Instrumentation for Fluorescence Spectroscopy

Abstract

The successful application of fluorescence methods requires an understanding of the instrumentation. Considerable attention to the experimental details is necessary. There are two reasons for this. First, fluorescence is a highly sensitive method. The gain or amplification of the instruments can invariably be increased to obtain observable signals. However, these signals may not originate with the fluorophore of interest. Instead, one may observe interference due to background fluorescence from the solvents, light leaks in the instrumentation, stray light scattered by turbid solutions, Rayleigh and/or Raman scatter, to name a few. Secondly, there is no ideal spectrofluorometer and these instruments do not yield true excitation or emission spectra. This is because of the nonuniform spectral output of the light sources and the wavelength-dependent efficiency of the monochromators and of the photomultiplier tubes. The polarization or anisotropy of the emitted light can also affect the measurement of fluorescence intensities. To obtain reliable spectral data one needs to be aware of and control these numerous factors. In this chapter we will discuss the properties of the individual components in a spectrofluorometer and those properties of samples which affect the measured parameters. In our discussion of these instrumental factors we will stress the acquisition of excitation and emission spectra. However, similar concerns are important in the measurement of fluorescence lifetimes and anisotropics, which will be described in Chapters 3 and 5.