Instrumentation for Fluorescence Spectroscopy

  • Joseph R. Lakowicz


The successful application of fluorescence methods requires considerable attention to experimental details and a good understanding of the instrumentation. There are numerous potential artifacts which can distort the data. Fluorescence is a highly sensitive method. The gain or amplification of the instruments can usually be increased to obtain observable signals, even if the sample is nonfluorescent. These signals seen at high amplification 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 passing through the optics, light scattered by turbid solutions, Rayleigh scatter, and/or Raman scatter, to name a few sources of interference. Furthermore, there is no ideal spectrofluorometer, and 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 detectors (photomultiplier tubes). The polarization or anisotropy of the emitted light can also affect the measured 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 how these properties affect the observed spectral data.


Emission Spectrum Fluorescence Spectroscopy Xenon Lamp Stray Light Exciting Light 
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Copyright information

© Springer Science+Business Media New York 1999

Authors and Affiliations

  • Joseph R. Lakowicz
    • 1
  1. 1.University of Maryland School of MedicineBaltimoreUSA

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