Stanford University in collaboration with DVS Sciences. This method couples flow cytometry with ICP-MS.140 In this technique, cells are labeled with isotopic reporter, typically antibodies linked to monoisotopic transition elements. This high throughput technique has the ability to analyze 1000 cells per s, and is currently capable of measuring 34 parameters simultaneously (i.e., the binding of the 31 isotopic reporters, cell viability, DNA content, and relative cell size). Bendall et al. used this method to study hematopoiesis, the formation of blood.141 In bone marrow, hematopoietic stem cells give rise to a wide variety of cellular blood components through several differentiation pathways. Differentiation is a transitional process. This technique is capable of tracking a cells stage of maturation

the cellular localization and concentration of pollutants in both plant and human cells. Tartivel et al. studied the localization of the pollutant bromotoluene in Hedera helix plants and soil.128 In this experiment, bromotoluene was detected in the plant cell walls in both the leaves and roots. The technique has also been used to track halogenated pollutants in human cells. Gutleb et al. found that the flame retardant tetrabromobisphenol A was homogenously distributed and surfactant perfluorooctanesulfonic acid was heterogeneously distributed in the cell membrane of H295R cells.129 In addition, the pollutants polybrominated diphenyl-ethers and its hydroxylated metabolites, 4-OH-BDE69 and 4-OH-BDE121, were also tracked in GH3.TRE cells. Wedlock et al. have tracked the subcellular distribution of gold after treatments from a possible chemotherapeutic agent, [Au(d2pype)2]Cl, in tumor cells.130 Energy filtered transmission electron microscopy was used to visualize changes in the cellular morphology and NanoSIMS analysis was used to detect drug distribution. Changes in the subcellular morphology were observed after treatment (i.e., the cells were round and vacuolated, and the chromatin was more condensed). Aggregations of gold were detected in the non-DNA containing regions of the nucleus and the nuclear membrane and in the cytoplasm. The gold was colocalized with the sulfur-rich regions in the nucleus and cytoplasm. This finding suggests that thiolcontaining proteins should be considered to be a possible target or part of a mechanism of action for the gold compounds. Overall, the experiment shows the power of NanoSIMS as a platform for studying the pharmacokinetics, specifically the subcellular distribution, of metal-based drugs. Morono et al. found a diverse population of microbes alive in the sediment obtained from the 460 000-year-old deep sea floor.131 In this proof-of-life experiment, the microbes were incubated in isotopically labeled 13C and 15N and the uptake within individual cells was monitored with NanoSIMS. Microbes of various size and morphology were detected, and their respective metabolically activity was also observed. Surprisingly, the study showed that subsea floor microbial cells preferentially incorporate nitrogen into their biomass from ammonia. The uptake of various isotopically tagged metabolites was quantitatively monitored and used to characterize the energy requirements of the microbes. The high resolution of NanoSIMS was used to examine the lipid environment surrounding proteins in the cell membrane. In order to elucidate the roll of lipids in the functionality of membrane bound proteins, Wilson et al. in the Kraft lab developed fluorine-functionalized colloidal gold immunolabels to locate specific membrane proteins using the high-spatial resolution of NanoSIMS.132 A gold nanoparticle was functionalized with fluorinated thiols linked to secondary antibodies. In a proof-of-concept experiment, the particles were functionalized with the primary antibody, mouse antihemagglutinin monoclonal, which specifically binds to the protein membrane influenza hemagglutinin in the plasma membranes of transfected fibroblast cells. The gold and fluorine signal and surrounding location were analyzed. Parallel detection of a specific protein and the surrounding lipid content were carried out with this method. NanoSIMS has also found utility in clinical investigations. Galle et al. imaged podocytes, specialized cells found wrapped around capillaries in the Bowman capsule in kidney tissue, in human renal biopsies.133 These cells are responsible for filtering the blood. The malfunction of these cells results in protein-rich urine, a disorder known as proteinuria. The SIMS analysis 535

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Figure 4. Microfluidic devices for single cell manipulation. (A) Design and operation of an integrated microfluidic device for single cell gene expression analysis (Adapted with permission from ref 150. Copyright 2011 National Academy of Sciences.); (B) a microfluidic platform for cell culture and sample preparation with two orthogonal single cell resolution techniques: flow cytometry and fluorescence microscopy (Adapted with permission from ref 164. Copyright 2012 Royal Society of Chemistry.); (C) an integrated microfluidic approach for whole-genome molecular haplotyping of single cells (Adapted with permission from ref 177. Copyright 2011 Nature Publishing Group).

including microwells, electrokinetic trapping, and optical tweezers among others. Microwell or Microhurdle-Based Single Cell Trapping and Analysis. White et al. developed an integrated microfluidic device for single K562 cell gene analysis by combining single cell capture, single cell lysis, and reverse transcription quantitative PCR (RT-qPCR) on a chip (Figure 4A).150 A capture efficiency of 89% was obtained by microhurdle trapping in the capture chamber. Using this device, miRNA expression in single K562 cells, coregulation of a miRNA, and one of its target transcripts during differentiation in embryonic stem cells, as well as single nucleotide variant detection in primary lobular breast cancer cells were measured. The results demonstrated that this lab-on-a-chip device has combined all single cell-processing steps into an integrated platform and could provide a solid foundation for building advanced microfluidic single cell transcription analysis. Hosokawa et al. described a microwell-based single cell trapping microfluidic device for single cell cytotoxicity assays integrated with a microfluidic chemical gradient generator.151 A single cell capturing efficiency of 88 ± 6% was achieved by this device within 30 s. This chip was used to assess cytotoxicity by exposing the cells to a concentration gradient of potassium cyanide (KCN) formed in the chemical gradient generator. The LC50 at 2 h post KCN treatment was calculated as 9.7 μM. The entrapped cells were affected by KCN in a concentrationdependent manner, demonstrating the feasibility of this device to quantitatively assess chemical cytotoxicity as well as to trace cytotoxicity over time.

through the differentiation process based on the collection of proteins on its surface. Cells are clustered by their chemical signatures using spanning-tree progression analysis of densitynormalized events (SPADE). The branches of the SPADE tree represent the different cell lineages, and each node represents the mature blood cell. Single cell mass cytometry provides a platform to characterize cells during healthy development, in disease states, and after pharmaceutical intervention. This technique has also been used to characterize CD8+ T-cell populations,142 to discriminate between bacterial species,143 for massive cell multiplexing via isotopic bar-coding,144 and to investigate the possibility of cancer stem cells.145

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LAB-on-a-CHIP FOR SINGLE CELL MANIPULATION AND ANALYSIS Advances in microfabrication techniques have given rise to integrated microfluidics or ‘‘lab-on-a-chip’’ devices and systems. These chips have a number of properties that make them ideally suited to the analysis of individual cells. Capture and/or analysis of single cells with these lab-on-a-chip approaches by several single cell manipulation strategies have been carried out including microwell or microhurdle-based docking, electrokinetic or hydrodynamic single cell focusing or injection techniques, trapping with optical tweezers, droplet trapping, chemical patterning, etc. Several recent reviews have been published over the last two years.146−149 Single Cell Trapping and Analysis on a Chip. An area of interest in chip-based single cell assays has been the development of new cell trapping schemes. Here, we review several approaches 536

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microchip can monitor 96 independent samples with up to 96 qPCR probes (equivalent to 9216 reactions) in a single experiment, which can be completed within 2−3 days. Lecault et al. reported a microwell- or microchamber-based single cell trapping device for live-cell imaging studies of nonadherent cells in culture, with precise control of the growth conditions.159 Single cell loading efficiencies of 10−30% in one of the 1600 chambers enable 160−480 cells to be distributed individually for clonal analyses. Hematopoietic stem cells were exposed to time-varying concentrations of a signaling molecule, Steel factor, which initiates the end of the cell quiescence. Precise studies of the mechanism controlling cell growth and fate decision can be carried out with this method. Live-cell imaging studies were also reported with a microwellbased device for single T cell trapping. Single primary CD4+ T cells were trapped and activated by antigen-coated microbeads in deep microwells. Live cell imaging of individual T cells can be performed for up to 72 h.160 This approach was also applied to the dynamic monitoring of gene expression using a fluorescent reporter gene and to the study of cell−cell interactions between regulatory and effector T cells. Torres et al. described the immobilization of lipid bilayers and tethered ligands on the surface of dense arrays of microwells for study of surface ligand− T cell interactions.161 The detection of secreted cytokines from the activated human T cell clones by fluorescence microscopy can be carried out with this approach. With the same microchip, Choi et al. used immobilized antibodies with covalently attached fluorescent oligomers to detect cytokines released from single cells.162 In this case, a hybridization chain reaction technique was applied to amplify signals resulting from sandwich immunoassays that can be amplified to increase detection sensitivity up to 200fold. Ma et al. reported a high-throughput single cell barcoded chip for detection of different cytokine secreted from single cells.163 This microchip contains 1040 microwells with immobilized DNA-barcoded antibodies to more than 10 cytokines, with quantitation standards at protein copy number resolution built into the antibody barcodes. From one to forty cells can be trapped in each microwell, and their cytokine secretions are assessed with a fluorescence immune-sandwich assay. The chip was successfully used to profile cytokine secretion from tumor antigen-specific cytotoxic T cells and might be useful for the profiling of other immune pathways. Electrokinetic or Hydrodynamic Flow-Based Single Cell Trapping and Analysis. Wu et al. developed a microfluidic device for cell culture, stimulation, and downstream hydrodynamic flow focusing followed by single cell analysis by multicolor fluorescence and scattering imaging (Figure 4B).164 This device can be used for global profiling of TLR4 signaling events at different time scales (from seconds to hours) and subcellular locations (from cell surface to nucleus). First, realtime imaging of TLR4 receptor activation by lipopolysaccharide can be monitored by live-cell fluorescent microscopy in microfluidic culture, and then, immunohistochemical analysis of other proteins can be performed. Cells are then released via enzymatic cleavage and focused into a single cell line by two sheath flows and interrogated individually by a five-channel (2 scattering, 3 fluorescent) detector. The results demonstrate that the microfabricated device and multichannels imaging technique can be combined for dynamic measurements of an entire pathway in a single cell experiment. Zhang et al. developed an automated microchip electrophoresis system using electrokinetically gated single cell injection

Park et al. also described a microwell-based single cell docking process inside a microfluidic channel, in which cells were individually captured in the microwells using several back and forth sweeps of a cell-containing solution plug.152 A 90% cell capturing efficiency was achieved by optimizing the microwell sizes, microchannel sizes, and cell loading densities. Highthroughput automated fluorescent microscopy was combined with this system to study cellular responses of the MAPK signaling pathways in the budding yeast, Saccharomyces cerevisiae. Real-time cellular responses of the mating MAPK pathway were realized at various concentrations of mating pheromone at single cell resolution. Cao et al. reported a quantum dot-based immunofluorescent approach using a microwell-based single cell trapping device to quantify the glycan expressions and the changes on the K562 cells after 2-deoxy-D-glucose treatment.153 The microfluidic platform comprises an array of microwells allowing a 70% single cell capturing efficiency. Compared with the organic fluorescence probe used in conventional immunofluorescent staining, the quantum dot-based fluorescence probe provides higher brightness and stability against photobleaching. Eyer et al. developed a microhurdle-based microfluidic device for single cell trapping and lysis.154 Cell lysates can be retained in the microchamber without loss of the intracellular contents as the microchamber can be closed pneumatically after the lysis buffer is introduced. The concentration levels of the coenzymes NADH and NADPH or the expression levels of the enzyme glucose-6phosphate dehydrogenase in single U937 histiocytic lymphoma cells have been determined by fluorescence imaging. The integration of cell trapping, cell treatment, labeling, and lysis permits fast analysis of single adherent cells. Chen et al. developed a novel microfluidic platform combining cell-recognizable aptamer modified microwells for single cell trapping and fluorescence microscopy for single cell imaging.155 The single cell capture efficiency of this method is 88.2%, which is much higher than for aptamer modified flat surfaces and nonaptamer modified microwells. Cellular carboxylesterases were studied by time-course measurements of cellular fluorescence kinetics at the single cell level. The results revealed the diversity in the amount of intracellular carboxylesterase in targeted single normal and tumor cells. Van den Brink et al. reported a single cell trapping strategy yielding 85% efficiency. This is obtained with a microhurdle within the interface of connecting channels between the main channel and multiple side channels.156 The cell permeabilization and electroosmotic flow (EOF)-based extraction of the cell content has been studied using calcein-loaded cells, which are visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content. Li et al. described single cell trapping by microhurdles in a microfluidic channel.157 They studied the drug accumulation of daunorubicin in a single multidrug resistant leukemia cell, as well as multidrug resistance modulation in the same cell. Rapid samesingle cell analysis for drug accumulation has been demonstrated by use of a short typical experiment cycle (2200 s). Citri et al. described a protocol for high-throughput gene expression profiling from single neuronal cells using a commercial Fluidigm’s Biomark high-throughput quantitative PCR (qPCR) chip.158 This chip uses a pressure-regulated microfluidic circuit in order to perform mixing of nanoliter volumes of samples and probes within individual chambers. After loading and mixing are completed, thermal cycling is performed, coupled to the imaging of the chip at the end of each cycle. This 537

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of agarose droplets can be generated in less than 1 h by this method. After droplet collection, single cell RT-PCR and DNA or RNA staining, fluorescence microscopy, and flow cytometry was applied to detect and quantify agarose beads containing DNA clones. Two different cell lines (Kato III cell and MDAMB-231 cell) were studied by this single cell RT-PCR method, and the results confirmed the expression level differences between the two cell lines. Rane et al. reported the application of microfluidic droplet techniques for encapsulation of a single cell in one droplet and subsequent single cell lysis.172 In this experiment, a peptide nucleic acid (PNA) molecular beacon was applied to carry out intradroplet single cell content 16S rRNA detection by the increased fluorescence upon hybridization with complementary targets following the FRET principle. Iino et al. reported the application of surface droplet techniques to assess drug efflux activity in individual Escherichia coli cells.173 Single cell femtoliter droplet arrays were formed using a cover glass with hydrophilic-in-hydrophobic micropatterned surface. First, the medium and the cells were put on the cover glass. Fluorinated oil was then added on the substrate surface to cause single cell droplet formation by medium and bacteria retained in the hydrophilic SiO2 glass surface, while the hydrophobic surface was covered with oil. Different efflux activity of fluorescein-di-β-D-galactopyranoside at wild-type and efflux pump gene deletion mutant E. coli strains was shown. Moon et al. reported a single cell trapping technique with a drop-on-demand patterning platform.174 Single drop containing single cell was generated and patterned on the surface with an ejector using a valve driven by a controlled air pressure pulse. The application for total RNA analysis was used to demonstrate that total RNA quality from the droplet-based isolated cells was similar to that of the control set obtained by the manual pipet method. The stem cell-related markers including Kit and Notch1 were found in both the printed cells and nonprinted control groups. This fact indicates that this technique can be used to provide useful biological information from single stem cells. Chen et al. reported droplet merging techniques for the detection of oxygen consumption in single islets.175 In this device, two kinds of droplets (CaCl2 droplets or alginate droplet containing islets and oxygen-sensitive dye) from two inlet channels collide and merge into each other to form bigger droplets, and then after the 3 min transit time through the main channel, calcium causes the alginate surrounding the islet to polymerize, thereby encasing both islet and oxygen-sensitive dye in a thin alginate layer. The oxygen-dependent dye was used to report changes in oxygen consumption in only 0.5 min. This approach was shown to be not harmful to the function or viability of islets over the course of two days. Chemical Patterning-Based Single Cell Trapping and Analysis. Collins et al. reported methodology combining biomolecules and biocompatible polymers patterned at subcellular scales for single cell capture.176 First, cell adhesive fibronectin was patterned on the glass substrates; tip-based lithography was then used to precisely readdress these protein patterns with microdomains of polymers carrying different materials to be exposed to the cells. A maximum number of 576 single patterned cells can be achieved by this patterning technique, and the introduction of calcein AM, calcein Red AM, or quantum dots was used to demonstrate that precise control of the cellular microenvironment is possible with this method.

techniques combined with single cell lysis and electrophoretic separation to rapidly analyze the intracellular hydrogen peroxide concentration with laser induced fluorescence.165 The whole process was carried out in 60 s, and the average content of hydrogen peroxide in single HepG2 cells was monitored with this method. Phillips et al. also described a microchip electrophoresis system for the study of fluorescein and fluorescein carboxylate that are preloaded into single cells.166 Hydrodynamic flow was applied for single cell flow focusing and single cell injection. The intracellular content was studied by mechanical lysis from a lasergenerated cavitation bubble, electrophoresis separation, and fluorescence detection. With this method, 30 cells could be analyzed in about 1 min, which proved to be a comparatively high throughput. Xu et al. described a microchip electrophoresis system for the analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes.167 A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed for hydrodynamic focusing and introduction of single cells. By this technique, the average cell throughput for the separation of ROS and GSH in single cells was about 38 cells per min. Galla et al. reported a microchip electrophoresis system for the separation and detection of the protein content from a GFPlabeled single Spodoptera f rugiperda (Sf9) insect cell with two color UV/vis-LIF detection.168 This microfluidic chip has a reduced UV fluorescence background (83% reduction) by use of PDMS with carbon black pigments as additives compared to a PDMS chip. Single cell protein content separation and analysis confirmed that UV-LIF can be used to detect more proteins based on autofluorescence compared to only one single peak measured by the visible-LIF. Optical Tweezer-Based Single Cell Trapping and Analysis. Werner et al. described the study of intracellular pH dynamics in single yeast cells trapped by refractive multiple optical tweezers combined with microfluidics and optical microscopy.169 More than 200 yeast cells can be trapped into a high-density array in a microfluidic channel. Using this system, they demonstrated the temporal decrease of intracellular pH of optically trapped yeast cells induced by the trapping laser within 14 min and the temporal changes of intracellular pH upon exposure to 10 mM glucose. These results demonstrated the applicability of the array cytometer for studying dynamic responses in single cells. Nelson et al. reported a nanopore-based microfluidic device, which can be used for single molecule transfection into a single cell.170 The microfluidic device consists of two-level microchannels separated by a membrane layer featuring a nanopore. A single cell was trapped using optical tweezers from a laminar flow in the microfluidic channel and positioned over the nanopore. Some 20 kbp YOYO intercalated dsDNA was introduced by electrophoresis and electroporation into the cell via the nanopore. Fluorescence imaging was used to show the process of the dsDNA migration from the cell surface to cell nuclei. These results demonstrated the potential for transfecting a single cell by electroporation using a nanopore. Droplet-Based Single Cell Trapping and Analysis. Zhang et al. reported a microfluidic device for performing single copy, emulsion reverse transcription polymerase chain reaction (RTPCR) within agarose droplets.171 Highly monodispersed and size tunable droplets can be produced in this microfluic device with two-aqueous inlet channels (one for the cell, one for the RT− PCR and cell lysis reagents) and two oil inlet channels. Millions 538

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Other Techniques for Single Cell Analysis. Fan et al. developed a microfluidic device capable of separating and amplifying homologous copies of each chromosome from a single human metaphase cell (Figure 4C).177 The microfluidic device consists of a valve-controlled single cell trapping region, a cell lysis and chromosome release region, a chromosome partitioning region with 48 partitions, an amplification region for each partition, and a product retrieval region for subsequent analysis. Combined with a single-nucleotide polymorphism array, direct deterministic phasing was verified with three lymphoblastoid cell lines and this work had several practical applications, including direct observation of recombination events in a family trio, deterministic phasing of deletions in individuals, and direct measurement of the human leukocyte antigen haplotypes of an individual. These results demonstrated that this method could be used as a molecular-based, wholegenome haplotyping technique amenable for personal genomics. Wang et al. reported the formation and detection of individual telomere fibers from single cells by the fluorescence in situ hybridization (FISH) method in a microchannel.178 A straight microfluidic channel was used to immobilize a single interphase cell nucleus and introduce a proteinase K and YOYO-1 dye solution flow to stretch the released DNA. A millimeter-long band of stretched DNA molecules was generated from a single nucleus, and FISH results confirmed the individual telomere fiber could be resolved from single cells. Ainla et al. fabricated a multifunctional pipet which can deliver liquid into an open volume while simultaneously rerouting the liquid back into the pipet.179 This kind of injection creates a hydrodynamically confined flow (HCF) volume achieved by means of automatic positive and negative pressure control to spaced adjacent channels. This HCF volume, connected to the tip of the device via a channel, can be positioned to stimulate and analyze a single cell or other object of interest. Single cells were pretreated with a cell-penetrating fluorescent species, which can be converted into a calcium chelator inside the cell. A highly fluorescent complex was formed and detected after calcium stimulation with this multifunctional pipet at the single cell level. This kind of multifunctional pipet was also applied to individually electroporate the membrane for local analyte delivery to single cells.180 Kim et al. developed a tunable multicolor imaging system coupled with a microfluidic channel for the determination of drug-induced cardiotoxicity via simultaneous quantitative monitoring of intracellular sodium ion concentration, potassium ion channel permeability, and apoptosis/necrosis in the H9c2 cell line.181 In this system, a straight microchannel (3.8 mm width, 17 mm length, 400 μm height) was used for the culture of adherent cells. Multiple fluorophores (spectral region ranging from 500 to 630 nm) were used to screen the cells. Single cell imaging and quantitative multivariate cellular analysis was achieved. Zhou et al. reported a laser scanning cytometry approach for the rapid and automated fluorescence imaging of cells cultured on a microchip.182 High throughput analysis of single cell signaling pathways and rapid screening of cellular responses to an array of extracellular proteins patterned on a chip was achieved by this approach. The results demonstrate the capability of this device for high-throughput single cell analysis. McKenna et al. reported a 384-microfluidic channel system coupled with a high-speed scanning photomultiplier-based detector combined to a low-pixel-count, to perform onedimensional imaging of cells.183 This approach overcomes the

low throughput of CCD-based imaging and achieves cell imaging in up to 384 fluidic channels simultaneously. This study also examined expressed GFP fusion proteins on yeast cells. Bai et al. reported a microhurdle-based single cell trapping microfluidic approach for the detection of oxygen evolution in single cells using amperometry and electrochemical impedance spectroscopy.184 In this study, needle-shaped tips containing a localized platinum microelectrode were positioned on immobilized cells through the use of AFM-related techniques, which do not affect cell physiology while minimizing the formation of toxic hydrogen peroxide. The activity of oxygen evolution of a Peperomia chloroplast and the dependency of the oxygen concentration on light intensity was confirmed.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

§ These authors contributed equally to this work. M.E.K., R.T., M.K.P., and J.W. worked on the fluorescence, electrochemistry, mass spectrometry, and lab-on-a-chip sections, respectively.

Notes

The authors declare no competing financial interest. Biographies Andrew G. Ewing is currently Professor of Analytical Chemistry at both Chalmers University of Technology and the University of Gothenburg in Sweden and is Chair of Analytical Chemistry at Chalmers. He is also Director of the Chalmers/GU Focus for Bioanalytical Chemistry. He was previously Professor at Penn State University. He received his B.S. from Saint Lawrence University in Canton, NY and his Ph.D. at Indiana University in Bloomington, IN. He was a postdoc in the laboratory of Royce Murray at the University of North Carolina for 13 months. His research interests have been in single cell analysis and neuroanalytical chemistry. Michael E. Kurczy received a B.S. in chemistry from Salem State College in 2003 and earned his Ph.D from Penn State in 2009. Since finishing his degree, he has been a postdoc in the Chemical and Biological Engineering Department at Chalmers University of Technology in Gothenburg, Sweden working with Ann-Sofie Cans. His research interests include studying lipid membranes in living and model systems using mass spectrometry imaging and electrochemical methods. Melissa K. Passarelli is currently a postdoctoral researcher in the Department of Chemistry at the University of Gothenburg. She graduated with her B.S. from Union College, Schenectady, New York in 2005. She obtained her Ph.D. degree in Chemistry from the Pennsylvania State University in 2011 under the advisement of Nicholas Winograd. Her research interests include lipid profiling, cell differentiation, and imaging mass spectrometry, specifically secondary ion mass spectrometry (SIMS). Raphaël Trouillon is currently a postdoctoral researcher in the Department of Chemistry at the University of Gothenburg. He graduated with his B.Eng and M.Eng. from the Ecole Polytechnique (France) in 2006 and with his M.Sc. from the Department of Bioengineering of the Imperial College London (UK) in 2007. He obtained his Ph.D. degree in Biomedical Engineering from the Imperial College in 2010. His research interests include electrochemistry applied to life sciences and cells-on-a-chip. Jun Wang received his bachelor degree from Shandong Medical University in 1998 and obtained his master's degree in 2001 from Liaoning University. He graduated with his Ph.D. in 2006 at the Dalian Institute of Chemical Physics, Chinese Academy of Sciences. He has 539

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worked in Yoshinobu Baba’s Lab at Nagoya University, Japan, as a postdoctoral researcher from 2007 to 2011. He then joined the group of Andrew Ewing at Gothenburg University. His research interests mainly focus on the study of neuron cell network by the combination of array microelectrode and microfluidic technique. He is also interested in electrochemical sensing.

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ACKNOWLEDGMENTS We acknowledge the many co-workers that have come before and whose works we cite in this Review. We acknowledge support from the European Research Council (ERC Advanced Grant), the Knut and Alice Wallenberg Foundation in Sweden, the Swedish Research Council (VR), and the USA National Institutes of Health (NIH).

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Analytical Chemistry

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dx.doi.org/10.1021/ac303290s | Anal. Chem. 2013, 85, 522−542