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measuring the different lifetimes of
endogenously fluorescent compound
NADH in its free and bound states,
one can distinguish cells with normal metabolism and ones with abnormally high metabolic rates—a hallmark of cancer. Being able to do so on
a flow cytometry platform would allow
rapid discrimination of cancer cells
out of large samples of normal background cells. NMSU researchers are
also now sorting cells using the fluorescence lifetime as a single parameter. 12 Therefore, sorting out cells with
altered metabolism based on fluorescence decay times is near.
An even broader goal is to use fluorescence lifetime as a separate dimension to
massively increase the multiplexing capabilities of flow cytometers (see Fig. 5b). By
using fluorescence lifetime as a parameter, spectrally overlapping emissions can
be distinguished. Even just doubling the
number of channels available for detection—a rather conservative estimate of
the potential benefit of multiplexing
with fluorescence lifetime—would be a
huge boon to immunologists and other
cell biology researchers currently forced
to work with a limited number of tags on
the single cramped dimension of the visible spectrum.
Another tantalizing benefit potentially achievable with fluorescence lifetime multiplexing is the elimination
of spectral spillover compensation. By
trading some spectral channels for lifetime channels, the remaining spectral channels can be spaced further
apart, making compensation unnecessary. As any flow cytometrist will attest,
compensation is a source of unending
headaches for operators and researchers alike. Eliminating spectral overlap would radically simplify the performance of even complex flow cytometry
protocols, speeding up workflow, reducing operating costs, and improving the
quality of results. And that’s something
everyone can get behind! «
The authors recognize the contribu-
tions to the Danube I and II projects
from Mark Naivar at DarklingX, LLC,
with custom data acquisition module
design and development and the Kytos
data analysis software.
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12. B. Sands et al., PLoS ONE, 9,
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Houston, and J. P. Houston,
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Acta , 1823, 215–226 (2012).
15. J. Houston and S. Leavesley, co-chairs, C Y TO 2014: XXIX Congress of
the International Society for Advancement of Cytometry, Workshop #7 (Ft.
Lauderdale, FL; 2014).
16. P. Jenkins, J. Houston, W. Peria, B.
Sands, and R. Brent, CYTO 2014:
XXIX Congress of the International
Society for Advancement of Cytometry, Paper #97 (Ft. Lauderdale, FL;