Assignment of the Number of Equivalent Reference Fluorophores to Dyed Microspheres

A procedure will be described to assign to each dyed microsphere a number called the Equivalent number of Reference Fluorophores (ERF). The ERF unit gives the number of reference fluorophores in solution which produce the same fluorescence signal as a single dyed microsphere. In the first step, fluorescence measurements were carried out on serial dilutions of a solution of reference fluorophores. The resulting fluorescence intensities and the corresponding concentrations were used to calibrate the response of the fluorometer. The calibration consisted of establishing a linear relation between the intensities and concentrations. In the second step, the fluorescence intensity from a suspension of microspheres was measured in order to determine the equivalent concentration of reference fluorophores which gave the same fluorescence intensity as the suspension of microspheres. This was performed by utilizing the calibration line obtained in the first step. In the third step, a flow cytometer and a light obscuration apparatus were used to measure the total concentration of microspheres in the suspensions used for the fluorescence measurements. In addition to the total microsphere concentration, the flow cytometer also enabled the measurement of the concentration of a sub population of microspheres which are used to calibrate the fluorescence scale of a flow cytometer. The fourth step utilized the data collected in steps one, two, and three to assign a value of ERF to individual microspheres. The set of microspheres with assigned ERF values will be used to establish a linear fluorescence scale in each channel of a flow cytometer. The discussion will emphasize the estimate of uncertainties in each step of the assignment process.