Abstract
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, ε, φ, and %T to fluorescence calibration standards, where ε is the absorption coefficient of the fluorophore, φ 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.