It was also worth it for Tannishtha Reya, who studies stem cell fate and cancer at Duke University Medical Center in Durham, North Carolina. "Imaging is just so powerful, I can't get enough of it," she laughs. It took Reya more than a year to develop an imaging system able to track individual haematopoietic stem cells (HSCs); her greatest challenge was getting the cells to stay put long enough to photograph them. (The solution? A layer of stromal cells to slow their movement.) Once the apparatus was completed, Reya used it to compile videos of symmetric and asymmetric divisions in HSCs, showing for the first time that HSCs undergo both types of division and that the balance between the two can be influenced by microenvironmental cues and subverted by oncogenes3. Single-cell analysis, including imaging and gene expression, can reveal information population studies cannot Fluidigm Corporation For now, single-cell imaging capabilities remain sequestered in labs lucky enough to have the know-how, stamina and funding to develop and maintain them. "Technically, it's extremely hard to do," says McKay. For the technology to spread, he says, the process will need to be simplified. Fortunately, another single-cell technology has proven more portable and attainable — it can be bought from a local sales representative.

Tracking expression

The quantitative real-time polymerase chain reaction (qRT-PCR) remains the experiment of choice for analyzing cellular gene expression. But for single cells or small populations of cells, the extensive DNA amplification needed in conventional RT-PCR techniques often introduces noise and bias. In addition, genes with abundant transcripts are more likely to be detected, and typically, only a few genes can be analyzed at a time.

Enter the microfluidics chip.

In 1999, Stephen Quake and colleagues at Stanford University in California founded the company Fluidigm, based in San Francisco, to commercialize the integrated fluidic circuits technology developed in his laboratory4. Often referred to as a 'lab on a chip', the bottle cap–sized devices covered in silicon circuits require only minute amounts of samples and reagents to carry out a variety of experiments, including the measurement of gene expression. One of Fluidigm's products, a microfluidics chip developed by the company's chief scientific officer Mark Unger and his team, has an extensive miniature plumbing system in which up to 96 samples from a single cell can be tested against 96 genes, giving 9,126 individual qPCR reactions in 75 minutes. "The volume requirements are so low that we've done as many as 1,000 genes off an individual cell," says Gajus Worthington, cofounder and chief executive of the company. "Most researchers using the tool are typically looking at between 50 and 100 genes," he notes, citing such scientists as Irving Weissman at Stanford, Shinya Yamanaka at Kyoto University in Japan and Toshio Suda at Keio University in Japan.

Suda, who admired the technology while visiting a colleague's lab at Stanford in 2006, uses the Fluidigm chip to examine subpopulations of HSCs, which, although highly purified, can vary widely in function and gene expression. He recently used the chip to analyze key genes involved in the expression of the cell-adhesion molecule N-cadherin in HSCs, a controversial area of research, and his results are currently in press.

Single-cell analysis, including imaging and gene expression, can reveal information population studies cannot. (Fluidigm Corporation)

Mylene Yao, who studies early mammalian embryos at the Stanford School of Medicine and who is a past consultant for Fluidigm, heard of the chip from colleagues. With collaborators, her team used it to investigate the role of Oct4, a pluripotency regulator in embryonic stem cells, and found it to be a critical regulator of the early embryo's gene network5. Because of the time and cost involved in isolating enough two-cell mouse embryos for conventional RT-PCR, "we wouldn't even have attempted what we did", says Yao. "The chip allowed us to validate a large number of genes in a very short time with a limited amount of biological materials." During the review process for their paper, reviewers expressed curiosity about the new technology rather than scepticism or resistance, Yao recalls, which could be attributed to its extensive use in other Stanford labs and the quality of the data, which is highly reproducible.

But for all its positives, the chip is still restricted to gene-expression studies and cannot do all the work for you. "Microfluidics allows you to multiplex an experiment and make it more efficient," says Peter Zandstra, chair of stem cell bioengineering at the University of Toronto, who doesn't currently use the technology, "but you still need to know what genes to look for."

Such technologies might be on the horizon. In the upcoming issue of Nature Methods, Applied Biosystems and collaborators, including M. Azim Surani of the University of Cambridge, describe a technique for high-throughput whole-transcriptome analysis of single cells. Using a single mouse blastomere, researchers were able to detect the expression of 75% more genes than they were able to using microarray techniques, and they were able to pinpoint 1,753 previously unknown splice junctions6. The protocol, which uses current off-the-shelf reagents, will be made available to customers shortly after publication, says Kaiqin Lao, principal scientist in Applied Biosystems' molecular cell biology unit and coauthor of the paper.

The technology is an interesting and valuable step forward, says Zandstra. However, he notes, "we still do not know, of all the genes and splice variants that this technology will output, which are functionally predictive of a particular stem cell state." It is possible, he adds, that by comparing results from stem cells with those from closely related non-stem cells, one might pinpoint the gene candidates worth further investigation.