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Keywords:

  • single-cell analysis;
  • metabolomics;
  • single-cell separation;
  • metabolite detection;
  • mass spectrometry;
  • metabolite imaging

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

Single-cell analysis is a promising method for understanding not only cellular physiology but also biological mechanisms of multicellular organisms. Although neighboring cells in multicellular organisms originate from the same genomic information, different circumstances around cells or epigenetic differences have different influences on each cell, leading to differing expression of genes, and thus differing levels and dynamics of metabolites, in single cells. However, single-cell analysis is a tough challenge, even with recent technologies, because of the small size of single cells. Unlike genes, metabolites cannot be amplified, and therefore metabolite analysis is another issue. To analyze such a tiny quantity of metabolites in a single cell, various techniques have been tried and developed. Especially in mass spectrometry, marked improvements in both detection sensitivity and ionization techniques have opened up the challenge for the analysis of metabolites in single cells. In this review, we discuss the method for metabolite detection at the level of single cells and recent advancements in technology.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

Cellular heterogeneity may result from several factors: genetic, epigenetic or phenotypic differences; morphological, biochemical or functional changes; positional, exogenous or endogenous mutations; and physical, chemical or biological effects from the environment (Wang and Bodovitz, 2010; Rubakhin et al., 2011; de Souza, 2011). These differences may be systematic or stochastic (Wang and Bodovitz, 2010; Rubakhin et al., 2011). This means that individual cells have unique gene expression and metabolite dynamics. Therefore, single-cell analysis is necessary to understand the biological and physiological properties of single cells and multicellular organisms, including humans and plants. However, because of the small size of single cells, their separation and isolation remain a challenging task. After pioneering work in single-cell analyses using the pressure probe (Tomos and Leigh, 1999; Tomos and Sharrock, 2001), various technical developments have improved the quality of separation of a single cell and the detection of metabolites in a single cell. Several devices such as microwells have been developed for single-cell separation (Lindström and Andersson-Svahn, 2010, 2011) (Figure 1). On the other hand, improvements in the spatial resolution of microscopes and high-resolution imaging technology enable the visualization of gene expression in single cells without isolating them (Itzkovitz and van Oudenaarden, 2011; Maiuri et al., 2011; Schroeder, 2011; Tsuyama et al., 2011). Laser microdissection is also an effective method for isolating specific single cells from multicellular organisms, including plants (Kehr, 2003; Nelson et al., 2006; Moco et al., 2009.) The isolation of a single chloroplast from leaves of tobacco by this method has been reported by Meimberg et al. (2003), demonstrating the high spatial resolution of laser microdissection. Coincidentally, easily isolated cells such as neurons, stem cells and guard cells, have been used as model systems for single-cell analysis (Kehr et al., 1998; Grant et al., 2000; Zhang et al., 2004; Lapainis et al., 2009; Eberwine and Bartfai, 2011; Oikawa et al., 2011; Schroeder, 2011; Zhao et al., 2011).

image

Figure 1.  Methods for isolating single cells. Organisms consisting of multiple cells are prepared as an aggregate of single cells (a), and then separated into single cells by flow cytometry (b), microscopy (c) or microdevices (d).

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Analysis of gene expression has already reached the single-cell level (Karrer et al., 1995; Roy et al., 2008; Wang and Bodovitz, 2010; Eberwine and Bartfai, 2011; Itzkovitz and van Oudenaarden, 2011; Maiuri et al., 2011; Tang et al., 2011; Wallden and Elf, 2011). Recently, new technologies, including ‘lab-on-a-chip’ (Marcus et al., 2006; Whitesides, 2006), mRNA sequencing (Tang et al., 2009; Wang et al., 2009), and quantitative PCR (Taniguchi et al., 2009), have been developed and applied to quantitative large-scale and reproducible analyses. Next-generation DNA sequencing technology could improve single-cell genomics (Zhang et al., 2006). Metabolite analysis of single cells requires highly sensitive instruments, because metabolites cannot be amplified, in contrast to DNA and RNA. Recent technical developments in nuclear magnetic resonance (NMR), mass spectrometry (MS), chromatography and electrophoresis have enabled us to detect metabolites at very low concentrations in single cells (Amantonico et al., 2010a; Heinemann and Zenobi, 2011; Rubakhin et al., 2011). In addition, the direct infusion of single cells separated by microfluidic devices or micropipette into MS suggests the possibility of highly sensitive metabolite analysis in single cells (Moco et al., 2009; Heinemann and Zenobi, 2011; Rubakhin et al., 2011; Tsuyama et al., 2011).

A major difference between animal and plant cells is the autonomy of individual cells. In the case of animal cells, the behavior of isolated cells is similar to that expected in vivo. This is very rare in plant cell biology. This is the main advantage of animal cells in single-cell analysis. However, recent technological developments of single-cell isolation, such as laser microdissection, enable us to perform single-cell analyses of plant cells, thereby providing detailed biological explanations.

Here, we discuss the recent developments in the detection of metabolites in single cells.

Detection of Metabolites in Single Cells

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

The volume of a single cell is on the level of pl, which is not sufficient to detect most of the metabolites in a cell using a conventional detector, except for several abundant metabolites such as sugars (Lindström and Andersson-Svahn, 2010), or pigments such as anthocyanins (Miyanaga et al., 2000; Ceoldo et al., 2005). The most common technology for metabolite detection in single cells is MS. The detection limit of recent mass spectrometers is on the order of zeptomolar (10−21) or yoctomolar (10−24) concentrations (Anderson et al., 2002; Whitmore et al., 2007). Here, we discuss several devices, including MS, for the detection of metabolites in single cells. Moreover, we describe the application of metabolomics to single-cell analysis.

Nuclear Magnetic Resonance

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

Nuclear magnetic resonance (NMR) spectroscopy is an excellent instrument for the non-invasive and quantitative detection of metabolites (Rubakhin et al., 2011). It has recently been used for metabolite analysis of single cells (Grant et al., 2000; Lee et al., 2006; Reckel et al., 2007; Motta et al., 2010). Köckenberger, (2001) has reveiwed the non-invasive metabolite analysis in plant tissue using micro-imaging based on NMR. Although the spatial resolution was not so high, NMR signals of one or several nuclei of metabolites clearly showed the localization of the metabolites in plant tissues. For example, spectroscopic images of a dried fennel mericarp showed the distribution of protons in methylene (chemical shift: δ = 1.3 ppm), a methly group (δ = 1.8 ppm), and protons in the methoxy group and aromatic/olefinic protons of anethole (δ = 3.8 and 7.0 ppm, respectively) (Rumpel and Pope, 1992). This study demonstrated the technical feasibility of NMR spectroscopy of single cells. Because it is not highly sensitive, NMR spectroscopy tends to provide low coverage for metabolite detection. However, the detection limits of NMR spectroscopy have been enhanced by small-volume NMR probes, which allow the characterization of cell-sized samples (Olson et al., 1998; Krojanski et al., 2008).

Electrochemical Detection

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

The high sensitivity of electrochemical detectors makes them suitable for single-cell analysis (Amantonico et al., 2010a). In fact, they can be used as intra- or extracellular probes for the label-free detection of metabolites in the cell and those released outside the cell. However, only electroactive substances can be analyzed, which makes the electrochemical method only applicable to targeted studies of metabolites in single cells. A series of microscale electrochemical methods was applied to monitor various physiological processes (Huang and Kennedy, 1995; Cannon et al., 2000). Microelectrode measurements can be used to investigate both the intracellular pools of ions and membrane transport processes of single live cells (Miller et al., 2001). Microelectrodes can detect these processes in the surface layers of root and leaf of intact plants. Careful manipulation of the plant minimizes disruption, and therefore the information obtained from these measurements most probably represents the ‘in vivo’ situation. Compartmental concentrations of inorganic metabolite ions have been measured using several different methods, and the results obtained for the cytosol are comparable. Ion-selective microelectrodes have been used to measure the activities of ions in the apoplast, cytosol and vacuole of single cells. Furthermore, Yasukawa et al. (2002) detected ascorbic acid and H2O2 by combining a pl electrochemical analytical chamber and enzyme-linked assays.

Amperometric detection has also been used in combination with microfluidics (Xia et al., 2005). In this method, cell injection, loading and cell lysis, and electrokinetic transportation and detection of intracellular species were integrated in a microfluidic chip by using an electric field. Then, the cell was lysed by a direct current electric field of 220 V cm−1. The analyte of interest inside the cell was electrokinetically transported to the detection end of the separation channel, and was electrochemically detected. In this study, ascorbic acid in single wheat callus cells was directly detected at a carbon fiber disk bundle electrode. This technique can be applied to a variety of electroactive species within single cells.

Fluorescence

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

Fluorometric assays are generally based on the presence of fluorescent tags or probes. Only very few wheat metabolites can be directly analyzed in single cells using autofluorescence. In many cases, fluorometric assays can be applied to the targeted analysis of small-molecule compounds in cells and cell extracts (Secrist et al., 1972; Chang and Yeung, 1995; Fuller et al., 1998; Fehr et al., 2002; Williams, 2004; Cohen et al., 2008; Berg et al., 2009; Moro et al., 2010). The advantages of fluorescence detection of intracellular metabolites include high sensitivity, the ability to perform studies on concentration dynamics, the non-destructive nature of the method and high-throughput detection. Single-cell sugar analyses were performed by isolating cell sap from individual epidermal cells using microcapillary action combined with an enzyme assay using a photomultiplier (Kehr et al., 1998). Zhang et al. (2004) detected the cytosolic H2O2 by using 2′,7′-dichlorofluorescin in single guard cell protoplasts of Vicia faba. Following the introduction of a fluorescent probe into the cell, a readout can be obtained with an established technique such as fluorescence microscopy (Fehr et al., 2002; Okumoto et al., 2005). The fluorescent probes described above are expressed in living cells (Davey and Kell, 1996). Although nanosensor probes can, in principle, be developed for different analytes, fluorescence detection limits the number of compounds that can be simultaneously detected. Therefore, the fluorescence method is not appropriate to a comprehensive analysis such as metabolomics. Although current studies involving the application of fluorescent probes to compounds make use of fluorescence microscopy, such assays in combination with flow cytometry or fluorescence-activated cell sorting may enable studies of intracellular metabolites in a large number of cells.

Fluorescence detection is often used in combination with sample preparation steps. These can include single-cell lysis and separation of analytes by capillary electrophoresis (CE) (Amantonico et al., 2010a). In an early study by Kennedy et al. (1989), amino acids were readily analyzed in extracts from individual neurons obtained from snails. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection is a highly sensitive method for detecting metabolites in a very small sample, such as single cells. CE-LIF was used to separate and detect doxorubicin and at least five metabolites from NS-1 cells treated with 25 mm doxorubicin (Anderson et al., 2002). Using 10 mm borate and 10 mm sodium dodecyl sulfate (pH 9.3) as the separation buffer, a 488-nm argon-ion laser line for fluorescence excitation and a 635 ± 27.5-nm bandpass filter for detection, the limit of detection (signal-to-noise, S/N = 3) for doxorubicin was 61 ± 13 zmol. This low limit of detection allowed for the detection of a larger number of metabolites than previously reported. Furthermore, a yoctomole analysis of ganglioside metabolism was recently performed with CE-LIF (Whitmore et al., 2007). Unfortunately, there have been few reports of this excellent technique applied to plant studies. Chen et al. (2005) detected nine amino acids in individual wheat embryonic protoplasts by using CE-LIF. In this study, the fluorescent reagent for chemical derivertization was introduced into living protoplasts by electroporation. In addition, a special osmotic buffer was used to keep the osmotic balance of protoplasts. Under these experimental conditions they found the concentrations of detected amino acids were at the millimolar level.

Secondary Ion Mass Spectrometer

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

Secondary ion MS (SIMS) uses a highly focused beam of energetic primary ions to bombard the sample surface, sputtering secondary ions for mass analysis (Rubakhin and Sweedler, 2010). Time-of-flight SIMS provides outstanding lateral resolution, which is a unique way to study the spatial localization of chemical species with a molecular mass below ∼1000 Da in single cells. SIMS is the method of choice for analyzing the lipid composition of cellular membranes, because of its high sensitivity for many lipids and their fragments. As an innovative study of molecular mechanisms of cellular membrane composition, SIMS revealed the time-dependent chemical dynamics of membranes in the unicellular organism Tetrahymena thermophila (Kurczy et al., 2010), showing that the formation of membrane lipid structures followed structural changes that occurred during the mating process. Although SIMS has never been applied to plant cells, future technical developments may enable the analysis of protoplasts or cultured cells of plants.

Laser Desorption Ionization Mass Spectrometry

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

Laser desorption ionization includes several MS techniques. In an early example, a laser beam with a 5-μm diameter was used as the chemical probe of thin tissue slices: various metabolites were detected in different regions of single cells (Kaufmann et al., 1978). Several powerful analyte desorption/ionization approaches have been developed afterwards and applied to detect metabolites in single cells. Northen et al. (2007) introduced nanostructure-initiator mass spectrometry (NIMS), a tool for spatially defined mass analysis. NIMS uses ‘initiator’ molecules trapped in nanostructured surfaces or ‘clathrates’ to release and ionize intact molecules adsorbed on the surface. This surface responds to both ion and laser irradiation. The lateral resolution (ion-NIMS: about 150 nm), sensitivity, matrix-free and reduced fragmentation of NIMS permits the direct characterization of peptide microarrays, the direct mass analysis of single cells and tissue imaging. Recently, Hölscher et al. (2009) reported matrix-free laser desorption/ionization mass spectrometric imaging of highly localized UV-absorbing secondary metabolites in plant tissues at single-cell resolution. The highest spatial resolution achieved in this study was at the level of a single cell (approximately 10 μm). Their methods enabled us to image plant secondary metabolites, such as flavonoids, including kaempferol and its glycoside, in Hypericum and Arabidopsis thaliana at the cellular level. Jun et al. (2010) also applied laser desorption ionization mass spectrometry to image surface metabolites of A. thaliana. The high spatial and high mass resolution analysis allowed the direct identification of lipid metabolites on root surfaces at the level of single cells.

MALDI MS

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

Matrix-assisted laser desorption/ionization (MALDI) MS is a powerful analytical approach for simultaneously detecting a broad mass range of analytes, including metabolites, peptides and proteins. It is also applicable to a wide range of biological sample types. MALDI MS can be used to detect and characterize compounds that are responsible for the biologically essential activities of cells. In single-cell analyses, MALDI MS has often used for metabolite imaging (van Hove et al., 2010; Setou et al., 2010; Kaspar et al., 2011). Amantonico et al. (2010b) made an attempt to validate a single-cell mass spectrometric method for detecting changes in metabolite levels occurring in populations of unicellular organisms using MALDI MS. Some metabolites involved in central metabolism (ADP, ATP, GTP and UDP-glucose) could readily be detected in single cells of Closterium acerosum. The analytical capabilities of this approach were characterized using standard compounds. A combination of UV MALDI time-of-flight (TOF) MS and a pressure probe has been applied for the direct analysis of plant metabolites in single-cell cytoplasm extract (Gholipour et al., 2008). In this study, single-cell cytoplasm sap (1–10 pl) was extracted from tulip leaf and bulb by using a pressure-probe glass microcapillary tip, analyzed by UV-MALDI-TOF MS for underivatized carbohydrate content, and then several sugars were detected by using various matrices.

Video MS

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

Metabolite analysis using ambient MS can also be performed directly without analyte separation (Cooks et al., 2006). For example, live video MS is one such method that uses a metal-coated micropipette to visually sample selected areas of individual live cells. These samples are then directly electrosprayed from a micropipette into a mass analyzer. Tejedor et al. (2009) reported a direct and quick method for the molecular analysis of single live plant cells viewed under a video microscope in their native environment using a nano-electrospray tip and MS. This method has been successful in identifying specific molecules in live plant single-cell analysis by MS, and introduces the possibility of comparing different cell types from different tissues with morphological evidence from undamaged plants.

Laser Ablation Electrospray Ionization MS

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

Physical contact between the sampling apparatus and the cell is eliminated in laser ablation electrospray ionization (LAESI) MS. In LAESI, the native water content of cells efficiently absorbs the energy of mid-infrared (IR) light, resulting in sample ablation (Nemes and Vertes, 2007). The ablated material is then captured by charged electrosprayed droplets to convert the chemical constituents of the sample into gas-phase ions that are subsequently detected by MS. To investigate metabolic variations in cell populations LAESI MS was recently used for the in situ analysis of individual cells at atmospheric pressure (Shrestha and Vertes, 2009). Single-cell ablation was achieved by delivering mid-IR laser pulses through the etched tip of a GeO2-based glass fiber. Metabolic analysis was performed on single cells and small cell populations in the epidermis of Allium cepa and Narcissus pseudonarcissus bulbs, as well as single eggs of Lytechinus pictus. Of the 332 peaks detected for A. cepa, 35 were assigned to metabolites using accurate ion masses and tandem MS analyses. Recently, the same group has performed in situ cell-by-cell imaging of molecules such as cyanidin and hexose in onion epidermal cells (Shrestha et al., 2011).

CE-MS

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

Capillary electrophoresis (CE) is a superior method for separating single cells, as mentioned above. As is the case for LIF, CE can also be combined with MS for the metabolomic profiling of single cells (Lapainis et al., 2009). Detection limits are in the low nanomolar range [i.e. <50 nm (<300 amol)] for a number of cell-to-cell signaling molecules, including acetylcholine (ACh), histamine, dopamine and serotonin. The utility of this set-up for single-cell metabolomic profiling was demonstrated with identified neurons from Aplysia californicasthe: the R2 neuron and metacerebral cell (MCC). Single-cell electropherograms were reproducible, and detected a large number of metabolites: more than 100 compounds yielded signals of over 104 counts from the injection of only 0.1% of the total content of a single MCC. This method could be applied to plant cells by optimizing the experimental conditions of single-cell isolation.

Metabolomics of Single Cells

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

Metabolomics, a comprehensive metabolite analysis, becomes an important method for understanding biological and physiological features of organisms (Saito and Matsuda, 2010). Figure 4 shows a recent increase in the number of publications on metabolomics. Additionally, several reviews on metabolomics of single cells have recently been published (Moco et al., 2009; Amantonico et al., 2010a; Ebert et al., 2010; Wang and Bodovitz, 2010; Heinemann and Zenobi, 2011; Rubakhin et al., 2011). Schad et al. (2005) reported the metabolic profiling of vascular bundles of Arabidopsis thaliana by using laser microdissection and gas chromatography-mass spectrometry (GC-MS). In this study, 68 metabolites were identified and more than half of them were shown to be enriched or depleted in vascular bundles, as compared with the surrounding tissues. In addition, metabolic profiling of A. thaliana epidermal cells, including pavement, basal and trichome cells, was performed using microcapillaries for single cell sampling and a GC-MS-based metabolomic analysis technique (Ebert et al., 2010). The 117 detected metabolites were localized depending on the cell type. However, because these methods required 100 respective vascular bundles or 200 trichome cells for metabolite detection, they were not exactly ‘single-cell’ analyses. Unfortunately, metabolomic analysis of a single cell type or at the single-cell level is quite rudimentary. Various limitations in the present analytical approaches make it impossible to conduct routine analyses of many metabolites in all single cells, in contrast to the case of conventional metabolomics. Therefore, the next challenge is to develop more sensitive and comprehensive detection and quantification of various metabolites in single cells. These approaches will become an innovative method rudimentary in systems biology research. The data obtained from single-cell metabolomic studies will aid in the establishment of cellular metabolic models that will not be biased by averaging the metabolite concentrations over multiple cells.

Giant Single Cells

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

As single-cell analyses must be applicable to general cells, the small size of these cells has become a bottleneck in this area of research. Therefore, several types of cells, including stem cells and neurons, are often used in single-cell analysis studies, because these cells are larger than general cells, and are easily isolated and cultured. Hence, single-cell analysis of stem cells and neurons reveals the heterogeneity of these cells. However, the size of these cells is still small, and necessitates microscopic experiments for single-cell analysis. Recently, we reported on single vacuole metabolomics using a giant internodal cell of the alga Chara australis (Oikawa et al., 2011). This cell grows up to 20 cm in length, with a volume of over 50 μl. Using this cell, metabolite analysis of a single organelle is also possible. We used a metabolomic method to elucidate the localization and dynamics of 125 known metabolites isolated from the vacuole and cytoplasm of the single cell (Figure 2). The number of metabolites in the vacuole and cytoplasm fluctuated asynchronously under various stress conditions, suggesting that metabolites are spatially regulated within the cell. By projecting the metabolomic data onto the pathway map, we also found that metabolites in the central metabolic pathway, including the glycolytic and pentose phosphate pathways, were mainly localized in the cytoplasm (Figure 3). This suggests that the continuous reactions of these pathways occur in the cytoplasm, and that there is no storage in the vacuole. On the other hand, most metabolites at the end of these pathways were detected in vacuolar-type clusters (Figure 3), suggesting that the final biosynthesis products in cells are stored in the vacuole.

image

Figure 2.  Hierarchical cluster analysis (HCA) based on changes in metabolite levels of an internodal cell of Chara australis at different time points under changing light conditions. HCA revealed that metabolites could be divided into two major clusters consisting of vacuole-type and cytoplasm-type metabolites. These two clusters were again divided into two respective clusters: cluster 1 (blue); cluster 2 (light blue); cluster 3 (orange); and cluster 4 (red). The yellow and blue colors correspond to high and low relative metabolite levels, respectively. (Reprinted from Oikawa et al., 2011, copyright American Society of Plant Biologists.)

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image

Figure 3.  Metabolic pathway map. Circles represent a single metabolite and the circle colors correspond to the clusters described in Figure 2: cluster 1 (blue); cluster 2 (light blue); cluster 3 (orange); and cluster 4 (red). Grey circles represent metabolites that were not detected in this study or are not annotated because of a lack of standard compounds. Metabolites shown in the lower right frame could not be projected onto known metabolic pathways. (Reprinted from Oikawa et al., 2011, copyright American Society of Plant Biologists.)

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Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References

The number of published articles on ‘single-cell analysis’ is increasing at a rapid rate (Figure 4). Although only papers published up to October were taken into account for the total number of publications in 2011, this number is already larger than that of the entire year of 2010. This indicates an increasing interest in single-cell analysis.

image

Figure 4.  The increase in the number of publications on single-cell analysis and metabolomics. The total number of publications was obtained by searching the PubMed website (http://www.ncbi.nlm.nih.gov/pubmed) year by year, using the keywords ‘single-cell analysis’ and ‘metabolomics’, respectively.

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Stochastic events, including gene expression, enzyme activities and metabolite fluctuations, occurring in every individual cell, confer individual characteristics to single cells. However, this heterogeneity of single cells cannot be uncovered in conventional studies using multiple cells in bulk as analytes. It is absolutely certain that the increased spatial resolution at the level of single cells reveals the true biology and physiology of multicellular organisms. Furthermore, single-cell analysis can reduce biological noise (Wang and Bodovitz, 2010). This leads to fundamental improvements in experimental design and data analysis for application in single cells. Stem cells, for example, hold great potential for regenerative medicine because they can self-renew and differentiate along different lineages. However, embryonic stem cells, adult stem cells and induced pluripotent stem cells are all heterogeneous populations (Takahashi et al., 2007; Graf and Stadtfeld, 2008; Chan et al., 2009). Single-cell analysis can target specific populations, and therefore elucidate signaling pathways and networks associated with self-renewal and differentiation, such as studies on cancer and neurons (O’Dowd and Smith, 1996; Bao et al., 2006; Clarke et al., 2006; He et al., 2010). In plant sciences, dedifferentiated cells, such as callus, are routinely used. Single-cell analysis may be applied to reveal the differences between each dedifferentiated cell, leading to the understanding of plant development mechanisms. Although greater improvements in the sensitivity of single-cell analysis are necessary, analysis of exudates from individual cells has the potential to uncover chemical communications between cells.

Current methods for single-cell imaging, transcriptomics and genomics are more sophisticated than those for the metabolite analysis of single cells. Although the technologies for measuring subsets of metabolites are potent, they typically detect only a handful of analytes present at the highest concentrations. There is no question that an increase in the sensitivity of MS and higher resolution separations will result in improved analyte coverage. Perhaps just as importantly, eliminating losses during sample preparation and more efficient analyte extraction will aid such studies. A few reports have been published on metabolite analysis of single plant cells (Kehr et al., 1998; Tejedor et al., 2009). Even in these early studies, the number of detected metabolites was very small. Considering the advantages and potential of single-cell analysis in plant physiology studies, the application of single-cell analysis in plant sciences will accelerate. Furthermore, the techniques used in single-cell analysis could apply to subcellular metabolite analysis. These improvements in spatial resolution may uncover novel facts yet to be discovered.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Detection of Metabolites in Single Cells
  5. Nuclear Magnetic Resonance
  6. Electrochemical Detection
  7. Fluorescence
  8. Secondary Ion Mass Spectrometer
  9. Laser Desorption Ionization Mass Spectrometry
  10. MALDI MS
  11. Video MS
  12. Laser Ablation Electrospray Ionization MS
  13. CE-MS
  14. Metabolomics of Single Cells
  15. Giant Single Cells
  16. Conclusion
  17. References