Long-term STED imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes

Pablo Carravilla,Anindita Dasgupta,Gaukhar Zhurgenbayeva,Dmytro I Danylchuk,Andrey S Klymchenko,Erdinc Sezgin,Christian Eggeling

doi:10.1016/j.bpr.2021.100023

Abstract

Understanding the plasma membrane nanoscale organization and dynamics in living cells requires microscopy techniques with high spatial and temporal resolution that permit for long acquisition times and allow for the quantification of membrane biophysical properties, such as lipid ordering. Among the most popular super-resolution techniques, stimulated emission depletion (STED) microscopy offers one of the highest temporal resolutions, ultimately defined by the scanning speed. However, monitoring live processes using STED microscopy is significantly limited by photobleaching, which recently has been circumvented by exchangeable membrane dyes that only temporarily reside in the membrane. Here, we show that NR4A, a polarity-sensitive exchangeable plasma membrane probe based on Nile red, permits the super-resolved quantification of membrane biophysical parameters in real time with high temporal and spatial resolution as well as long acquisition times. The potential of this polarity-sensitive exchangeable dye is showcased by live-cell real-time three-dimensional STED recordings of bleb formation and lipid exchange during membrane fusion as well as by STED-fluorescence correlation spectroscopy experiments for the simultaneous quantification of membrane dynamics and lipid packing that correlate in model and live-cell membranes.

Highlights

Living organisms meticulously adapt their membrane physical and chemical properties [1]
To assess whether NR4A's exchangeable nature influenced its sensitivity to lipid packing, we quantified the lipid packing resolution as measured by the generalized polarization (GP) difference (DGP) between Lo and Ld environments in phase-separated Giant unilamellar vesicles (GUVs) (Fig. 1 D) and compared it with Laurdan, a good standard for sensitivity
We found that the lipid packing and bilayer dynamics anticorrelate in model and live-cell membranes, suggesting that the slow diffusion coefficients commonly measured in plasma membranes can be ascribed to the high degree of lipid packing of this structure, which arises from its distinct lipid composition [56], as previously suggested for highly packed viral membranes [59]

Summary

Introduction

Living organisms meticulously adapt their membrane physical and chemical properties [1]. The STED phenomenon is reversible and, in theory, does not induce enhanced dye destruction (but rather a reduction) [18], photobleaching rates are above those of conventional microscopy, especially at high STED laser powers required to achieve the highest spatial resolution [19,20]. This higher photobleaching rate seems to be associated with transitions to the more reactive higher excited singlet and triplet states [21,22] through absorption of STED photons [19,20,23]. Photobleaching remains one of the major complications of STED microscopy, despite big efforts to avoid it, such as the development of novel photostable dyes [24,25], separation of excitation pulses [26], the use of high scanning rates [27,28], or adaptive illumination modes [29]

Methods

Results

Conclusion