Abstract A method of calibrating a flow cytometer or fluorescent microscope is based on a set of highly uniform microbeads associated with a fluorescent dye in such a way that the microbeads have the same excitation and emission spectral properties as the samples which are to be measured. The calibration values of the microbeads are plotted against the relative fluorescence intensity peak channel for each microbead in the set. From this calibration plot, the relative fluorescence intensity peak channel of the sample is translated into equivalent soluble fluorescent dye molecules per sample particle. The calibration values of the standard microbeads are determined against solutions of the dyes. In cases where the background scatter of the bulk microbeads suspensions is too high for a direct determination against the solutions, a different set of microbeads with low background scatter is calibrated against the dye solutions and used to make an initial calibration of a flow cytometer or fluorescent microscope, which in turn, is used to calibrate the uniform microbead standards. A novel method of making the microbead standards is also disclosed.
The method of making a calibration microbead standard to minimize aggregation and maximize yield comprising the steps: (a) homogenizing a highly insoluble (<10 -3 g/l) compound of molecular weight <1000 in a surfactant solution to obtain a first homogenate; (b) swelling small seed microbeads, 0.1-3u in diameter, with said first homogenate; (c) homogenizing an aqueous surfactant solution containing a large stabilizing alkaline halide salt with a mixture of oil soluble polymerizable monomers within which is dissolved 0.5-5% by weight an oil soluble initiator to obtain a second homogenate; (d) swelling the seed microbeads a second time with said second homogenate; and (e) placing said twice swollen microbeads under conditions appropriate to cause said initiator to polymerize the monomers in the swollen microbeads.
Claim 2
Independent Claim
122 words
The method of making a calibration microbead standard to minimize aggregation and maximize yield comprising the steps: (a) homogenizing a highly insoluble (<10 -3 g/l) compound of molecular weight <1000 in a surfactant solution to obtain a first homogenate; (b) swelling small seed microbeads, 0.1-3u in diameter, with said first homogenate; (c) homogenizing an aqueous surfactant solution containing a large stabilizing alkaline halide salt with a mixture of oil soluble polymerizable monomers within which is dissolved 0.5-5% by weight an oil soluble initiator to obtain a second homogenate; (d) swelling the seed microbeads a second time with said second homogenate; and (e) placing said twice swollen microbeads under conditions appropriate to cause said initiator to polymerize the monomers in the swollen microbeads.
Claim 3
Dependent Claim
13 words
The method of claim 6 wherein said highly water soluble compound is 1-chlorododecane.
Claim 4
Dependent Claim
31 words
The method of claim 6 wherein said stabilizing alkaline halide salt is potassium chloride, said oil soluble monomers methyl methacrylate and glycidyl methacrylate, and said oil soluble initiator is benzoyl peroxide.