&lt;title&gt;Background correction in multiharmonic Fourier transform fluorescence lifetime spectroscopy&lt;/title&gt;

Swift, Kerry M.; Mitchell, George Weston

doi:10.1117/12.44188

Cited by 3 publications

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“…It could be quite important in some cases to correct for background, and methods of on-line background subtraction are available. 15 Other techniques of capitalizing on the multiplex advantage of the well plate format can be imagined. For example, time-resolved diode array detection should be possible for the multiple wells of a row or column simultaneously.16…”

Section: Discussionmentioning

confidence: 99%

<title>High throughput screening using dynamic fluorescence</title>

Swift

Matayoshi

1995

SPIE Proceedings

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Multifrequency phase-modulation data for fluorescence lifetime and ardsotropy decay standards have been obtained for liquid samples in 96-well microtiter plates. We coupled the bifurcated fiber optic probe of a fluorescence plate reader to a phase and modulation lime-resolving fluorometer. Laser excitation was used and multifrequency data could be acquired quickly over a range of modulation frequencies from 15 to greater than 150 MHz, at each well position, through the use of multiharmonic Fourier (MHF) methods. We evaluated the system's performance for UV-excited fluorescence, with the goal of screening quality or properties of proteins or peptides by steady-state or dynamic intrinsic fluorescence intensity or anisotropy using the high throughput 96-well plate format.

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Section: Discussionmentioning

confidence: 99%

<title>High throughput screening using dynamic fluorescence</title>

Swift

Matayoshi

1995

SPIE Proceedings

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

et al. 1991

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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 ihe total signal intensity.KEY WORDS: Frequency domain; background; lifetime; dynamic polarization. INTRODUC~ONPhase fluorometry has become a widely utilized technique for performing time-resolved fluorescence measurements. As this technique becomes increasingly applied to complex samples at low concentrations, notably biological samples, unavoidable background fluorescence has come to significantly limit its overall sensitivity. Fluorescent contaminants in reagents, parasitic light, and scattered light (Rayleigh and Raman) are among the most common sources of background. 153background is attributable to a single exponential decay, then a multicomponent analysis, in which one component can be clearly assigned to the background, is adequate to recover the sample's time-resolved parameters. Usually background contributions are more complex, though, rendering this approach inappropriate.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]. In their method, the fraction of the total steady-state signal attributable to the background must be determined in a separate experiment under identical instrument conditions. 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. Moreover, in their approach a rigorous error propagation analysis cannot be achieved during data acquisition but must be performed sepa-1053-0509/91/0900-0153506.50/0 9 1991 Plenum Publishing Corporation 154 Reinhart, Marzola, Jameson, and Gratton rately. Consequently the method is not a true on-line procedure.Another approach to blank correction was recently proposed by Swift and Mitchell [2] specifically for the case of parallel data acquisition. In parallel phase fluoro...

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Time-resolved measurements of luminescence

Collier

McShane

2013

Journal of Luminescence

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<title>Background correction in multiharmonic Fourier transform fluorescence lifetime spectroscopy</title>

Cited by 3 publications

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<title>High throughput screening using dynamic fluorescence</title>

<title>High throughput screening using dynamic fluorescence</title>

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

Time-resolved measurements of luminescence

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