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2011 ; 699
(ä): 67-84
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Quantum dots for quantitative flow cytometry
#MMPMID21116979
Buranda T
; Wu Y
; Sklar LA
Methods Mol Biol
2011[]; 699
(ä): 67-84
PMID21116979
show ga
In flow cytometry, the quantitation of fluorophore-tagged ligands and receptors
on cells or at particulate surfaces is achieved by the use of standard beads of
known calibration. To the best of our knowledge, only those calibration beads
based on fluorescein, EGFP, phycoerythyrin and allophycocyanine are readily
available from commercial sources. Because fluorophore-based standards are
specific to the selected fluorophore tag, their applicability is limited to the
spectral region of resonance. Since quantum dots can be photo-excited over a
continuous and broad spectral range governed by their size, it is possible to
match the spectral range and width (absorbance and emission) of a wide range of
fluorophores with appropriate quantum dots. Accordingly, quantitation of site
coverage of the target fluorophores can be readily achieved using quantum dots
whose emission spectra overlaps with the target fluorophore.This chapter focuses
on the relevant spectroscopic concepts and molecular assembly of quantum dot
fluorescence calibration beads. We first examine the measurement and
applicability of spectroscopic parameters, ?, ?, and %T to fluorescence
calibration standards, where ? is the absorption coefficient of the fluorophore,
? is the quantum yield of the fluorophore, and %T is the percent fraction of
emitted light that is transmitted by the bandpass filter at the detector PMT. The
modular construction of beads decorated with discrete quantities of quantum dots
with defined spectroscopic parameters is presented in the context of a
generalizable approach to calibrated measurements of fluorescence in flow
cytometry.
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