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Fluorescent microspheres, beads and particles applications including blood flow determination, tracing, fluorimetry, in vivo imaging and calibration of imaging and flow cytometry instruments. Because our fluorescent dyes are incorporated in the bead and not just on the surface, they are relatively immune to photo bleaching and other environmental factors. Spheres are in difference sizes and fluorescences, high intensity, FITC, GFP, red, green, yellow, light yellow, sky blue, blue, orange, deep-red, Nile-red, purple, mcherry. The diameter of the spheres is usually higher than 50nm and in the micrometer um range.