The Single-Cell Scene Single-cell studies are appearing in many journals, but do we really need to look at one cell at a time?
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or centuries, scientists have been grinding up whole organs or large chunks of tissue to discover how our bodies function. Nucleic acid and protein science has advanced over the years because of this research, and even today many tissues and cells are routinely homogenized for biochemical and genetic analyses. If researchers have made great strides studying large populations of cells, why develop new analytical methods to probe single cells? “The main advantage with studying single cells is that you can tap into the heterogeneity that’s present in a cell population,” says Sheri Lillard at the University of California, © 2004 AMERICAN CHEMICAL SOCIETY
Katie Cottingham
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FIGURE 1. Biopsy of a human embryo, which involves the removal of a single cell for PCR analysis. (Adapted with permission. Copyright 2004 Humana Press.)
Riverside. She points out that a homogenate provides an average picture across all the cells in a sample. But if the same analysis were performed on individual cells, one might see that an analyte is differentially expressed in various cells within the same tissue or that a drug may activate one cell while inhibiting its neighbor’s activity. Nancy Allbritton at the University of California, Irvine, agrees: “People are finding very complex behaviors in cells that you completely overlook in populations.” According to Alan Thornhill at the Mayo Clinic, another advantage of single-cell analysis is that one can study rare samples in which only one or a few cells are available. He says that this is sometimes the case for forensics samples. In his own work, Thornhill diagnoses genetic disorders in embryos at the 6–10-cell stage with single-cell PCR (Figure 1). Only one or two cells may be biopsied for DNA analysis without permanently damaging the embryo. Researchers also say that single-cell techniques allow them to determine the locations of molecules within cells. Various signaling proteins, for example, move from the cytoplasm into the nucleus when a cell is activated. But some experts warn that, in some situations, zeroing in on a single cell can give an inaccurate picture of what is going on. “By doing [single-cell analysis], you have problems of representation,” Thornhill says. “Is this cell or series of cells representative of what I really want to look at?” Variability may be caused by something other than the process being studied. For instance, two cells may express different proteins because they happen to be in different stages of division, not because of an applied stimulus. Richard Caprioli at the Vanderbilt University School of Medicine says this problem of representation can be explained in a hypothetical story in which a Martian visits Earth, takes a picture of a single person, and claims the photo shows exactly what human beings look like. Caprioli says that if “human being” means having a head, two arms, and two legs, then that is generally an accurate description. “But if you want to say, ‘Now you see the bridge on the nose, and you see that the hair is always blonde,’ that doesn’t give you the right picture,” he says. 236 A
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Focusing on one cell may not only give a scientist the wrong picture, it can result in missed opportunities. “Cells actually talk to each other,” says Ed Yeung at Iowa State University. “So if you have an isolated cell, it, of course, would never form a tumor, even though it may be cancerous.” Yeung explains that the cooperative effects of cells may be overlooked if single cells are studied in isolation. For this reason, he advocates both tissue-level and single-cell-level studies to get the whole picture of a process. Caprioli also doesn’t see this as an “either/or” proposition. “There’s no right or wrong here. It’s basically, ‘What do you want to do?’” he says. His research team takes both approaches, depending on the research question. He says that sometimes looking at a single cell is not necessary and that more information can be gleaned from thin tissue slices.
The techniques Although chemists are relatively new to the single-cell scene, biologists have been studying single cells for some time. Since the late 1800s, biologists have used stains and dyes to observe the morphologies of individual cells within tissue slices. In the early 20th century, fluorescent labeling enabled the visualization of many subcellular structures. Flow cytometry, another common biological technique, sorts single cells and detects fluorescent tags on proteins, carbohydrates, lipids, and nucleic acids. Recently, biologists have adapted DNA extraction and PCR procedures for genotyping single cells. The method involves a crude DNA preparation, in which a cell is placed into a tube and lysed. PCR reagents are added directly to the mixture in the tube. Thornhill says that protocol is simpler than the standard extraction to obtain DNA from a population of cells. Because the sample is repeatedly transferred into different tubes to purify the DNA in the standard procedure, some DNA can be lost. Sample loss is not a luxury that a single-cell researcher can afford. Although biological methods have yielded important data for both clinical diagnostics and basic research over the years, limitations remain. Immunohistochemistry and flow cytometry rely on fluorescently labeled antibodies and other dyes to tag specific target molecules, and PCR requires the addition of primers based on known genomic sequences. “The analytical chemists have gotten into the act, trying to make approaches work where you don’t have to know what you’re looking for,” says Jonathan Sweedler at the University of Illinois. For example, chemists can often examine several members of a broad category of analyte, such as peptides, lipids, or nucleic acids. Thus, most chemical techniques are much more general in scope. According to Caprioli, chemical techniques complement biological methods. “The neat part about this technology is it doesn’t do away with anything; it’s not meant to replace anything else,” he says. “It’s a new way to get information about cells and tissues.” One can choose from several techniques, depending on the application at hand. Electrochemistry can provide temporal and spatial information on the release of chemicals from individual
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cells. Extracellular events, in addition to those occurring inside a cell, can be monitored. Neuroscientists often use carbon microelectrodes to track the transport of neurotransmitters and exocytosis (Figure 2). MS can provide accurate masses, which can be used to identify analytes. In TOFsecondary ion MS (TOF-SIMS), an ion beam hits the sample and a mass spectrometer measures the secondary ions and neutrals that are sputtered from the surface. Small molecules, such as phospholipids and cholesterol, are currently studied with this method. Andrew Ewing and his collaborator Nick Winograd at the Pennsylvania State University say that most of the molecules their team observes with TOF-SIMS are
