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Most often associated with stunning micrographs, quantum dots have been widely used in the imaging field because of the sensitivity they afford. But Mario Roederer at the US National Institutes of Health Vaccine Research Center, and his colleagues, adopted these fluorochromes to improve the ability to resolve subsets of immune cells.

The complexity of the immune system translates into a combination of different proteins on the surface of each cell type, and these markers can be identified by fluorescent antibody detection and subsequent flow cytometry analysis—a process known as immunophenotyping.

“What we find is that we can't adequately describe the functionalities of the immune compartment by just making a single measurement like 'Do they make gamma interferon?' or 'Do they divide or not?', but it's the combination of all these measurements that will start resolving interesting subsets,” says Roederer. With organic fluorochromes, Roederer and colleagues had been able to achieve 12-color flow cytometry. Addition of quantum dots, with their limited spectral overlap (Fig. 1), allowed them to resolve 18 colors to date.

Figure 1: Emission spectra of various quantum dots.
figure 1

Blue diamonds mark the wavelengths for long-pass dichroic filters, and band-pass filters are shown as gray bars. Image reprinted with permission from Nature Medicine.

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Selection of the fluorophore panel for a given experiment, however, remains a trial-and-error process. Based on their extensive experience working with the fluorochromes, Roederer and colleagues match the brightest reagents with less-abundant markers, keeping in mind that spectral overlap reduces sensitivity. Yet to develop a large panel of fluorophores, they test several different colors of each of the antibodies they want to use. “We start by mixing several different combinations of six or eight antibodies and see how that performs, and then we start layering in additional reagents and continually revising the panel,” explains Roederer.

In new work reported in Nature Medicine, they used quantum dot–conjugated antibodies in combination with organic fluorochrome–conjugated antibodies to assay the phenotype of antigen-specific T cells. In this 17-color analysis, they were able to identify the expected markers, but also found new phenotypic differences between the populations.

As in imaging, the addition of quantum dots to the palette of fluorescent tags affords greater sensitivity. As Roederer puts it, “Historically, we've been limited to three or four different tags, but now we are working our way to where we have several dozen, and that gives us just much more flexibility in designing and implementing experiments where we want to detect fluorescent tags.”