Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles

Coucke, Quinten; Parveen, Nagma; Fernández, Guillermo Solís; Qian, Chen; Hofkens, Johan; Debyser, Zeger; Hendrix, Jelle

doi:10.1016/j.bpr.2023.100122

Cited by 2 publications

(5 citation statements)

References 66 publications

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“…Further explanation of phasor theory for TD-and FD-FLIM can be found in the ISS phasor tech note (89), in a paper on three-probe multiplexed lifetime phasor by Rahim et al (90), in an extensive paper by Ranjit et al (91) and in our own work on particle-based phasor analysis which includes a broad theoretical explanation (11). The phasor method is interesting as it allows the 'sorting' of FLIM pixels rather than applying a strenuous fitting algorithm.…”

Section: Phasor Approach For Flimmentioning

confidence: 96%

“…One of the most common FRET applications involve protein-protein interactions, which is referred as intermolecular FRET. By labeling each interaction partner with a donor or acceptor, FRET enables the detection of changes in fluorophore distance when the two proteins come closer to each other (11)(12)(13)(14)(15)…”

Section: The Versatility Of Fretmentioning

confidence: 99%

“…Assuming every photon is assigned a single phasor, a rotating phasor covers all TCSPC time bins (N bins) in a full rotation of the universal circle, or 2𝜋. This means that the universal circle contains N points, each separated by 2𝜋/N radians (11). When every photon from the decay histogram is transformed into the phasor space and allocated to a point on the universal circle, the pixelphasor is the averaged location.…”

Section: Phasor Approach For Flimmentioning

confidence: 99%

“…In any case, a phasor analysis does not require more photons than conventional NLS fitting. Therefore it can be applied to experiments with relative high FRET dynamics or dim fluorescent samples such as FRET biosensors with fluorescent proteins (1,11,92).…”

Section: Evaluating Fret In the Phasor Plotmentioning

confidence: 99%

“…Such referencing requires an easily obtained measurement of a dye with a well-known monoexponential lifetime, which will yield the IRF in its measurement. More details on referencing for successful application of the phasor can be found in Ranjit et al (141), Michalet et al (142) and Coucke et al (11).…”

Section: Imaging Artifactsmentioning

confidence: 99%

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Navigating FRET Imaging Techniques for Biosensor Applications

Coucke,

Vorsselmans,

Aytekin

et al. 2024

Preprint

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This tutorial paper is designed for biologists and bioscientists who want to integrate Förster Resonance Energy Transfer (FRET) in their research. It provides a bird’s eye view of the current FRET landscape, covering the factors that determine its occurrence and its effect on both the donor and acceptor fluorescence signal. This tutorial also provides practical guidance on the various methods used to measure the FRET phenomenon, including intensity-based FRET techniques, such as acceptor photobleaching and ratiometric FRET, as well as FLIM-based techniques, including fitting strategies and the phasor approach. Each technique is discussed in terms of its advantages and disadvantages, equipping researchers with the knowledge to select the most suitable approach for their objectives. Additionally, we present an overview for multiplexed FRET imaging and discuss its current potential and availability. The software packages available for analyzing FRET are also presented. This tutorial concludes with a discussion on the future of FRET experiments and the importance of improved hardware to support FRET biosensor readouts. Through this paper, biologists and bioscientists will gain a comprehensive understanding of FRET, its underlying principles, and the different techniques available for its quantification. Practical insights are shared to navigate the common obstacles and nuances of experimental design. This knowledge will facilitate the effective incorporation of FRET-based sensors into their research.

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Section: Phasor Approach For Flimmentioning

confidence: 96%

Section: The Versatility Of Fretmentioning

confidence: 99%

Section: Phasor Approach For Flimmentioning

confidence: 99%

Section: Evaluating Fret In the Phasor Plotmentioning

confidence: 99%

Section: Imaging Artifactsmentioning

confidence: 99%

See 3 more Smart Citations

Navigating FRET Imaging Techniques for Biosensor Applications

Coucke,

Vorsselmans,

Aytekin

et al. 2024

Preprint

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The Quantitative In Vivo Assessment of Diabetic and Non‐Diabetic Skin Wound Healing Using Phasor‐FLIM Approach

Zuhayri,

Lepekhina,

Nikolaev

et al. 2024

Journal of Biophotonics

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A quantitative assessment of wound status in a murine model was developed using phasor plot presentation of fluorescence lifetime imaging microscopy (FLIM) data. The quantitative assessment is based on calculating Bhattacharyya distance between g coordinates of FLIM data phasor plot density distributions of wound and healthy skin. The approach was validated for both diabetic and non‐diabetic mice wounds, including during low‐dose photodynamic therapy (LDPDT). Analysis revealed a shift in the FLIM data phasor plot g coordinates, suggesting altered metabolic processes involved in wound healing. Bhattacharyya distances in the LDPDT groups were closer to zero compared to the control group, which was not treated by LDPDT. Bhattacharyya distances in the non‐diabetic LDPDT groups were closer to zero compared to the diabetic LDPDT groups that is consistent with the literature regarding the positive role of LDPDT in accelerating wound healing and the role of diabetes mellitus in impairing wound healing.

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Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles

Cited by 2 publications

References 66 publications

Navigating FRET Imaging Techniques for Biosensor Applications

Navigating FRET Imaging Techniques for Biosensor Applications

The Quantitative In Vivo Assessment of Diabetic and Non‐Diabetic Skin Wound Healing Using Phasor‐FLIM Approach

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