Background Suppression in Frequency-domain Fluorometry

Overview

Journal Anal Biochem

Publisher Elsevier

Specialty Biochemistry

Date 1999 Dec 28

PMID 10610691

Citations 3

Authors

J R Lakowicz

I Gryczynski

Z Gryczynski

M L Johnson

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

A robust method for autofluorescence-free immunofluorescence using high-speed fluorescence lifetime imaging microscopy.

Hwang W, McPartland T, Jeong S, Evans C Sci Rep. 2025; 15(1):5503.

PMID: 39953137 PMC: 11828918. DOI: 10.1038/s41598-025-89142-6.

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

Rich R, Stankowska D, Maliwal B, Sorensen T, Laursen B, Krishnamoorthy R Anal Bioanal Chem. 2012; 405(6):2065-75.

PMID: 23254457 PMC: 3566262. DOI: 10.1007/s00216-012-6623-1.

Phase differential enhancement of FLIM to distinguish FRET components of a biosensor for monitoring molecular activity of Membrane Type 1 Matrix Metalloproteinase in live cells.

Eichorst J, Huang H, Clegg R, Wang Y J Fluoresc. 2011; 21(4):1763-77.

PMID: 21519891 PMC: 3637990. DOI: 10.1007/s10895-011-0871-x.

Real-time background suppression during frequency domain lifetime measurements.

Herman P, Maliwal B, Lakowicz J, Maliwal B Anal Biochem. 2002; 309(1):19-26.

PMID: 12381357 PMC: 6945983. DOI: 10.1016/s0003-2697(02)00213-0.

References

Nederlof P, van der Flier S, Wiegant J, Raap A, Tanke H, Ploem J . Multiple fluorescence in situ hybridization. Cytometry. 1990; 11(1):126-31. DOI: 10.1002/cyto.990110115. View

Barrow D, Lentz B . The use of isochronal reference standards in phase and modulation fluorescence lifetime measurements. J Biochem Biophys Methods. 1983; 7(3):217-34. DOI: 10.1016/0165-022x(83)90031-3. View

Guo X, Castellano F, Li L, Lakowicz J . A long-lifetime Ru(II) metal-ligand complex as a membrane probe. Biophys Chem. 1998; 71(1):51-62. DOI: 10.1016/s0301-4622(97)00135-x. View

Szmacinski H, Lakowicz J, Johnson M . Fluorescence lifetime imaging microscopy: homodyne technique using high-speed gated image intensifier. Methods Enzymol. 1994; 240:723-48. PMC: 6897572. DOI: 10.1016/s0076-6879(94)40069-5. View

Watkins A, Ingersoll C, Baker G, Bright F . A parallel multiharmonic frequency-domain fluorometer for measuring excited-state decay kinetics following one-, two-, or three-photon excitation. Anal Chem. 1998; 70(16):3384-96. DOI: 10.1021/ac9803481. View

Lakowicz J, Jayaweera R, Joshi N, Gryczynski I . Correction for contaminant fluorescence in frequency-domain fluorometry. Anal Biochem. 1987; 160(2):471-9. DOI: 10.1016/0003-2697(87)90078-9. View

Lakowicz J, Szmacinski H, Nowaczyk K, Johnson M . Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci U S A. 1992; 89(4):1271-5. PMC: 48431. DOI: 10.1073/pnas.89.4.1271. View

Dickson E, Pollak A, Diamandis E . Ultrasensitive bioanalytical assays using time-resolved fluorescence detection. Pharmacol Ther. 1995; 66(2):207-35. DOI: 10.1016/0163-7258(94)00078-h. View

Speicher M, Gwyn Ballard S, Ward D . Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet. 1996; 12(4):368-75. DOI: 10.1038/ng0496-368. View

10.

Lakowicz J, Cherek H, Balter A . Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry. J Biochem Biophys Methods. 1981; 5(3):131-46. DOI: 10.1016/0165-022x(81)90012-9. View

11.

Szmacinski H, Lakowicz J . Fluorescence lifetime-based sensing and imaging. Sens Actuators B Chem. 2021; 29(1-3):16-24. PMC: 8049533. DOI: 10.1016/0925-4005(95)01658-9. View

12.

Lakowicz J, Laczko G, Cherek H, Gratton E, Limkeman M . Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data. Biophys J. 1984; 46(4):463-77. PMC: 1435017. DOI: 10.1016/S0006-3495(84)84043-6. View

13.

Lakowicz J, Cherek H . Phase-sensitive fluorescence spectroscopy: a new method to resolve fluorescence lifetimes or emission spectra of components in a mixture of fluorophores. J Biochem Biophys Methods. 1981; 5(1):19-35. DOI: 10.1016/0165-022x(81)90030-0. View

14.

Terpetschnig E, Szmacinski H, Lakowicz J . Long-lifetime metal-ligand complexes as probes in biophysics and clinical chemistry. Methods Enzymol. 1997; 278:295-321. DOI: 10.1016/s0076-6879(97)78016-9. View

15.

Prober J, Trainor G, Dam R, Hobbs F, Robertson C, Zagursky R . A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science. 1987; 238(4825):336-41. DOI: 10.1126/science.2443975. View

16.

Reinhart G, Marzola P, Jameson D, Gratton E . A method for on-line background subtraction in frequency domain fluorometry. J Fluoresc. 2013; 1(3):153-62. DOI: 10.1007/BF00865362. View

17.

Guo X, Castellano F, Li L, Szmacinski H, Lakowicz J, Sipior J . A long-lived, highly luminescent Re(I) metal-ligand complex as a biomolecular probe. Anal Biochem. 1998; 254(2):179-86. PMC: 6915065. DOI: 10.1006/abio.1997.2413. View