TY - JOUR
T1 - Correction for contaminant fluorescence in frequency-domain fluorometry
AU - Lakowicz, Joseph R.
AU - Jayaweera, Ranjith
AU - Joshi, Nanda
AU - Gryczynski, Iganzy
PY - 1987/2/1
Y1 - 1987/2/1
N2 - We describe a general method to correct for contaminant fluorescence when using the technique of frequency-domain fluorometry. The method can be applied regardless of the origin of the background signal, from scattered light, impurity fluorescence, or both. The procedure requires measurement of the frequency-dependent phase and modulation of the background at enough frequencies to approximate the decay law of the background. We also describe a general method to propagate the uncertainties in the measured phase and modulation values into the corrected values. This propagation is necessary to ensure proper weighting of the frequency-dependent data in the least-squares fitting algorithms. The practical usefulness of this correction method is demonstrated using frequency-domain data for one and two component mixtures which were deliberately contaminated with scattered light and/or other fluorophores.
AB - We describe a general method to correct for contaminant fluorescence when using the technique of frequency-domain fluorometry. The method can be applied regardless of the origin of the background signal, from scattered light, impurity fluorescence, or both. The procedure requires measurement of the frequency-dependent phase and modulation of the background at enough frequencies to approximate the decay law of the background. We also describe a general method to propagate the uncertainties in the measured phase and modulation values into the corrected values. This propagation is necessary to ensure proper weighting of the frequency-dependent data in the least-squares fitting algorithms. The practical usefulness of this correction method is demonstrated using frequency-domain data for one and two component mixtures which were deliberately contaminated with scattered light and/or other fluorophores.
KW - background correction
KW - fluorescence spectroscopy
KW - frequency-domain fluorometry
KW - scattered light
KW - time-resolved fluorescence
UR - http://www.scopus.com/inward/record.url?scp=0023114591&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(87)90078-9
DO - 10.1016/0003-2697(87)90078-9
M3 - Article
C2 - 3578773
AN - SCOPUS:0023114591
VL - 160
SP - 471
EP - 479
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -