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Fluorescence Spectroscopy: Instrumentation

المؤلف:  Wilson, K., Hofmann, A., Walker, J. M., & Clokie, S. (Eds.)

المصدر:  Wilson and Walkers Principles and Techniques of Biochemistry and Molecular Biology

الجزء والصفحة:  8th E , P481-482

2026-07-23

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Fluorescence spectroscopy works most accurately at very low concentrations of emitting fluorophores. UV/vis spectroscopy, in contrast, is least accurate at such low concentrations. One major factor adding to the high sensitivity of fluorescence applications is the spectral selectivity. Due to the Stokes shift, the wavelength of the emitted light is different from that of the exciting light. Another feature makes use of the fact that fluorescence is emitted in all directions. By placing the detector perpendicular to the excitation pathway, the background of the incident beam is reduced.

The schematics of a typical spectrofluorimeter are shown in Figure 1. Two monochromators are used, one for tuning the wavelength of the exciting beam and a second one for analysis of the fluorescence emission. Due to the emitted light always having a lower energy than the exciting light, the wavelength of the excitation monochromator is set at a lower wavelength than the emission monochromator. The better fluorescence spectrometers in laboratories have a photon-counting detector yielding very high sensitivity. Temperature control is required for accurate work as the emission intensity of a fluorophore is dependent on the temperature of the solution.

Fig1. Schematics of a spectrofluorimeter with ‘T’ geometry (90°). Optical paths are shown as blue lines. Inset: Geometry of front-face illumination.

Two geometries are possible for the measurement, with the 90° arrangement most commonly used. Pre- and post-filter effects can arise owing to absorption of light prior to reaching the fluorophore and the reduction of emitted radiation. These phenomena are also called inner filter effects and are more evident in solutions with high concentrations. As a rough guide, the absorption of a solution to be used for fluorescence experiments should be less than 0.05. The use of microcuvettes containing less material can also be useful. Alternatively, the front-face illumination geometry ( Figure 1 inset) can be used, which obviates the inner filter effect. Also, while the 90° geometry requires cuvettes with two neighbouring faces being clear (usually, fluorescence cuvettes have four clear faces), the front-face illumination technique requires only one clear face, as excitation and emission occur at the same face. However, front-face illumination is less sensitive than 90° illumination.