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Quantifying Fluorescence Lifetime Responsiveness of Environment-Sensitive Probes for Membrane Fluidity Measurements

posted on 2024-03-08, 17:35 authored by Erdinc SezginErdinc Sezgin, Franziska RagallerFranziska Ragaller, Ellen Sjule, Yagmur Balim Urem, Jan SchlegelJan Schlegel, Rojbin El, Dunja Urbancic, Iztok Urbancic, Hans Blom

It contains data sets from Ragaller et al, Journal of Physical Chemistry B, 2024 (

It contains emission spectra, decay curves, lifetime images and excel sheets of the data.

Abstract: The structural diversity of different lipid species within the membrane defines its biophysical properties such as membrane fluidity, phase transition, curvature, charge distribution and tension. Environment-sensitive probes, which change their spectral properties in response to their surrounding milieu, have greatly contributed to our understanding of such biophysical properties. To realize the full potential of these probes and to avoid misinterpretation of their spectral responses, a detailed investigation of their fluorescence characteristics in different environments is necessary. Here, we examined fluorescence lifetime of two newly developed membrane order probes, NR12S and NR12A, in response to alterations in their environments such as degree of lipid saturation, cholesterol content, double bond position and configuration and phospholipid headgroup. As comparison, we investigated lifetime sensitivity of the membrane tension probe Flipper in these environments. Applying fluorescence lifetime imaging microscopy (FLIM) in both model membranes and biological membranes, all probes distinguished membrane phases by lifetime, but exhibited different lifetime sensitivities to varying membrane biophysical properties (e.g. cholesterol). While the lifetime of Flipper is particularly sensitive to membrane cholesterol content, NR12S and NR12A lifetime is moderately sensitive to both cholesterol content and lipid acyl chains. Moreover, all probes exhibit longer lifetimes at longer emission wavelengths in membranes of any complexity. This emission-wavelength dependency results in varying lifetime resolution at different spectral regions, highly relevant for FLIM data acquisition. Our data provides valuable insights on how to perform FLIM with these probes and highlights both their potential and limitations.

Data usage

Researchers are welcome to use the data contained in the dataset for any projects. Please cite this item upon use or when published. We encourage reuse using the same CC BY 4.0 License.

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png or tif files for lifetime images

Excel files for graphs

Software to open files

.xlsx - Microsoft Excel

.tif, .png - Fiji (



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