Correlative nanophotonic approaches to enlighten the nanoscale dynamics of living cell membranes

Abstract

Dynamic compartmentalization is a prevailing principle regulating the spatiotemporal organization of the living cell membrane from the nano- up to the mesoscale. This non-arbitrary organization is intricately linked to cell function. On living cell membranes, dynamic domains or ‘membrane rafts' enriched with cholesterol, sphingolipids and other certain proteins exist at the nanoscale serving as signaling and sorting platforms. Moreover, it has been postulated that other local organizers of the cell membrane such as intrinsic protein interactions, the extracellular matrix and/or the actin cytoskeleton synergize with rafts to provide spatiotemporal hierarchy to the membrane. Elucidating the intricate coupling of multiple spatial and temporal scales requires the application of correlative techniques, with a particular need for simultaneous nanometer spatial precision and microsecond temporal resolution. Here, we review novel fluorescence-based techniques that readily allow to decode nanoscale membrane dynamics with unprecedented spatiotemporal resolution and single-molecule sensitivity. We particularly focus on correlative approaches from the field of nanophotonics. Notably, we introduce a versatile planar nanoantenna platform combined with fluorescence correlation spectroscopy to study spatiotemporal heterogeneities on living cell membranes at the nano- up to the mesoscale. Finally, we outline remaining future technological challenges and comment on potential directions to advance our understanding of cell membrane dynamics under the influence of the actin cytoskeleton and extracellular matrix in uttermost detail.

Multimodal Investigation into the Interaction of Quinacrine with Microcavity-Supported Lipid Bilayers

“…Alternative approaches that build membranes supported over aqueous micropores to improve the fluidity without the use of tethers while maintaining stability have evolved. Most importantly, in the case of buffer-filled pore-suspended bilayers, they offer the advantage of a relatively deep aqueous reservoir in contact with the proximal leaflet, which SLBs do not have. − We recently reported on such microcavity array-supported lipid bilayers (MSLBs) made from polystyrene sphere-templated gold and PDMS substrates, which we used to explore drug–membrane − and protein–membrane − interactions.…”

Plasmonics for advance single-molecule fluorescence spectroscopy and imaging in biology

“…Whilst the first examples simply started with gold nanoparticles and nanorods, thanks to the rapid progress in numerical simulations and nanofabrication, high-performance antennas configurations could be developed and implemented in large scale to enhance the capabilities of FCS measurements. These configurations allowed to controllable confine excitation light to sizes between 10 and 50 nm with a temporal resolution in the order of the μs and enable single-molecule detection at concentrations range as high as 10 μM (Punj et al, 2013a;Winkler and García-Parajo, 2021). However, as the modulation of the fluorescence signal is usually dictated by the overlap between the molecular and the plasmon transition bands, the choice of fluorescent tags suitable for a specific antenna configuration might be restricted.…”

Broadband Plasmonic Nanoantennas for Multi‐Color Nanoscale Dynamics in Living Cells

“…[6][7][8][9][10] Such findings were revealed owing to the recent development of different optical techniques aimed at improving both spatial and temporal resolution beyond that of conventional diffraction-limited optical methods. [10][11][12][13][14][15] Yet, monitoring dynamic multi-molecular interactions in living cells at the nanoscale remains challenging.…”

“…[6][7][8][9][10] Such findings were revealed owing to the recent development of different optical techniques aimed at improving both spatial and temporal resolution beyond that of conventional diffraction-limited optical methods. [10][11][12][13][14][15] Yet, monitoring dynamic multi-molecular interactions in living cells at the nanoscale remains challenging.With the advancement of single-molecule and super-resolution approaches, multiple techniques have been implemented aiming to reduce the illumination volume set by diffraction, thus enabling single-molecule dynamic studies at high labeling conditions in living cells on the nanoscale. For instance, stimulated emission depletion microscopy (STED) [10] and metallic nanoapertures [16][17][18] reduce the illumination area down to 50-200 nm in diameter.…”