Using Laurdan and Spectral Phasor Analysis to Study Erythrocytes Membrane Solubilization

Abstract

Nanoscale organization of the membranes of living cells plays crucial roles in numerous vital processes, including during the activation of T-cells and their formation of the immunological synapse. However, the exact nature and function of reorganization of lipids during this key initiating event remain unclear. To gain further insight into this process, we employed two techniques that probe complementary properties of the membranes at the molecular level: 1) super-resolution STED-FCS to reveal detailed picture of the diffusion of the lipids, with additional information on spatial heterogeneity provided by the scanning mode; and 2) spectral (super-resolution STED) imaging with environment-sensitive membrane probes, i.e. fluorescence microspectroscopy, to map differences in local molecular order within the lipid bilayer. Using these methods, we monitored diffusion properties of lipids and molecular order of the plasma membrane of (Jurkat) T-cells over space and time upon their activation, revealing marked differences in lipid diffusion and molecular order. For the most informative and robust description of the latter, we systematically analyzed the benefits and pitfalls of the established representations, i.e. phasors, generalized polarization, and lineshape description.

Correlated flickering of erythrocytes membrane observed with dual time resolved membrane fluctuation spectroscopy under different d-glucose concentrations

“…For the GP value calculation, two-photon excitation is needed to minimize the severe photobleaching of the dye by one-photon excitation. This technique has been used before to measure membrane fluidity 46 , 47 , 51 .…”

“…Human RBCs were obtained from the same healthy donor volunteer and stored at 4 °C. RBCs were centrifuged at 4000 rpm (1800 g), rinsed, and suspended to the original hematocrit (40% vol/vol) in PBS-saline buffer 48 , 51 . RBCs (at final hematocrit of 5% vol/vol) were incubated for 24 h at 37 °C in d -glucose at three concentrations (5.5, 12.5, and 25 mM).…”

“…Intensity images were acquired in a two-photon microscope Zeiss LSM780 NLO using a 63X oil immersion objective (Plan-Apochromat). A Ti: Sapphire laser (Coherent, Chameleon) with 80 MHz repetition rate and tuned at 780 nm was used to excite the sample 48 , 51 , and the emission signal was received in two channels (412–464 nmand482–534 nm). The image size was 134.82 µm 2 of 1024 × 1024 pixels.…”