PHOSPHO FLOW CYTOMETRY: SINGLE-CELL SIGNALING NETWORKS IN NEXT-GENERATION DRUG DISCOVERY AND PATIENT STRATIFICATION - Flow Cytometry in Drug Discovery and Development - page 316

15.7 NEXT-GENERATION CYTOMETRY
Current cytometers, based on fluorescence, can measure up to 17 parameters per
cell simultaneously [27]. However, even through the use of time-delayed
excitation and detection, novel fluorophores such as quantum dots, and upcon-
version, extension to more than 20 parameters seems to be unlikely. In addition,
more than 10-12 parameter experiments, possible on some commercial instru-
ments, require skilled experimental design to account and correct for spectral
overlap. Often called “compensation,” spectral overlap causes emission from
one fluorophore to be measured in multiple detector channels, convoluting
analysis and reducing the quantitative nature of the experiments (Figure 15.9).
Now a new platform, mass cytometry, offers single-cell analysis capabilities
potentially approaching 100 parameters without fluorescent agents or interfer-
ence from spectral overlap (Figure 15.9). This platform utilizes nonbiological,
rare earth metal isotopes as reporters. By exploiting the resolution, sensitivity,
and dynamic range of elemental mass spectrometry, but on a timescale that
allows the measurement of 1000 individual cells per second, this device
potentially offers a much simplified alternative for ultrahigh content cytometric
analysis.
FIGURE 15.9 Comparison of traditional fluorescence-basedcytometryandmass-based
cytometry. In fluorescence-based cytometry (a), the emission spectra of the various
fluorophores overlap one another significantly. Although bandpass filters can be used to
minimize compensation between fluorophores, they are not able to eliminate fluorescence
spillover completely. The breadth of the emission spectra limits the number of fluorophores
that can realistically be used in any one experiment. In the mass-based cytometry on the
other hand (b), the mass labels appear at only one discrete mass value when measured. With
a well-tuned mass cytometer, compensation is not required. Signals from one mass label
will only interfere with another mass label in cases of oxidation ( þ 16Da) or from
impurities in the isotopic labels themselves. The 30 elemental metal isotope labels plus
2 DNA intercalators (Ir-191 and Ir-193) shown in this plot are available at extremely high
purity and do not produce overlapping oxides, essentially enabling compensation-free
analysis of all 30 parameters plus DNA content. (See the color version of the figure in the
Color Plates section.)