Obtaining High Precision From Seemingly Inadequate Spot Sizes Using High Laser Power

In practice, high-precision fluorescence measurements have been made with beam geometries and core sizes that result in uneven illumination; this becomes possible when the illumination intensity is sufficiently high to produce photon saturation and bleaching of the dye being measured (van den Engh and Farmer,

1992). If the intensity at the edges of the core meets this criterion, the maximum possible fluorescence signal will be obtained from particles at the edge, and particles subjected to the higher illumination intensity on the core axis will not produce correspondingly stronger signals. In order to benefit from saturation, it is generally necessary to employ laser powers of hundreds of milliwatts for excitation; in most modern commercial instruments, in which 10 to 20 mW is a more typical figure, achieving high measurement precision requires a design in which illumination intensity is relatively uniform over the width of the core.

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