A method for on-line background subtraction in frequency domain fluorometry

Gregory D Reinhart,David M Jameson,Enrico Gratton,Pasquina Marzola

doi:10.1007/bf00865362

Abstract

Time-resolved fluorescence determinations on biochemical samples are often complicated by contributions from background. In the time-correlated single-photon counting method, background subtraction is a routine procedure. A limitation of frequency domain fluorometry, however, arises from the difficulty of performing this operation. This limitation has become increasingly significant as frequency domain methods are being applied to evermore complex biological systems using the higher-frequency capabilities of modern instrumentation. We have devised a method for such a correction in the frequency domain, regardless of the complexity of the background decay, based on measurement of the background phasor and subsequent subtraction from the sample phasor. This method is applicable to both lifetime and dynamic polarization measurements, and it can be readily implemented on commercially available frequency domain fluorometers. Decay curves may be accurately recovered from samples containing background contributions ranging from less than 5% to greater than 90% of the total signal intensity.

Highlights

Rather we have chosen a few specific examples with the purpose of illustrating the method, verifying its validity, comparing the on-line method with a posteriori data analysis, and demonstrating that such a posteriori data analysis is fundamentally inapplicable in some cases
The procedure outlined above for background correction represents a true on-line method that can be routinely implemented during frequency domain data acquisition
Our background correction method works if the background phasor can be accurately determined

Summary

Introduction

A method to correct frequency domain data for background signals, whether they originate from contaminant fluorescence or scattered light, has been reported by Lakowicz et al [1]. This measurement is necessitated by the fact that, in their method, the signal acquisition is based on phase and modulation data alone, so that the AC contributions of the background must be calculated a posteriori from the independently determined DC values.

Results

Conclusion