Fluorescence Made Easier: Fluorescence Techniques for the Novice Episode 3:. Avoiding Those Pesky Artifacts: Sources of Error in Spectra and Steady State Measurements

  • Richard B. ThompsonEmail author
  • Suzanne F. Scarlata
Part of the Reviews in Fluorescence book series (RFLU)


For those contemplating employing fluorescence methods in their experiments, there are some potential artifacts and sources of error that can invalidate results. With a little care, these artifacts can be avoided to produce the kind of results that will make the reviewers jump for joy (and accept your paper). We will cover some sources of error arising from issues arising from the sample itself, and basic problems with fluorometers. At the end we have a handy checklist for when your spectrum comes out looking really messed up.


Raman scattering Rayleigh scattering Inner filter effects Instrument troubleshooting Artifacts Sample purity 



RBT wishes to belatedly thank Peter M. Torgerson (among many who taught him fluorescence) for the concept of the Golden Rules.


  1. 1.
    Chen RF, Knutson JR (1988) Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes: energy transfer to nonfluorescent dimers. Anal Biochem 172(1):61–77CrossRefGoogle Scholar
  2. 2.
    Cheong WF, Prahl SA et al (1990) A review of the optical properties of biological tissues. IEEE J Quantum Electron 26:2166–2185CrossRefGoogle Scholar
  3. 3.
    Engstrom RW (1980) RCA photomultiplier handbook. RCA Solid State Division, LancasterGoogle Scholar
  4. 4.
    Hamamatsu Photonics K.K., E. T. D (2016) Photomultiplier tubes and related products. Hamamtsu Photonics K.K, Iwata CityGoogle Scholar
  5. 5.
    Moore JH, Davis CC et al (1983) Building scientific apparatus: a practical guide to design and construction. Addison-Wesley Publishing Co, ReadingGoogle Scholar
  6. 6.
    Ntziachristos V (2006) Fluorescence molecular imaging. Annu Rev Biomed Eng 8:1–33CrossRefGoogle Scholar
  7. 7.
    Plant AL (1986) Mechanism of concentration quenching of a xanthene dye encapsulated in phospholipid vesicles. Photochem Photobiol 44(4):453–459CrossRefGoogle Scholar
  8. 8.
    Scarlata S, Thompson RB (2017) Fluorescence made easier: fluorescence techniques for the novice. Episode 2: Using fluorescence anisotropy or polarization to view intermolecular associations. In: Geddes CD (ed) Reviews in fluorescence 2016. Springer, Cham, pp 9–15CrossRefGoogle Scholar
  9. 9.
    Thompson RB (1994) Red and near-infrared fluorometry. In: Lakowicz JR (ed) Topics in fluorescence spectroscopy Vol. 4: Probe design and chemical sensing, vol 4. Plenum Press, New York, pp 151–181CrossRefGoogle Scholar
  10. 10.
    Thompson RB, Scarlata S (2017) Fluorescence made easier: fluorescence techniques for the novice. Episode 1: The Basics. In: Geddes CD (ed) Reviews in fluorescence 2016. Springer, Cham, pp 1–8Google Scholar
  11. 11.
    Turro NJ (1978) Modern molecular photochemistry. Benjamin/Cummings Publishing Co, Menlo ParkGoogle Scholar
  12. 12.
    Wolfbeis OS (1985) The fluorescence of organic natural products. In: Schulman SG (ed) Molecular luminescence spectroscopy methods and applications: part I. Wiley-Interscience, New York, pp 167–370Google Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Maryland School of MedicineBaltimoreUSA
  2. 2.Department of Chemistry and BiochemistryWorcester Polytechnic InstituteWorcesterUSA

Personalised recommendations