Questions Driving Future Research

The future of single-cell analysis appears bright, and we expect that the major advances will occur as driven by the issued raised by the following key questions:

Instrumentation/sampling: Is true quantitation from single-cell samples within our grasp using current technologies? What can be done to improve cellular analyte extraction? Quantitation with mass spectrometry has involved the addition of selective labels. However, when assaying single-cell samples, where the amount of material is close to the detection limit and extra sample steps often cause sample losses that prevent detection, quantitation is especially difficult. Further work will involve the development of new sample handling procedures as well as an increased use of normalizing factors to account for variations in samples and sample preparation strategies. New and improved extraction and preparation protocols are required to increase detectability and to extend the mass range available for analysis.

Whole organ and organism studies: In light of the considerable success of single-cell MS, how can we prepare samples so that cells in entire three-dimensional regions of interest can be characterized at the cellular level? Through high-throughput analyses and the development of advanced molecular imaging, high-resolution chemical reconstructions can advance both medical and biological science. Using serial tissue slices, several three-dimensional chemical images have been constructed using MALDI MSI (Crecelius et al., 2005). By increasing the resolution and speed of data acquisition, analyses of entire organs, as well as small regions of interest on a cellular level, should be obtainable. Using SIMS, the application of depth profiling with cluster ion sources, particularly with C60 sources (Wucher et al., 2004), may also prove beneficial and allow for the three-dimensional chemical imaging of single cells on a submicron scale (Chandra, 2005).

Cellular classification in the CNS of "higher" animals: Is it possible to classify cells in vertebrate CNS (subtypes of neurons, glia, etc.) based on their mass fingerprints? Will such classifications change for specific neurons during learning or behavioral plasticity? The classification and analysis of single mammalian cells from tissues presents numerous challenges with respect to both instrument sensitivity and preparation protocols, but also represents the next step in single-cell analyses.

Response to applied stressors: How do the distributions of chemicals in an organism's central nervous system change after the application of a stressor such as a drug of abuse? In which cells are these changes manifested? This problem appears ideally suited for the combination of MSI and single-cell analysis. An understanding of chemical changes within a biological system may lead to advances in disease or disorder treatment and prevention, is the driving force behind many of the projects mentioned within this chapter, and presents an intriguing avenue for further research.

Biomarker assay: Can single-cell mass spectrometry be used to detect chemical signatures indicative of disease states? Will such detections be of any use in clinical settings for the virtual real-time diagnosis of these disease states (as in single-cell-sized biopsies)? Further information is required to address these questions. The capability to classify cancerous cells on the basis of mass spectra (Schwartz et al., 2004) indicates that such capabilities are feasible. In order to gain acceptance in the clinical community, reproducibility and throughput must be increased. This does, however, present an intriguing application of single-cell analysis because chemical changes often occur well before morphological changes may be observed; obviously, the earlier that one may detect changes in individual cells resulting from disease, the earlier the treatment may begin.

While the studies that have been performed using single-cell MS have certainly been exciting, they are merely the beginning. As the throughput, information content, and ease of use continue to improve, a greater range of experiments will highlight the complexities of biology with a combination of temporal, spatial, and chemical information provided by the next generation MS-based single-cell experiments.

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