Dataset Information

Unraveling the hidden temporal range of fast β₂-adrenergic receptor mobility by time-resolved fluorescence.

ABSTRACT: G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β₂-adrenergic receptor over a wide time range (nanosecond to second). Particularly, a translational mobility (10 µm²/s), one order of magnitude faster than membrane associated lateral mobility that explains membrane protein turnover and suggests a wider picture of the GPCR availability on the plasma membrane. And a so far elusive rotational mobility (1-200 µs) which depicts a previously overlooked dynamic component that, despite all complexity, behaves largely as predicted by the Saffman-Delbrück model.

SUBMITTER: Balakrishnan A

PROVIDER: S-EPMC8885909 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Unraveling the hidden temporal range of fast β<sub>2</sub>-adrenergic receptor mobility by time-resolved fluorescence.

Balakrishnan Ashwin A Hemmen Katherina K Choudhury Susobhan S Krohn Jan-Hagen JH Jansen Kerstin K Friedrich Mike M Beliu Gerti G Sauer Markus M Lohse Martin J MJ Heinze Katrin G KG

Communications biology 20220228 1

G-protein-coupled receptors (GPCRs) are hypothesized to possess molecular mobility over a wide temporal range. Until now the temporal range has not been fully accessible due to the crucially limited temporal range of available methods. This in turn, may lead relevant dynamic constants to remain masked. Here, we expand this dynamic range by combining fluorescent techniques using a spot confocal setup. We decipher mobility constants of β<sub>2</sub>-adrenergic receptor over a wide time range (nano ...[more]

PMID: 35228644

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Project description:The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8-7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence lifetime measurements of low quantum efficiency sub-nanosecond fluorophores.

Project description:SignificanceWide-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.AimTo 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).ApproachThe 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.ResultsThe FLT of fluorescein samples was found to decrease from 4.01±0.01 to 3.56±0.02 ns, whereas both r and θ were significantly increased from 0.053±0.012 to 0.252±0.003 and 0.15±0.05 to 11.25±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±0.011 to 0.150±0.013 and θ from 0.98±0.13 to 1.65±0.20 ns for the first CDs and from 0.280±0.008 to 0.310±0.004 and 5.55±1.08 to 7.95±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.ConclusionsThrough 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.

Dataset Information

Unraveling the hidden temporal range of fast β₂-adrenergic receptor mobility by time-resolved fluorescence.

Publications

Unraveling the hidden temporal range of fast β<sub>2</sub>-adrenergic receptor mobility by time-resolved fluorescence.

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Unraveling the hidden temporal range of fast β2-adrenergic receptor mobility by time-resolved fluorescence.

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Unraveling the hidden temporal range of fast β<sub>2</sub>-adrenergic receptor mobility by time-resolved fluorescence.

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Unraveling the hidden temporal range of fast β₂-adrenergic receptor mobility by time-resolved fluorescence.