Linear Behavior of the Phase Lifetime in Frequency-Domain Fluorescence Lifetime Imaging of FRET Constructs

Sumetsky, Daniel; Jiang, James; Ayad, Marina A.; Mahon, Timothy; Menaesse, Audrey; Cararo-Lopes, Marina M.; Patel, Mihir V.; Firestein, Bonnie L.; Boustany, Nada N.

doi:10.3389/fphy.2021.648016

Cited by 6 publications

(3 citation statements)

References 30 publications

(58 reference statements)

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“…Therefore, our FD-FLIM system demonstrates good sensitivity to the FRET efficiency range expected in our probes. Based on the relationship between phase lifetime and FRET efficiency 4 , our data are consistent with lower lifetime corresponding to higher FRET efficiency. Work is underway to use the known FRET efficiency of TSMod in CHO-K1 cells to create a calibration curve and convert the lifetime data to FRET efficiency using the method developed by Sumetsky et al 4 .…”

Section: Plasmid Expression and Lifetime Measurement In 2d Cellular M...supporting

confidence: 84%

Feasibility of imaging intracellular tension probes in multicellular aggregates

Hu¹,

Ong²,

Barroso³

et al. 2023

Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XIV

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Multicellular aggregates constitute important 3-dimenstional (3D) models for investigating cellular behaviors, including proliferation and migration, that are relevant to neoplastic growth and progression. Studies point to the role of the aggregates' biomechanical environment in modulating cell proliferation, segregation, and migration. To obtain measurements of mechanical responses at the cellular level, we investigate the use of molecular tension probes in multicellular aggregates. While these tension probes have been used in 2-dimensional monolayers of cells, the feasibility of their application in 3D multicellular aggregates has not been demonstrated yet. In this paper, we utilize a previously described, frequency-domain fluorescence lifetime microscope (FLIM), to test the feasibility of measuring FRET using the Vinculin Tension Sensor (Vinculin TS), in multicellular aggregates of CHO-K1 cells. Our data suggest that the probes can be expressed in the spheroids and that we can measure FLIM-FRET signal from calibration constructs that are diffusely distributed within the cells. When the cells express Vinculin TS, we were able to discern changes in fluorescence distribution, compared with the calibration constructs, with evidence of punctate staining suggestive of localization at adhesion sites. The fluorescence lifetime of puncta expressing Vinculin TS was 2.46 +/-0.12 ns, longer than 2.21 +/-0.103 ns, the lifetime of puncta expressing Tail-less Vinculin TS (VinTL), which lacks the actin-binding domain. The longer lifetime is consistent with higher tension across vinculin TS compared with the unloaded VinTL control. Work is underway to fully characterize these puncta.

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Section: Plasmid Expression and Lifetime Measurement In 2d Cellular M...supporting

confidence: 84%

Feasibility of imaging intracellular tension probes in multicellular aggregates

Hu¹,

Ong²,

Barroso³

et al. 2023

Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XIV

Self Cite

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“…The phasor lifetime calculation is advantageous for large datasets due to its fit-free nature and faster analysis times [ 31 , 36 ]. In applications related to Förster resonance energy transfer (FRET) or tracking lifetime changes, the phase lifetime

has been used as a metric for determining lifetime alteration [ 39 – 43 ]. To simplify our analysis, the lifetime we reported in this article is phase lifetime

at a laser repetition rate of 80 MHz.…”

Section: Methodsmentioning

confidence: 99%

Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina

Nguyen,

Chen,

Nguyen

et al. 2024

Biomed. Opt. Express

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Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.

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“… 31 Nonetheless, there are a variety of works that signify the use of FD measurements for the resolution of fluorescence intensity (FI) and FA decays thus probing cell behavior and monitoring changes in its microenvironment. 16 , 32 – 37 Furthermore, the measurement procedures and the data analysis in the FD method are significantly faster, and hence more suitable for clinical applications. 38 In both FD apparatuses, 27 , 31 the images at different polarizations are acquired using two sets of measurements.…”

Section: Introductionmentioning

confidence: 99%

Imaging the rotational mobility of carbon dot-gold nanoparticle conjugates using frequency domain wide-field time-resolved fluorescence anisotropy

et al. 2023

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Wide-field measurements of time-resolved fluorescence anisotropy (TR-FA) provide pixel-by-pixel information about the rotational mobility of fluorophores, reflecting changes in the local microviscosity and other factors influencing the fluorophore's diffusional motion. These features offer promising potential in many research fields, including cellular imaging and biochemical sensing, as demonstrated by previous works. Nevertheless, θ imaging is still rarely investigated in general and in carbon dots (CDs) in particular.Aim: To extend existing frequency domain (FD) fluorescence lifetime (FLT) imaging microscopy (FLIM) to FD TR-FA imaging (TR-FAIM), which produces visual maps of the FLT and θ, together with the steady-state images of fluorescence intensity (FI) and FA (r ). Approach:The proof of concept of the combined FD FLIM/ FD TR-FAIM was validated on seven fluorescein solutions with increasing viscosities and was applied for comprehensive study of two types of CD-gold nano conjugates. Results:The FLT of fluorescein samples was found to decrease from 4.01 AE 0.01 to 3.56 AE 0.02 ns, whereas both r and θ were significantly increased from 0.053 AE 0.012 to 0.252 AE 0.003 and 0.15 AE 0.05 to 11.25 AE 1.87 ns, respectively. In addition, the attachment of gold to the two CDs resulted in an increase in the FI due to metal-enhanced fluorescence. Moreover, it resulted in an increase of r from 0.100 AE 0.011 to 0.150 AE 0.013 and θ from 0.98 AE 0.13 to 1.65 AE 0.20 ns for the first CDs and from 0.280 AE 0.008 to 0.310 AE 0.004 and 5.55 AE 1.08 to 7.95 AE 0.97 ns for the second CDs. These trends are due to the size increase of the CDs-gold compared to CDs alone. The FLT presented relatively modest changes in CDs.Conclusions: Through the combined FD FLIM/ FD TR-FAIM, a large variety of information can be probed (FI, FLT, r , and θ). Nevertheless, θ was the most beneficial, either by probing the spatial changes in viscosity or by evident variations in the peak and full width half maximum.

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Linear Behavior of the Phase Lifetime in Frequency-Domain Fluorescence Lifetime Imaging of FRET Constructs

Cited by 6 publications

References 30 publications

Feasibility of imaging intracellular tension probes in multicellular aggregates

Feasibility of imaging intracellular tension probes in multicellular aggregates

Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina

Imaging the rotational mobility of carbon dot-gold nanoparticle conjugates using frequency domain wide-field time-resolved fluorescence anisotropy

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