Time-resolved fluorescence determinations on biochemical samples are often complicated by con- tributions 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 modem 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 fluororneters. 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.

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

MARZOLA, Pasquina;
1991

Abstract

Time-resolved fluorescence determinations on biochemical samples are often complicated by con- tributions 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 modem 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 fluororneters. 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.
Frequency domain; background; lifetime; dynamic polarization
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11562/476568
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 34
  • ???jsp.display-item.citation.isi??? ND
social impact