STED super-resolution imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes

Abstract

Understanding the plasma membrane nano-scale organisation and dynamics in living cells requires microscopy techniques with high temporal and spatial resolution and long acquisition times, that also 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 resolution, 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 dyes is showcased by live-cell real-time 3D-STED recordings of bleb formation and lipid exchange during membrane fusion, as well as by STED-fluorescence correlation spectroscopy (STED-FCS) experiments for the simultaneous quantification of membrane dynamics and lipid packing, which correlate in model and live-cell membranes.

Fluorescence sensors for imaging membrane lipid domains and cholesterol

“…The size distribution of the presumptive lipid-domain STED puncta showed peaks at ~ 50 and 150 nm lateral width. Other recent studies using FCS-STED nanoscopy corroborate the usefulness of Nile Red-based sensors in studies on lipid domain packing and dynamics with long acquisition times (Carravilla et al, 2021). See Chapter 10 in this Volume for a comprehensive treatment of orthogonal lipid sensors.…”

Imaging Membrane Order and Dynamic Interactions in Living Cells with a DNA Zipper Probe

“…We want to emphasize that a number of fluorescent probes have been developed for imaging membrane order, in most cases, based on their membrane partitioning phase preference or the solvatochromic effect (Figure S26) [62–68] . The DNA Zipper probes reported here represent a complementary approach for visualizing membrane order, i.e., via measuring membrane dynamic interactions.…”

When Weak Is Strong: A Plea for Low‐Affinity Binders for Optical Microscopy

“…[2] A further development is the redesign of Nile Red, rendering it sensitive to polarity, which allowed long-time live-cell super-resolution microscopy of endocytosis events. [11] Another target accessible with exchangeable small-molecule labels are amyloid fibers, which are bound by Thioflavin T [12] or the curcumin derivative CRANAD-2. [13] A second group are molecules that consist of a lowaffinity target-binding unit conjugated to a fluorophore.…”