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

Franziska Ragaller1, Ellen Sjule1, Yagmur Balim Urem1

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New membrane probes NR12S and NR12A, alongside Flipper, show distinct fluorescence lifetime responses to lipid environments. Understanding these sensitivities is crucial for accurate membrane biophysics studies using fluorescence lifetime imaging microscopy (FLIM).

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Area of Science:

  • Membrane biophysics
  • Fluorescence spectroscopy
  • Lipidomics

Background:

  • Membrane structural diversity dictates biophysical properties like fluidity and tension.
  • Environment-sensitive probes are vital for studying these properties, but require detailed characterization.
  • Understanding probe behavior in different lipid environments is key to accurate interpretation of spectral responses.

Purpose of the Study:

  • To investigate the fluorescence lifetime characteristics of novel membrane order probes (NR12S, NR12A) in various lipid environments.
  • To compare the environmental sensitivity of NR12S and NR12A with the membrane tension probe Flipper.
  • To provide insights for optimizing fluorescence lifetime imaging microscopy (FLIM) data acquisition with these probes.

Main Methods:

  • Utilized fluorescence lifetime imaging microscopy (FLIM) on both model and biological membranes.
  • Examined probe responses to changes in lipid saturation, cholesterol content, double bond configuration, and phospholipid headgroups.
  • Compared the fluorescence lifetime sensitivity of NR12S, NR12A, and Flipper across diverse membrane compositions.

Main Results:

  • All probes distinguished membrane phases based on fluorescence lifetime.
  • Flipper's lifetime was highly sensitive to cholesterol content.
  • NR12S and NR12A lifetimes showed moderate sensitivity to both cholesterol and lipid acyl chains.
  • Probe lifetimes increased with emission wavelength, impacting spectral resolution in FLIM.

Conclusions:

  • NR12S and NR12A offer valuable insights into membrane order and biophysical properties.
  • Probe selection and data acquisition strategies in FLIM should consider their specific environmental sensitivities and wavelength-dependent lifetimes.
  • This study highlights the potential and limitations of NR12S, NR12A, and Flipper for advanced membrane studies.