cytometry, (4) microengraving, and (5) single-cell barcoding
microchip. In this chapter, we examine the advantages and limita-
tions of each technique (see Fig. 1) and summarize single-cell
protein copy numbers enabled by these approaches (seeTable1).
Future research opportunities are highlighted based on four mea-
surement considerations, which are absolute quantification, detec-
tion resolution, multiplexing capability, and throughput.2 Flow Cytometry
Currently, fluorescent flow cytometry is the golden standard in
single-cell protein analysis where single cells stained with
fluorescence-labeled antibodies are flushed in a capillary tube with
fluorescent levels quantified by photomultiplier tubes [44, 45](see
Fig.1a). After its initial invention, key technical developments
include the detection of multiple fluorescence simultaneous and
the inclusion of fluorescence-labeled calibration beads for absolute
quantitative measurement [46]. Leveraging commercially available
flow cytometry, quantitative evaluations of surface antigen expres-
sions of single cells were realized, contributing dramatically to a
variety of diseases.
Although well-established, fluorescent flow cytometry still has
several key limitations, which severely compromise its functional-
ities in the field of single-cell protein analysis. Fluorescent flow
cytometry can only provide an absolute quantitation of surface
proteins of single cells leveraging calibration beads, while it cannot
quantify intracellular proteins due to the lack of calibration
approaches [47–50]. In addition, fluorescent flow cytometry can-
not also effectively quantify secreted proteins at the single-cell level,
and it cannot simultaneously quantify more than 20 types of pro-
teins due to fluorescent spectrum overlap.3 Mass Cytometry
Since flow cytometry has the limitation of simultaneously charac-
terizing multiple fluorescent signals due to the emission spectral
overlaps, mass spectroscopy was proposed where rare earth metal
isotope tags were used to replace fluorescence labels and 100 of
signals can be detected simultaneously [51–55]. When performing
a mass cytometry experiment, cells of interest are first stained with
antibodies previously conjugated to stable heavy-metal isotopes.
Then cells are loaded into a nebulizer, which places cells into
droplets for introduction into the mass cytometer. Upon entering
the instrument, cells travel through an argon plasma, in which
covalent bonds are broken to produce charged free atoms. The
resulting ion cloud is passed through a quadrupole to discard294 Beiyuan Fan et al.