posted on 2024-02-28, 12:37authored byFranziska Ragaller, Ellen Sjule, Yagmur Balim Urem, Jan Schlegel, Rojbin El, Dunja Urbancic, Iztok Urbancic, Hans Blom, Erdinc Sezgin
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 avoid misinterpretation of their spectral responses, a
detailed investigation of their fluorescence characteristics in different
environments is necessary. Here, we examined the fluorescence lifetime
of two newly developed membrane order probes, NR12S and NR12A, in
response to alterations in their environments such as the degree of
lipid saturation, cholesterol content, double bond position and configuration,
and phospholipid headgroup. As a comparison, we investigated the 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 the membrane
cholesterol content, the NR12S and NR12A lifetimes are moderately
sensitive to both the cholesterol content and lipid acyl chains. Moreover,
all of the probes exhibit longer lifetimes at longer emission wavelengths
in membranes of any complexity. This emission wavelength dependency
results in varying lifetime resolutions at different spectral regions,
which are highly relevant for FLIM data acquisition. Our data provide
valuable insights on how to perform FLIM with these probes and highlight
both their potential and limitations.