Background suppression in frequency-domain fluorometry.
Gated detection is often used in time-domain measurements of long-lived fluorophores for suppression of interfering short-lived autofluorescence. However, no direct method has been available for gated detection and background suppression when using frequency-domain fluorometry. We describe a direct method for real-time suppression of autofluorescence in frequency-domain fluorometry. The method uses a gated detector and the sample is excited by a pulsed train. The detector is gated on following each excitation pulse after a suitable time delay for decay of the prompt autofluorescence. Under the same experimental conditions a detectable reference signal is obtained by using a long lifetime standard with a known decay time. Because the sample and reference signals are measured under identical excitation, gating and instrumental conditions, the data can be analyzed as usual for frequency-domain data without further processing. We show by simulations that this method can be used to resolve single and multiexponential decays in the presence of short lifetime autofluorescence.
Lakowicz
J R
JR
University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, Maryland, 21201, USA.
Gryczynski
I
I
Gryczynski
Z
Z
Johnson
M L
ML
D005453:Fluorescence; D005470:Fluorometry / Q000379:methods*; D007700:Kinetics; D008962:Models, Theoretical; D012044:Regression Analysis; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors