Reflected light enhancing intensity

The reflectance, R, is defined as the ratio of the reflected light intensity to the intensity of the incident beam. Usually, one determines the change in reflectance, A/ , induced by some parameter, such as the electrode potential. Experimentally, one measures only the intensity of the reflected beam, 4. So if the incident intensity remains constant, the reflected beam gives hJl/R = A4/4. Experimental results are presented as plots of A/J/R vs. the parameter of interest, such as the frequency of the incident light or electrode potential. Modulation schemes, wherein the beam is chopped or the potential is modulated, are used to enhance the signal-to-noise ratio. 

In addition, color film has an inherent artifact in areas of overexposure, in which the color of the film becomes yellow, no matter what the color of the exposing light. This produces apparent areas of enhanced concentration, which may or may not reflect the true intensity of label, especially in areas in which a low-level background image is added to the specific signal. This can lead to misinterpretations about selective areas of concentration of label. 

FIAs can be based on steady-state intensity measurements without probe amplification, owing to the sensitivity of detection that is possible with fluorescence instrumentation, which exceeds that of spectrophotometers by two or three orders of magnitude. A sensitive fluorometer has been described for an estradiol assay(36) in which the limit of estradiol detection is 3 x KT11 M. Estradiol antibody labeled with rhodamine B is reacted with estradiol samples. Unreacted labeled antibody is removed with Sepharose-estradiol-casein beads, and the remaining fluorescence is directly proportional to the analyte concentration. The detection limit of rhodamine B on the same fluorometer is 5 x 1(T12 M. This instrument uses a 0.75 mW green helium-neon (HeNe) laser to irradiate the sample from above, at the air-liquid interface, to increase the light path and to decrease surface reflections. The sample compartment has a top-mounted photon trap, and a mirror mounted on the side of the sample compartment opposite the PMT to enhance detection. 

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