Pathogen Detection Using Frequency Domain Fluorescent Lifetime Measurements

Yahav, Gilad; Gershanov, Sivan; Salmon‐Divon, Mali; Ben‐Zvi, Haim; Mircus, Gabriel; Goldenberg-Cohen, Nitza; Fixler, Dror

doi:10.1109/tbme.2018.2814597

Cited by 12 publications

(13 citation statements)

References 55 publications

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“…Consistent with our previous studies, findings of this FD-FLT study, as presented using boxplots ( Figure 3A); reveal that the infected samples present pronounced prolonged median FLTs with wider distribution (range of 2.30-4.54 ns) in respect to the controls (range 2.30-2.92 ns) [30]. Nonetheless, as the median FLT values and their distributions overlap between the bacterial and viral infected samples, this approach is insufficient to detect the type of infection.…”

Section: Resultssupporting

confidence: 92%

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The squared distance approach to frequency domain time‐resolved fluorescence analysis

Yahav

Diamandi

Preter

et al. 2019

Journal of Biophotonics

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A frequency‐domain (FD) analysis of fluorescence lifetime (FLT) is a unique and rapid method for cellular and intracellular classifications that can serve for medical diagnostics purposes. Nevertheless, its data analysis process demands nonlinear fitting algorithms that may distort the resolution of the FLT data and hence diminish the classification ability of the method. This research suggests a sample classification technique that is unaffected by the analysis process as it is based on the squared distance (D2) between the raw frequency response data (FRD). In addition, it presents the theory behind this technique and its validation in two simulated data sets of six groups with similar widely and closely spaced FLT data as well as in experimental data of 43 samples from bacterial and viral infected and non‐infected patients. In the two simulated tests, the classification accuracy was above 95% for all six groups. In the experimental data, the classification of 41 out of 43 samples matched earlier report and 29 out of 31 agreed with preliminary physician diagnosis. The D2 approach has the potential to promote FD‐time resolved fluorescence measurements as a medical diagnostic technique with high specifity and high sensitivity for many of today's conventional diagnostic procedures.

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Section: Resultssupporting

confidence: 92%

“…Simulation I represents a general case of three widely spaced FLTs. Simulation II represents common closely FLT values, which correspond to FLTs of DAPI bound to DNA of leukocytes in different situations .…”

Section: Resultsmentioning

confidence: 99%

The squared distance approach to frequency domain time‐resolved fluorescence analysis

Yahav

Diamandi

Preter

et al. 2019

Journal of Biophotonics

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“…Over the past decades, the ORR has been widely used as an intrinsic indicator of redox states in cells and tissues [26][27][28]. In addition to redox ratio imaging, fluorescence lifetime imaging microscopy (FLIM) has also been used for mapping of cellular metabolism [29][30][31] and detection of pathogens [32]. We found that the ORR of BAT increases significantly after NE injection in mice, which is correlated with the temperature rise in BAT induced by thermogenesis [14].…”

Section: Introductionmentioning

confidence: 86%

“…This may provide opportunities to investigate the metabolic characteristics of beige fat at subcellular resolution in vivo, which has not been conducted in live animal model. In addition to redox ratio imaging, fluorescence lifetime imaging microscopy (FLIM) has also been used for mapping of cellular metabolism [29][30][31] and detection of pathogens [32]. NADH and FAD exhibit distinct fluorescence lifetimes (FLTs) in free and protein-bound forms, and the free-to-bound ratios of NADH or FAD can be used to discriminate different cellular metabolic states [33][34][35][36].…”

Section: Introductionmentioning

confidence: 99%

In vivo study of metabolic dynamics and heterogeneity in brown and beige fat by label‐free multiphoton redox and fluorescence lifetime microscopy

Wei

Qin

et al. 2019

Journal of Biophotonics

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In this work, the metabolic characteristics of adipose tissues in live mouse model were investigated using a multiphoton redox ratio and fluorescence lifetime imaging technology. By analyzing the intrinsic fluorescence of metabolic coenzymes, we measured the optical redox ratios of adipocytes in vivo and studied their responses to thermogenesis. The fluorescence lifetime imaging further revealed changes in protein bindings of metabolic coenzymes in the adipocytes during thermogenesis. Our study uncovered significant heterogeneity in the cellular structures and metabolic characteristics of thermogenic adipocytes in brown and beige fat. Subgroups of brown and beige adipocytes were identified based on the distinct lipid size distributions, redox ratios, fluorescence lifetimes and thermogenic capacities. The results of our study show that this label-free imaging technique can shed new light on in vivo study of metabolic dynamics and heterogeneity of adipose tissues in live organisms. K E Y W O R D Sbeige fat, fluorescence lifetime, optical redox ratio, thermogenesis

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“…FLI can be measured in frequency domain (frequency modulated techniques) and in time domain (timeresolved techniques) 18,19 . Many time-domain FLI techniques use time-correlated single-photon counting (TCSPC), a technique generally used in scanning confocal set-ups 20 (with exceptions, see e.g.…”

Section: Introductionmentioning

confidence: 99%

Single Photon, Time-Gated, Phasor-based Fluorescence Lifetime Imaging Through Highly Scattering Medium

Ankri

Basu

Ulku

et al. 2019

Preprint

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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 autofluorescence (AF), which decreases the signal to noise ratio and (iii) broadening of the decay signal, which can result in incorrect lifetime estimation. Here, we report the use of a large-frame time-gated single-photon avalanche diode (SPAD) imager, SwissSPAD2, with a very short acquisition time (in the milliseconds range) and a wide-field microscopy format. We use the phasor approach to convert each pixel's data into its local lifetime. The phasor transformation provides a simple and fast visual method for lifetime imaging and is particularly suitable for in vivo FLI which suffers from deformation of the fluorescence decay, and makes lifetime extraction by standard fitting challenging. We show, for single dyes, that the phasor cloud distribution (of pixels) increases with decay broadening due to scattering and decreasing fluorescence intensity. Yet, as long as the fluorescence signal is higher than the tissue-like phantom AF, a distinct lifetime can still be clearly identified with an appropriate background correction. Lastly, we demonstrate the detection of few hundred thousand A459 cells expressing the fluorescent protein mCyRFP1 through highly scattering phantom layers, despite significant scattering and the presence of the phantom AF.

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Pathogen Detection Using Frequency Domain Fluorescent Lifetime Measurements

Abstract: The FD-FLIM system can provide a high throughput diagnostic technique that does not require a physician.

Cited by 12 publications

References 55 publications

The squared distance approach to frequency domain time‐resolved fluorescence analysis

The squared distance approach to frequency domain time‐resolved fluorescence analysis

In vivo study of metabolic dynamics and heterogeneity in brown and beige fat by label‐free multiphoton redox and fluorescence lifetime microscopy

Single Photon, Time-Gated, Phasor-based Fluorescence Lifetime Imaging Through Highly Scattering Medium

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