Technology
Avoiding fluorescence
Avoiding fluorescence
Fluorescence
Many materials are coloured. Often, these materials absorb light in a specific part of the visible spectrum. But most of the light is transmitted or reflected; this light fraction can be observed as the complementary colour of the absorbed light.
The absorbed light excites electrons of the material to higher energy levels for a very short time. Usually, the electrons return quickly to the ground state by emitting light of the same energy or wavelength, respectively. However, in some materials this return needs by far more time and is accompanied by loosing part of its energy due to internal molecular processes. As a result, light is emitted at longer wavelengths compared to the absorbed light. This process is called fluorescence.
Competing processes of Raman scattering and fluorescence
If Raman scattering is excited close to an electronic transition, it sometimes coincides with fluorescence. Since fluorescence is typically much more intense, with a broad emission, already very low amounts of fluorescent species can cover the Raman scatter of a material. Without Raman scattering enhancement techniques, detection limits are in the range of about 0.1% to 1%. Since fluorescence is typically several orders of magnitude stronger than Raman scattering, already fluorescent traces of impurities below the detection limit may avoid the detection of the Raman spectrum of the major material.
Strategies to avoid fluorescence
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Selecting the appropriate excitation wavelength
Selecting an excitation wavelength far away from any electronic transition of the sample generally avoids fluorescence. Fluorescence can be avoided most effectively by NIR FT Raman spectroscopy. -
Purifying the sample
This method is appropriate for samples where fluorescence originates from impurities. -
Photo-bleaching
Irradiating the sample with intense light for a few minutes, preferably by the Raman laser, decomposes fluorescent species. This method is applicable for solid samples where fluorescence originates from impurities. -
Confocal setup
Spatial filtering by the pinhole of a confocal microscope reduces the detected fluorescence from outside of the laser focus and detects the Raman scatter within the laser focus. Applicable to relatively small intense fluorescent samples. -
Shifting and subtraction technique
This method is applicable for low fluorescent samples. Two Raman spectra are collected subsequently at slightly different laser wavelengths. Subtracting the spectra from one another removes the unchanged fluorescence background. The Raman spectrum is reconstructed from the difference spectrum by a mathematical procedure. -
Baseline correction
Small intense fluorescent background is removed via a mathematical procedure. Typically applied for removing a remaining low-intense fluorescent background after photo-bleaching. -
Enhancement by RR and SERS
Raman scatter intensities are increased relatively to fluorescence by resonance Raman scattering (RR) and surface enhanced Raman scattering (SERS).
