Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events. Although several experiments indicate their existence, lipid nanodomains ('rafts') remain controversial owing to the lack of suitable detection techniques in living cells. The controversy is reflected in their putative size of 5-200 nm, spanning the range between the extent of a protein complex and the resolution limit of optical microscopy. Here we demonstrate the ability of stimulated emission depletion (STED) far-field fluorescence nanoscopy to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes approximately 70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently ( approximately 10-20 ms) trapped in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas. The non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells.

Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.

We have designed and synthesized a new functional (amino reactive) highly efficient fluorescent molecular switch (FMS) with a photochromic diarylethene and a rhodamine fluorescent dye. The reactive group in this FMS -N-hydroxysuccinimide ester- allows selective labeling of amino containing molecules or other materials. In ethanolic solutions, the compound displays a large fluorescent quantum yield of 52 % and a large fluorescence modulation ratio (94 %) between two states that may be interconverted with red and near-UV light. Silica nanoparticles incorporating the new FMS were prepared and characterized, and their spectroscopic and switching properties were also studied. The dye retained its properties after the incorporation into the silica, thereby allowing light-induced reversible high modulation of the fluorescence signal of a single particle for up to 60 cycles, before undergoing irreversible photobleaching. Some applications of these particles in fluorescence microscopy are also demonstrated. In particular, subdiffraction images of nanoparticles were obtained, in the focal plane of a confocal microscope.

I see red, I see red, I see red: Optically switchable compounds were synthesized whose visible fluorescence can be quenched down to 6 % with light at 360–400 nm and then restored with red light from a diode laser. The molecular switch offers potential applications in data storage and far‐field microscopy with spatial resolution far below the diffraction limit.