Fluorescence Notes

Sample Preparation:
  • Use UV-grade solvents and avoid using water that has been stored in plastic bottles as small amounts of fluorescent species can leach from plastic materials.
  • To avoid inner filter effects and other artifacts, keep the sample absorbance at the excitation wavelength <0.05-0.1 in a 1 cm pathlength cell.
  • The fluorescence of many fluorophores is pH dependent, so make sure your samples are buffered sufficiently.
  • Dust or aggregates will scatter light and interfere with fluorescence experiments. Filter or centrifuge all solutions to remove any turbidity (0.22µ filter or 10 min. spin at highest speed in microfuge). This is especially important in anisotropy measurements since scattered light is 100% polarized.
Experimental Setup on CSB Instrument:
  • The linear range of the Fluoromax-3 detector is from 1 - 3,000,000 cps. If your signal > 3,000,000 cps, the accuracy and linearity decrease. The error when intensity = 4,000,000 cps is ~5%, so Horiba Jobin-Yvon use this as the upper limit. If your signal exceeds that, you need to dilute your sample or narrow your slitwidths.
  • There's no real lower limit of detection--your data will just become noisier as the intensity drops. For emission scans, you can increase the integration time to effectively smooth the data.
  • The fluorescence of most fluorophores is sensitive to temperature. Since the temperature in MRB3 5141 is highly variable, it's recommended that you use the water bath for temperature control.
  • You cannot use the round microcuvette for anisotropy experiments. The CSB has a special square microcuvette (250uL viewed volume; ~220uL minimum volume) that can accommodate a small stir bar--please inquire.
Fluorescence Factoids:
  • The Rayleigh scattering peak occurs at the same wavelength as excitation and will increase markedly if there is dust or aggregate in the solution. The Rayleigh peak heights for your sample solution and buffer blank should be similar.
  • The Raman peak is a low intensity band of scattered radiation whose distance from the excitation band is a measure of the vibrational energy of the H-O bond in water. In general, it occurs at: 1/((1/λex)-(3.6 x 10-4)). At λex=280 nm, the water Raman occurs at 311 nm.
  • Quantum yields for Trp, Tyr, and Phe are 0.2, 0.1 and 0.04, respectively so Trp dominates when present.
  • Excitation at 280 nm excites both tryptophan and tyrosine. Excitation at 295 nm or higher selectively excites tryptophan.
  • λmax and Imax of tryptophan are dependent on the polarity of the environment of the chromophore(s) in a protein and typically varies from 308 nm (buried) to 352 nm (fully exposed).
  • The wavelength of Tyr fluorescence does not vary with polarity of the environment and occurs with a maximum at 303 nm. Tyrosinate is weakly fluorescent with a maximum around 345 nm. It is most easily observed at high pH, but can be seen at neutral pH's as well and can be mistaken for Trp.
  • Imidazole absorbs at the same wavelength as tryptophan.
  • Phosphate can quench tyrosine fluorescence.
  • Oxidized DTT absorbs light over a similar wavelength range as tryptophan. If you must use DTT as a reducing agent, add it just prior to starting the experiment.
FluorEssence Operational Notes