2019
DOI: 10.1021/acsphotonics.9b00874
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Single-Photon, Time-Gated, Phasor-Based Fluorescence Lifetime Imaging through Highly Scattering Medium

Abstract: Fluorescence lifetime imaging (FLI) is a powerful tool for in vitro and non-invasive in vivo biomolecular and cellular investigations. Fluorescence lifetime is an intrinsic characteristic of any fluorescent dye which, to some extent, does not depend on excitation intensity and signal level. However, when used in vivo with visible wavelength emitting fluorophores, FLI is complicated by (i) light scattering as well as absorption by tissues, which significantly reduces fluorescence intensity, (ii) tissue autofluo… Show more

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Cited by 18 publications
(24 citation statements)
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References 58 publications
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“…While these works used a finite number of gates, their large number (from ∼100 in Ref. 36 to 2800 in Ref. 16 ) implies that the calculated phasors are close to the continuous phasor discussed in this article.…”
Section: Application To Experimental Datasupporting
confidence: 52%
See 1 more Smart Citation
“…While these works used a finite number of gates, their large number (from ∼100 in Ref. 36 to 2800 in Ref. 16 ) implies that the calculated phasors are close to the continuous phasor discussed in this article.…”
Section: Application To Experimental Datasupporting
confidence: 52%
“…In both Refs. 36 and 16 , a local phasor calibration was used, the reason invoked being the non-uniformity of the detector’s response. This non-uniformity is detailed in Ref.…”
Section: Application To Experimental Datamentioning
confidence: 99%
“…To show the robustness of our method, we generate synthetic traces for immobilized molecules with: i) variable data set sizes, baseline values: lifetime between 1 ns and 10 ns which is the typical lifetime range of a fluorophore, 18,80 two species which is most frequent in related studies, 18,19,23 and fraction of molecules contributing photons from different species set at 50% : 50%.…”
Section: Methods Validation Using Synthetic Datamentioning
confidence: 99%
“…1,2 Some fluorescence approaches-such as confocal microscopy, 3 two-photon microscopy 4 and super-resolution widefield applications 5 -use constant illumination to provide information on chemical kinetics, [6][7][8] diffusional dynamics [9][10][11] or spatial locations of molecules. 12,13 Other methods use pulsed illumination [14][15][16][17][18] or illumination intensity modulated at a fixed frequency [18][19][20][21][22][23] where photon arrival times encode critical information, say, on the excited state lifetime or the number of different chemical species. This is the basis of lifetime imaging 13,[24][25][26][27] that has been used to reveal information on local pH, 28,29 oxygenation 28 and other cellular metabolic traits 23,30 affected by cellular microenvironments.…”
Section: Introductionmentioning
confidence: 99%
“…Time-correlated single-photon counting (TCSPC) is a technique by which a fast-pulsed light source, generally a laser, excites the molecule and a single-photon detector, e.g., a single-photon avalanche diode (SPAD), captures the photons resulting from the fluorescence decay. A histogram is constructed, timed with respect to the laser pulse, and photon time of arrivals can be computed directly using time-to-digital or time-to-amplitude converters [4]- [6] or indirectly by way of a global or rolling shutter, to achieve time gating [7], [8].…”
Section: Introductionmentioning
confidence: 99%