A fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore. This technology has particular importance in the field of biochemistry and protein studies, eg. in immunofluorescence and immunohistochemistry.

The choice of the type of fluorochrome to be used is fundamental for the sample to be analyzed and for the results to be obtained. The peculiarity of this substance is to absorb photons of a certain wavelength and as an effect exhibit fluorescence. Electrons passing from the state of excitation to the rest state lose energy. Consequence of this loss is the shift of the emission spectrum of fluorochromes towards longer wavelengths than the absorption spectrum (excitation).

Note that the wavelength varies inversely with the energy of the radiation. This phenomenon is known as Stokes’ law or Stokes shift. The size of the “shift” varies with the molecular structure, but can range from a few nanometers to over several hundred nanometers. For example, the Stokes shift for Fluorescein is about 20 nanometers, while that for HOECHST 33258 is 120 nanometers and that for Acridine Orange (+RNA) is about 200 nanometers. The use of optimal filters allows to increase the value of the shift and, consequently, to separate more easily the excitation light.

The curve that represents the emission, in the spectrum, of fluorochromes is usually lower or equal in intensity than the curve of excitation. In the representation graph the two curves have an almost specular shape. For example see the curves of the fluorochrome FITC in the figure below, which absorbs in the region of blue-green and produces a green-yellow emission. To achieve maximum luminous efficiency, fluorochromes are generally excited at the absorption peak. The selection of excitation and emission wavelengths is done by interference filters. In addition, the spectral response of the optical system is also dependent on other factors such as the transmission efficiency of the lens, the power and type of light source, the type of fluorochrome used.

Leave a Comment