Instrumentation for Fluorescence Spectroscopy

Abstract

The success of fluorescence experiments requires attention to experimental details and an understanding of the instru- mentation. There are also many potential artifacts that can distort the data. Light can be detected with high sensitivity. As a result, the gain or amplification of instruments can usually be increased to obtain observable signals, even if the sample is nearly nonfluorescent. These signals seen at high amplification may not originate with the fluorophore of interest. Instead, the interference can be due to back- ground fluorescence from the solvents, light leaks in the instrumentation, emission from the optical components, stray light passing through the optics, light scattered by tur- bid solutions, and Rayleigh and/or Raman scatter, to name a few interference sources. An additional complication is that there is no ideal spectrofluorometer. The available 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 detector tubes. The polarization or anisotropy of the emitted light can also affect the measured fluorescence intensities because the efficiency of gratings depends on polarization. It is important to understand and control these numerous factors. In this chapter we will discuss the prop- erties of the individual components in a spectrofluorometer, and how these properties affect the observed spectral data. These instrumental factors can affect the excitation and emission spectra, as well as the measurement of fluores- cence lifetimes and anisotropies. Additionally, the optical properties of the samples—such as optical density and tur- bidity—can also affect the spectral data. Specific examples are given to clarify these effects and the means to avoid them.