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Fluorescence Spectroscopy

Fluorescence spectroscopy
Fluorescence spectroscopy

Fluorescence spectroscopy capabilities utilizes both continuous wave (xenon lamp or solid-state lasers) or pulsed light source (Nd-YAG laser-pumped MOPO laser)  for measurements from UV (220 nm) up to near infrared (~ 1700 nm). These systems are integrated with flow through liquid cells, sample heating, liquid sample stirring and polarization control on both excitation and emission. The spectrometer offers efficient measurement—samples down to liquid helium temperature—while an integrated invert microscope allows measurement of solid samples with spatial resolution down to a few microns. The latter can provide sample analysis down to liquid helium temperature. All the temperature adjustment and polarization control are also available on the pulsed laser-induced time-resolved nanosecond fluorescence spectrometer. All fluorescence spectrometers can measure solid, liquid, solution suspension, and even gas samples. The suite of fluorescence instruments is designed to study humic substances in nature, complex reaction dynamics, such as enzymatic reactions, protein-protein interactions, aromatic hydrocarbon, and their adsorption on soils and sediments, and interfacial electron transfer processes.

Research application

  • Fluorescence spectroscopy supports the Terrestrial-Atmospheric Processes Integrated Research Platform by helping to understand complex nutrient dynamics, identify protein-protein reactions, and interactions between humic substances and other biomolecules within the soil matrix.  
  • The Biogeochemical Transformations Integrated Research Platform is assisted by fluorescence spectroscopy in the investigation of how nutrients, contaminants, aerosols, and other chemical compounds move and change in the environment. Fluorescence Spectroscopy enables researchers to gain new insights into the physiochemical effects of nutrient cycling in the environment, the interactions between metabolic pathways, and the dynamics of microbial communities and contaminants, such as aromatic hydrocarbons in spilled oil interaction with soil and sediment.
  • The Rhizosphere Function Integrated Research Platform utilizes fluorescence spectroscopy to characterize and model plant phenotypes at the molecular to organismal scale. For fluorescent metal ions and organic molecule, their uptake and transport can be tracked in real-time using the microscope-based spectrometer system. By investigating interactions at the molecular level, we are better able to understand, predict, and control plant traits at the plant-system scale.

Available instruments

  • A Horiba Fluorolog III spectrometer with NIR attachment.
  • A Nd-YAG laser pumped MOPO laser time-resolved fluorescence spectrometer is integrated with an inverted Nikon TE2000 optical microscope.
    • Technical specifications:
      • laser wavelength: 220 nm – 1800 nm
      • detector response: 440 nm-900 nm
      • time resolution: 5 ns
      • temperature range: 4.2 – 298 K

Tips for success

  • Fluorescence spectroscopy is a technique based on Beer’s Law, so sample concentration is critical to obtaining good fluorescence data. Fluorescence intensity is directly proportional to the excitation light and is linear when the sample absorbance is less than 0.05 AU in a 1 cm pathlength cell. If a sample is too concentrated, the emission light can be reabsorbed by the fluorophore attenuating the fluorescence signal at shorter wavelengths, resulting in apparent red shift emission. Excitation light may also not fully penetrate the full width of a highly concentrated sample, which will also lead to decreased fluorescence intensities. If solutions cannot be diluted, shorter pathlength or triangular cells can be used.
  • The fluorescence signal is proportional to the excitation light intensity and the slit sizes. This results in larger slits at higher intensity but also degrades the spectral resolution. Both the excitation and emission bandwidths should be varied to optimize a sample’s fluorescence signal and balance sensitivity and resolution needs.
  • For samples of weak fluorescence, increasing photomultiplier tube voltage will increase the signal level. However, care should be taken not to raise the photomultiplier tube voltage to above the specified voltage limit.
  • Scattered light can interfere with the fluorescence signal, particularly at shorter wavelengths. One way to know if the signal is due to scattered light is by changing the excitation wavelength. Scattered light tends to move along with the excitation wavelength while fluorescence spectra mostly not.

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