The biological functions of the cell membrane are influenced by the mobility of its constituents, which are thought to be strongly affected by nanoscale structure and organization. Interactions with the actin cytoskeleton have been proposed as a potential mechanism with the control of mobility imparted through transmembrane “pickets” or GPI-anchored lipid nanodomains. This hypothesis is based on observations of molecular mobility using various methods, although many of these lack the spatiotemporal resolution required to fully capture all the details of the interaction dynamics. In addition, the validity of certain experimental approaches, particularly single-particle tracking, has been questioned due to a number of potential experimental artifacts. Here, we use interferometric scattering microscopy to track molecules labeled with 20–40 nm scattering gold beads with simultaneous <2 nm spatial and 20 μs temporal precision to investigate the existence and mechanistic origin of anomalous diffusion in bilayer membranes. We use supported lipid bilayers as a model system and demonstrate that the label does not influence time-dependent diffusion in the small particle limit (≤40 nm). By tracking the motion of the ganglioside lipid GM1 bound to the cholera toxin B subunit for different substrates and lipid tail properties, we show that molecular pinning and interleaflet coupling between lipid tail domains on a nanoscopic scale suffice to induce transient immobilization and thereby anomalous subdiffusion on the millisecond time scale.

Lipid rafts are submicron proteolipid domains thought to be responsible for membrane trafficking and signaling. Their small size and transient nature put an understanding of their dynamics beyond the reach of existing techniques, leading to much contention as to their exact role. Here, we exploit the differences in light scattering from lipid bilayer phases to achieve dynamic imaging of nanoscopic lipid domains without any labels. Using phase-separated droplet interface bilayers we resolve the diffusion of domains as small as 50 nm in radius and observe nanodomain formation, destruction, and dynamic coalescence with a domain lifetime of 220 ± 60 ms. Domain dynamics on this timescale suggests an important role in modulating membrane protein function.droplet interface bilayer | iSCAT | lipid nanodomains | label-free imaging | light scattering C ell membranes compartmentalize into lipid domains that enable the selective recruitment of specific proteins (1). These "lipid rafts" have been proposed to control many membrane processes including apical sorting, protein trafficking, and the clustering of proteins during signaling. The dynamic formation and destruction of lipid nanodomains are thought to provide the central mechanism to regulate this wide range of essential processes (2-4). Although many methods now provide strong evidence to support their existence in vivo (5), the combination of nanoscopic size and dynamics on millisecond timescales has placed the direct observation of their behavior beyond the scope of existing techniques. Consequently, although we know they exist, frustratingly little is known regarding their function and dynamics (6).Recent advances in fluorescence nanoscopy provide the only time-dependent information on the behavior of lipid nanodomains (7-9). Stimulated emission depletion-fluorescence correlation spectroscopy has shown cholesterol-mediated hindered nanoscale diffusion of single labeled sphingomyelin lipids that is consistent with the lipid raft hypothesis and transient binding of lipids (9). Superresolution fluorescence microscopy has also revealed protein clusters in cell membranes with 0.5-s temporal resolution (7). All of these experiments, however, are limited in temporal resolution by fluorescence, and must infer lipid nanodomains from the addition of fluorescent labels.Macroscopic phase separation in artificial lipid bilayers has been widely used to help understand the biological implications of domain formation. Different lipid phases can be visualized using fluorescence microscopy with labels that preferentially partition into a specific phase (10-12). This approach is successful for micrometer-sized domains but inevitably fails on the tens to few hundreds of nanometers scale due to limitations in phase specificity, the limited residence time of a label within a specific nanoscopic domain, and the achievable optical resolution (13). The fluorescent probe is itself an additional component that can perturb phase behavior, either directly or through photooxidation (14, 15). As ...

Myosin 5a is a dual-headed molecular motor that transports cargo along actin filaments. By following the motion of individual heads with interferometric scattering microscopy at nm spatial and ms temporal precision we found that the detached head occupies a loosely fixed position to one side of actin from which it rebinds in a controlled manner while executing a step. Improving the spatial precision to the sub-nm regime provided evidence for an ångstrom-level structural transition in the motor domain associated with the power stroke. Simultaneous tracking of both heads revealed that consecutive steps follow identical paths to the same side of actin in a compass-like spinning motion demonstrating a symmetrical walking pattern. These results visualize many of the critical unknown aspects of the stepping mechanism of myosin 5 including head–head coordination, the origin of lever-arm motion and the spatiotemporal dynamics of the translocating head during individual steps.DOI: http://dx.doi.org/10.7554/eLife.05413.001

Observation techniques with high spatial and temporal resolution, such as single-particle tracking based on interferometric scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work, we have used lipid analogues tagged with a hybrid fluorescent tag–gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous supported lipid bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50  ⩽  t  ⩽  100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye tagged lipid analogues. These FCS measurements of hybrid fluorescent tag–gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2–3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially cross-linking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

The spatiotemporal organization and dynamics of the plasma membrane and its constituents are central to cellular function. Fluorescence-based single-particle tracking has emerged as a powerful approach for studying the single molecule behavior of plasma-membrane-associated events because of its excellent background suppression, at the expense of imaging speed and observation time. Here, we show that interferometric scattering microscopy combined with 40 nm gold nanoparticle labeling can be used to follow the motion of membrane proteins in the plasma membrane of live cultured mammalian cell lines and hippocampal neurons with up to 3 nm precision and 25 μs temporal resolution. The achievable spatiotemporal precision enabled us to reveal signatures of compartmentalization in neurons likely caused by the actin cytoskeleton.

The clearest dynamical signature of a roaming reaction is a very cold distribution of energy into the rotational and translational degrees of freedom of the roaming donor fragment (e.g. CO) and an exceptionally hot vibrational distribution in the roaming acceptor fragment (e.g. H2, CH4). These signatures were initially identified in joint experimental/theoretical investigations of roaming in H2CO and CH3CHO and are now used to infer the presence of roaming mechanisms in other photodissociation reactions. In this paper we construct a phase space theory (PST) model of triple fragmentation (3F) and show that the dynamical signature of 3F is similar to that of the roaming donor fragment. The PST model starts with a calculation of two-body fragmentation (2F) of a generic molecule, ABC into AB + C. Every AB fragment with sufficient energy to undergo subsequence spontaneous dissociation is allowed to dissociate and the PST distribution of energy into A + B products is calculated for every initial AB state. Using CH3CHO --> HCO + CH3 --> H + CO + CH3 as an example, we calculate that the energy disposal into the rotational and translational degrees of freedom of the 3F products is very low, and is similar to the dynamical signature expected for production of CO via a roaming mechanism. We compare the 3F PST model with published experimental data for photodissociation of CH3CHO and CH3OCHO at energies above the 3F threshold.

We report on the interrogation of a lead-insensitive dual-mode fiber sensor using a white-light interferometric technique with a multimode laser-diode source. An elliptical-core dual-mode fiber acts as a remote-sensing interferometer, and a second dual-mode fiber acts as a receiver interferometer. We establish the sensor design for optimized performance and demonstrate the capability of accurate detection of periodic strain signals in the presence of, e.g., temperature-induced low-frequency phase drifts.

Observation techniques with high spatial and temporal resolution, such as single-particle tracking (SPT) based on interferometric Scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye-tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work we have used lipid analogues tagged with a hybrid fluorescent tag – gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous Supported Lipid Bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50 ≤ t ≤ 100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye-tagged lipid analogues. These FCS measurements of hybrid fluorescent tag – gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2-3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially crosslinking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.