Abstract
Time-resolved fluorescence spectroscopy is presently regarded as a research tool in biochemistry, biophysics and chemical physics. However, advances in laser technology and the development of long-wavelength probes are resulting in the rapid migration of lifetime-based methods to the clinical chemistry lab and the patient's bedside. The advantages of lifetime-based fluorescence sensing are apparent from the various possible schemes for fluorescence sensing (Fig. 1). At present, most fluorescence assays are based on the intensity measurements, in which the intensity of the probe molecule changes in response to the analyte (far left). Intensity measurements are simple and accurate in the laboratory, but are often inadequate in real-world situations. The sample may be turbid, the optical surfaces maybe misaligned or imperfect, and the probe concentration may vary from sample to sample. The problems of intensity-based sensing can be avoided using wavelength-ratiometric probes, that is, fluorophores which display spectral changes in the absorption or emission spectrum upon binding with the analytes. In this case, the analyte concentration can be determined independently of the probe concentration by the ratio of intensities at two excitation or two emission wavelengths. However, few such wavelength-ratiometric probes are available.
© 1995 Optical Society of America
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