Fluorescence lifetime resolution with phase fluorometry

Ide, G; Engelborghs, Yves; Persoons, André

doi:10.1063/1.1137488

Cited by 31 publications

(9 citation statements)

References 5 publications

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“…The reason why image accumulation is needed is the considerably weak excitation light, which is limited by the efficiency of nonlinear wavelength conversion (typically 10%) in the PPLN crystal and signal loss (typically 1%) in the VIPA. Considering the total power of excitation light (242 μW) at the focus and the number of 2D image pixels (149 × 200 = 29,800 pixels), the excitation power per pixel was only 8 nW, which is much lower than that in the conventional PM method (typically on the order of microwatts) ( 10 , 11 ) or FIRE ( 20 – 22 ). Since the image SNR in the proposed system was shot noise–limited, as shown in Fig.…”

Section: Discussionmentioning

confidence: 98%

“…Fluorescence lifetime can be measured by the time-correlated single-photon counting (TC-SPC) method with pulsed excitation ( 4 , 8 , 9 ) or the phase measurement (PM) method with sinusoidal excitation (see Fig. 1, A and B , with the description in Materials and Methods) ( 4 , 10 , 11 ). Since both methods are based on point measurement, mechanical scanning of the focal point is necessary for obtaining an image using fluorescence lifetime.…”

Section: Introductionmentioning

confidence: 99%

“…Fluorescence lifetime can be measured by the time-correlated single-photoncounting method (TC-SPC) with pulsed excitation 4,8,9 or the phase measurement (PM) method with sinusoidal excitation 4,10,11 . Both methods are based on point measurement, and they have advantages and disadvantages, including data acquisition time.…”

mentioning

confidence: 99%

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Full-field fluorescence lifetime dual-comb microscopy using spectral mapping and frequency multiplexing of dual-comb optical beats

et al. 2021

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Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for quantitative fluorescence imaging because fluorescence lifetime is independent of concentration of fluorescent molecules or excitation/detection efficiency and is robust to photobleaching. However, since most FLIMs are based on point-to-point measurements, mechanical scanning of a focal spot is needed for forming an image, which hampers rapid imaging. Here, we demonstrate scan-less full-field FLIM based on a one-to-one correspondence between two-dimensional (2D) image pixels and frequency-multiplexed radio frequency (RF) signals. A vast number of dual-comb optical beats between dual optical frequency combs are effectively adopted for 2D spectral mapping and high-density frequency multiplexing in the RF region. Bimodal images of fluorescence amplitude and lifetime are obtained with high quantitativeness from amplitude and phase spectra of fluorescence RF comb modes without the need for mechanical scanning. The parallelized FLIM will be useful for rapid quantitative fluorescence imaging in life science.

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Section: Discussionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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Full-field fluorescence lifetime dual-comb microscopy using spectral mapping and frequency multiplexing of dual-comb optical beats

et al. 2021

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“…However, in practice the fitting sensitivity of modulation depth measurements is lower than that of phase delay [32]. Moreover, for our system, the modulation depth is also more sensitive to losses due to the detector frequency response and AOD diffraction efficiency, and thus less accurate than phase measurements.…”

Section: Methodsmentioning

confidence: 94%

Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy

Chan¹,

Diebold²,

Buckley³

et al. 2014

Biomed. Opt. Express

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Frequency domain fluorescence lifetime imaging is a powerful technique that enables the observation of subtle changes in the molecular environment of a fluorescent probe. This technique works by measuring the phase delay between the optical emission and excitation of fluorophores as a function of modulation frequency. However, high-resolution measurements are time consuming, as the excitation modulation frequency must be swept, and faster low-resolution measurements at a single frequency are prone to large errors. Here, we present a low cost optical system for applications in real-time confocal lifetime imaging, which measures the phase vs. frequency spectrum without sweeping. Deemed Lifetime Imaging using Frequency-multiplexed Excitation (LIFE), this technique uses a digitally-synthesized radio frequency comb to drive an acousto-optic deflector, operated in a cat's-eye configuration, to produce a single laser excitation beam modulated at multiple beat frequencies. We demonstrate simultaneous fluorescence lifetime measurements at 10 frequencies over a bandwidth of 48 MHz, enabling high speed frequency domain lifetime analysis of single- and multi-component sample mixtures.

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“…5 Self-referenced m ethods based on phase uorimetry have been described. These m ay use measurem ents at multiple frequencies 6,7 or m easurements of both phase and demodulation information at a single frequency. 8,9 Dual lifetime referencing (DLR) also exploits large lifetime differences between the reference and the analytesensitive molecule.…”

Section: Introductionmentioning

confidence: 99%

Self-Referencing Intensity Measurements Based on Square-Wave Gated Phase-Modulation Fluorimetry

et al. 2003

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An adaptation of square-wave gated phase-modulation (GPM) fluorimetry allows for self-referenced intensity measurements without the complexity of dual excitation or dual emission wavelengths. This AC technique utilizes square-wave excitation, gated detection, a reference emitter, and a sensor molecule. The theory and experimental data demonstrating the effectiveness and advantages of the adapted GPM scheme are presented. One component must have an extremely short lifetime relative to the other. Both components are affected identically by changes in intensity of the excitation source, but the sensor intensity also depends on the concentration of the analyte. The fluctuations of the excitation source and any optical transmission changes are eliminated by ratioing the sensor emission to the reference emission. As the concentration of the analyte changes, the corresponding sensor intensity changes can be quantified through several schemes including digitization of the signal and digital integration or AC methods. To measure pH, digital methods are used with Na3[Tb(dpa)3] (dpa = 2,6-pyridinedicarboxylic acid) as the long-lived reference molecule and fluorescein as the short-lived sensor molecule. Measurements from the adapted GPM scheme are directly compared to conventional ratiometric measurements. Good agreement between the data collection methods is demonstrated through the apparent pKa. For the adapted GPM measurements, conventional measurements, and a global fit the apparent pKa values agree within less than 2%. A key element of the adapted GPM method is its insensitivity to fluctuations in the source intensity. For a roughly 8-fold change in the excitation intensity, the signal ratio changes by less than 3%.

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Fluorescence lifetime resolution with phase fluorometry

Cited by 31 publications

References 5 publications

Full-field fluorescence lifetime dual-comb microscopy using spectral mapping and frequency multiplexing of dual-comb optical beats

Full-field fluorescence lifetime dual-comb microscopy using spectral mapping and frequency multiplexing of dual-comb optical beats

Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy

Self-Referencing Intensity Measurements Based on Square-Wave Gated Phase-Modulation Fluorimetry

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