Extending fluorescent microsphere methods for regional organ blood flow to13 simultaneous colors

Seven fluorescent microsphere colors can be used in a single experiment to estimate regional blood flow without correcting for spillover of emitted fl uorescence. To extend the method to 13 colors, we compared the accuracy of three methods for spillover correction. Fixed wavelength intensities were c orrected by matrix inversion, and synchronous scan spectra were corrected b y least squares fit of an overdetermined system of linear equations and by least squares fit of a sum of Gaussian and Lorentzian functions. Correction methods were validated in pigs and sheep by simultaneous injections of rad ioactive microspheres and fluorescent microspheres of 7, 10, and 13 differe nt colors. We induced extreme changes in flow to create regions with low fl uorescent signals bound on either side by high fluorescent signals. Blood f low was determined by radioactivity and by fluorescence using both fixed ex citation and emission wavelength pairs and synchronous scanning and then co rrected for spillover. Correlation between fluorescent intensity and radioa ctivity were excellent for all three correction methods [R-2 = 0.98 +/- 0.0 2 (mean +/- SD)]. Low-flow regions requiring large spillover correction had systematic errors for some color combinations in all methods. We conclude that for 13 fluorescent colors spillover error can be minimized so that all three correction methods provide accurate estimates of regional blood flow .