Heterogeneous nanoscopic lipid diffusion in the live cell membrane and its dependency on cholesterol.

Cholesterol plays a unique role in the regulation of membrane organization and dynamics by modulating the membrane phase transition at the nanoscale. Unfortunately, due to their small sizes and dynamic nature, the effects of cholesterol-mediated membrane nanodomains on membrane dynamics remain elusive. Here, using ultrahigh-speed single-molecule tracking with advanced optical microscope techniques, we investigate the diffusive motion of single phospholipids in the live cell plasma membrane at the nanoscale and its dependency on the cholesterol concentration. We find that both saturated and unsaturated phospholipids undergo anomalous subdiffusion on the length scale of 10-100 nm. The diffusion characteristics exhibit considerable variations in space and in time, indicating that the nanoscopic lipid diffusion is highly heterogeneous. Importantly, through the statistical analysis, apparent dual-mobility subdiffusion is observed from the mixed diffusion behaviors. The measured subdiffusion agrees well with the hop diffusion model that represents a diffuser moving in a compartmentalized membrane created by the cytoskeleton meshwork. Cholesterol depletion diminishes the lipid mobility with an apparently smaller compartment size and a stronger confinement strength. Similar results are measured with temperature reduction, suggesting that the more heterogeneous and restricted diffusion is connected to the nanoscopic membrane phase transition. Our conclusion supports the model that cholesterol depletion induces the formation of gel-phase, solid-like membrane nanodomains. These nanodomains undergo restricted diffusion and act as diffusion obstacles to the membrane molecules that are excluded from the nanodomains. This work provides the experimental evidence that the nanoscopic lipid diffusion in the cell plasma membrane is heterogeneous and sensitive to the cholesterol concentration and temperature, shedding new light on the regulation mechanisms of nanoscopic membrane dynamics.

Spinning disk interferometric scattering confocal microscopy captures millisecond timescale dynamics of living cells.

Interferometric scattering (iSCAT) microscopy is a highly sensitive imaging technique that uses common-path interferometry to detect the linear scattering fields associated with samples. However, when measuring a complex sample, such as a biological cell, the superposition of the scattering signals from various sources, particularly those along the optical axis of the microscope objective, considerably complicates the data interpretation. Herein, we demonstrate high-speed, wide-field iSCAT microscopy in conjunction with confocal optical sectioning. Utilizing the multibeam scanning strategy of spinning disk confocal microscopy, our iSCAT confocal microscope acquires images at a rate of 1,000 frames per second (fps). The configurations of the spinning disk and the background correction procedures are described. The iSCAT confocal microscope is highly sensitive-individual 10 nm gold nanoparticles are successfully detected. Using high-speed iSCAT confocal imaging, we captured the rapid movements of single nanoparticles on the model membrane and single native vesicles in the living cells. Label-free iSCAT confocal imaging enables the detailed visualization of nanoscopic cell dynamics in their most native forms. This holds promise to unveil cell activities that are previously undescribed by fluorescence-based microscopy.

From Dynamics to Membrane Organization: Experimental Breakthroughs Occasion a "Modeling Manifesto".

New experimental techniques, especially in the context of observing molecular dynamics, reveal the plasma membrane to be heterogeneous and "scale rich," from nanometers to microns and from microseconds to seconds. This is critical information, which shows that scale-dependent transport governs the molecular encounters that underlie cellular signaling. The data are rich and reaffirm the importance of the cortical cytoskeleton, protein aggregates, and lipidomic complexity on the statistics of molecular encounters. Moreover, the data demand simulation approaches with a particular set of features, hence the "manifesto." Together with the experimental data, simulations that satisfy these requirements hold the promise of a deeper understanding of membrane spatiotemporal organization. Several experimental breakthroughs in measuring molecular membrane dynamics are reviewed, the constraints that they place on simulations are discussed, and the status of simulation approaches that aim to meet them are detailed.

Glycosaminoglycans-Specific Cell Targeting and Imaging Using Fluorescent Nanodiamonds Coated with Viral Envelope Proteins.

Understanding virus-host interactions is crucial for vaccine development. This study investigates such interactions using fluorescent nanodiamonds (FNDs) coated with vaccinia envelope proteins as the model system. To achieve this goal, we noncovalently conjugated 100 nm FNDs with rA27(aa 21-84), a recombinant envelope protein of vaccinia virus, for glycosaminoglycans (GAGs)-specific targeting and imaging of living cells. Another recombinant protein rDA27(aa 33-84) that removes the GAGs-binding sequences was also used for comparison. Three types of A27-coated FNDs were generated, including rA27(aa 21-84)-FND, rDA27(aa 33-84)-FND, and hybrid rA27(aa 21-84)/rDA27(aa 33-84)-FND. The specificity of these viral protein-FND conjugates toward GAGs binding was examined by flow cytometry, fluorescence microscopy, and gel electrophoresis. Results obtained for normal and GAGs-deficient cells showed that the recombinant proteins maintain their GAG-targeting activities even after immobilization on the FND surface. Our studies provide a new nanoparticle-based platform not only to target specific cell types but also to track the fates of these immobilized viral proteins in targeted cells as well as to isolate and enrich GAGs-associated proteins on cell membrane.

A Rhizavidin Monomer with Nearly Multimeric Avidin-Like Binding Stability Against Biotin Conjugates.

Developing a monomeric form of an avidin-like protein with highly stable biotin binding properties has been a major challenge in biotin-avidin linking technology. Here we report a monomeric avidin-like protein-enhanced monoavidin-with off-rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head-group-biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24-meric avidin probe by fusing eMA to a multimeric cage protein. The 24-meric avidin and eMA were utilized to demonstrate how artificial clustering of cell-surface proteins greatly enhances the internalization rates of assembled proteins on live cells.

Shot-noise limited localization of single 20 nm gold particles with nanometer spatial precision within microseconds.

Single-particle tracking (SPT) is a powerful approach to investigate dynamics without ensemble average. Continuing effort has been made to track smaller particles with better spatial precision at higher speed. In this work, we demonstrate SPT of 20 nm gold nanoparticle (GNP) with 2 nm spatial precision up to 500 kHz by using microsecond interferometric scattering (µs-iSCAT) microscopy. The linear scattering signal from single GNPs is detected by a high-speed CMOS camera via interference. Through this homodyne detection, shot-noise limited sensitivity, and therefore optimal localization precision are achieved at high speed where considerable electronic noise is present. Using µs-iSCAT microscopy, we observe anomalous diffusion of GNPs labeled to lipid molecules in a supported bilayer membrane prepared on a glass substrate. The combination of nanometer spatial precision and microsecond temporal resolution provides the opportunity to study rapid motions of nano-objects on molecular scale with unprecedented clarity.

Probing chromatin condensation dynamics in live cells using interferometric scattering correlation spectroscopy.

Chromatin organization and dynamics play important roles in governing the regulation of nuclear processes of biological cells. However, due to the constant diffusive motion of chromatin, examining chromatin nanostructures in living cells has been challenging. In this study, we introduce interferometric scattering correlation spectroscopy (iSCORS) to spatially map nanoscopic chromatin configurations within unlabeled live cell nuclei. This label-free technique captures time-varying linear scattering signals generated by the motion of native chromatin on a millisecond timescale, allowing us to deduce chromatin condensation states. Using iSCORS imaging, we quantitatively examine chromatin dynamics over extended periods, revealing spontaneous fluctuations in chromatin condensation and heterogeneous compaction levels in interphase cells, independent of cell phases. Moreover, we observe changes in iSCORS signals of chromatin upon transcription inhibition, indicating that iSCORS can probe nanoscopic chromatin structures and dynamics associated with transcriptional activities. Our scattering-based optical microscopy, which does not require labeling, serves as a powerful tool for visualizing dynamic chromatin nano-arrangements in live cells. This advancement holds promise for studying chromatin remodeling in various crucial cellular processes, such as stem cell differentiation, mechanotransduction, and DNA repair.

Roadmap on Label-Free Super-Resolution Imaging.

Label-free super-resolution (LFSR) imaging relies on light-scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super-resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state-of-the-art in this field, and to discuss the resolution boundaries and hurdles which need to be overcome to break the classical diffraction limit of the LFSR imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction-limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super-resolution capability which are based on understanding resolution as an information science problem, on using novel structured illumination, near-field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere-assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.

Three-dimensional scanning microscopy through thin turbid media.

We demonstrate three-dimensional imaging through a thin turbid medium using digital phase conjugation of the second harmonic signal emitted from a beacon nanoparticle. The digitally phase-conjugated focus scans the volume in the vicinity of its initial position through numerically manipulated phase patterns projected onto the spatial light modulator. Accurate three dimensional images of a fluorescent sample placed behind a turbid medium are obtained.

Label-Free Dynamic Imaging of Chromatin in Live Cell Nuclei by High-Speed Scattering-Based Interference Microscopy.

Chromatin is a DNA-protein complex that is densely packed in the cell nucleus. The nanoscale chromatin compaction plays critical roles in the modulation of cell nuclear processes. However, little is known about the spatiotemporal dynamics of chromatin compaction states because it remains difficult to quantitatively measure the chromatin compaction level in live cells. Here, we demonstrate a strategy, referenced as DYNAMICS imaging, for mapping chromatin organization in live cell nuclei by analyzing the dynamic scattering signal of molecular fluctuations. Highly sensitive optical interference microscopy, coherent brightfield (COBRI) microscopy, is implemented to detect the linear scattering of unlabeled chromatin at a high speed. A theoretical model is established to determine the local chromatin density from the statistical fluctuation of the measured scattering signal. DYNAMICS imaging allows us to reconstruct a speckle-free nucleus map that is highly correlated to the fluorescence chromatin image. Moreover, together with calibration based on nanoparticle colloids, we show that the DYNAMICS signal is sensitive to the chromatin compaction level at the nanoscale. We confirm the effectiveness of DYNAMICS imaging in detecting the condensation and decondensation of chromatin induced by chemical drug treatments. Importantly, the stable scattering signal supports a continuous observation of the chromatin condensation and decondensation processes for more than 1 h. Using this technique, we detect transient and nanoscopic chromatin condensation events occurring on a time scale of a few seconds. Label-free DYNAMICS imaging offers the opportunity to investigate chromatin conformational dynamics and to explore their significance in various gene activities.

Digital phase conjugation of second harmonic radiation emitted by nanoparticles in turbid media.

We demonstrate focusing coherent light on a nanoparticle through turbid media based on digital optical phase conjugation of second harmonic generation (SHG) field from the nanoparticle. A SHG active nanoparticle inside a turbid medium was excited at the fundamental frequency and emitted SHG field as a point source. The SHG emission was scattered by the turbid medium, and the scattered field was recorded by off-axis digital holography. A phase-conjugated beam was then generated by using a phase-only spatial light modulator and sent back through the turbid medium, which formed a nearly ideal focus on the nanoparticle.

Second harmonic generation from nanocrystals under linearly and circularly polarized excitations.

We study second harmonic generation (SHG) from non-centrosymmetric nanocrystals under linearly polarized (LP) and circularly polarized (CP) excitations. Theoretical models are developed for SHG from nanocrystals under both plane-wave and focused excitations. We find that the focused excitation reduces the polarization dependency of the SHG signal. We show that the SHG response under CP excitation is generally inferior to the average of LP excitations over all orientations. We verify the theory by measuring the SHG polar responses from BaTiO3 nanocrystals with a scanning confocal microscope. The experimental data agrees well with the theory.

Imaging through turbid layers by scanning the phase conjugated second harmonic radiation from a nanoparticle.

We demonstrate imaging through a turbid layer by using digital phase conjugation of the second harmonic field radiated from a beacon nanoparticle. We show that the phase-conjugated focus can be displaced from its initial position by illuminating the same region of the turbid layer with an angular offset. An image is obtained by scanning the phase-conjugated focus through the turbid layer in a region around the nanoparticle. We obtain a clear image of the target by measuring the light transmitted through it when scanning the focused beam.

Nonlinear optical properties of core-shell nanocavities for enhanced second-harmonic generation.

A nonlinear optical plasmonic core-shell nanocavity is demonstrated as an efficient, subwavelength coherent light source through second-harmonic generation. The nonlinear optical plasmonic nanocavity incorporates a noncentrosymmetric medium, which utilizes the entire mode volume for even-order nonlinear optical processes. In previous plasmonic nanocavities, enhancement of such processes was only possible at the interface but symmetry prohibited in the body. We measured an enhancement of over 500 times in the second-harmonic radiation power. Calculations show that an enhancement of over 3500 times is achievable.

Three-dimensional harmonic holographic microcopy using nanoparticles as probes for cell imaging.

Luminescent markers play a key role in imaging techniques for life science since they provide a contrast mechanism between signal and background. We describe a new type of marker using second harmonic generation (SHG) from noncentrosymmetric BaTiO(3) nanocrystals. These nanoparticles are attractive due to their stable, non-saturating and coherent signal with a femtosecond-scale response time and broad flexibility in the choice of excitation wavelength. We obtained monodispersed BaTiO(3) nanoparticles in colloidal suspensions by coating the particle surface with amine groups. We characterized the SHG efficiency of 90-nm BaTiO(3) particles experimentally and theoretically. Moreover, we use the coherent SHG signal from BaTiO(3) nanoparticles for three-dimensional (3D) imaging without scanning. We built a harmonic holographic (H(2)) microscope which records digital holograms at the second harmonic frequency. For the first time, high-resolution 3D distributions of these SHG markers in mammalian cells are successfully captured and interpreted by the H(2) microscope.

Specular scattering probability of acoustic phonons in atomically flat interfaces.

We report a direct determination of the specular scattering probability of acoustic phonons at a crystal boundary by observing the escape of incident coherent phonons from the coherent state during reflection. In the sub-THz frequency range where the phonon wavelength is much longer than the lattice constant, the acoustic phonon-interface interaction is found to agree well with the macroscopic theory on wave scattering from rough surfaces. This examination thus quantitatively verifies the dominant role of atomic-scale corrugations in the Kapitza anomaly observed at 1-10 K and further opens a new path to nondestructively estimate subnanoscale roughness of buried interfaces.

High-speed imaging and tracking of very small single nanoparticles by contrast enhanced microscopy.

Nanoparticles have been used extensively in biology-related research and many applications require direct visualization of individual nanoparticles by optical microscopy. For long-term and high-speed measurements, scattering-based microscopy is a unique technique because of the stable and indefinite scattering signals. In scattering-based single-particle measurements, large nanoparticles are usually needed in order to generate sufficient signals for detection. However, larger nanoparticles introduce greater mass loading, experience stronger steric hindrance, and are more prone to crosslinking. In this work, we demonstrate coherent brightfield (COBRI) microscopy with enhanced contrast and show its capability of direct visualization of very small nanoparticles in scattering at a high speed. COBRI microscopy allows us to visualize and track single metallic and dielectric nanoparticles, as small as 10 nm, at 1000 frames per second. A quantitative relationship between the linear scattering cross section of the nanoparticle and its COBRI contrast is reported. Using COBRI microscopy, we further demonstrate the tracking of 10 nm gold nanoparticles labeled to lipid molecules in supported bilayer membranes, showing that the small nanoparticles may facilitate single-molecule measurements with reduced perturbation. Furthermore, the identical imaging sensitivities of COBRI and interferometric scattering (iSCAT) microscopy, the reflection counterpart of COBRI, is demonstrated at an equal illumination intensity. Finally, future improvements in the speed and sensitivity of scattering-based interference microscopy are discussed.

Characterization of Single-Protein Dynamics in Polymer-Cushioned Lipid Bilayers Derived from Cell Plasma Membranes.

Native cell-membrane-derived supported lipid bilayers (SLBs) are an emerging platform with broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is the proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact between the bilayer membrane and the supporting substrate, and thus, the mobility of the transmembrane proteins is maintained. However, little is known about how the polymer cushion affects the absolute mobility of membrane molecules. Here, we characterized the dynamics of single membrane proteins in polymer-cushioned lipid bilayers derived from cell plasma membranes and investigated the effects of polymer length. Three membrane proteins with distinct structures, i.e., a GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescent protein (GFP), and their dynamics were measured by fluorescent single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length on protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1000 to 5000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were augmented with the polymer length, whereas the diffusion coefficient of the GPI-anchored protein remained almost identical for different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 µm2/s approximately) for the cushioned membrane with the longest polymer length (molecular weight of 5000), indicating that at the microscopic length scale, the SLBs were fully suspended from the substrate by the polymer cushion. Transient confinements were observed for all three proteins, and increasing the polymer length reduced the tendency of transient confinement. The measured dynamics of membrane proteins were found to be nearly unchanged after the depletion of cholesterol, suggesting that the observed immobilization and transient confinement were not due to cholesterol-enriched membrane nanodomains (lipid rafts). Our single-molecule dynamics elucidate the biophysical properties of polymer-cushioned plasma membrane bilayers that are potentially useful for the future developments of membrane-based biosensors and analytical assays.

Monovalent and Oriented Labeling of Gold Nanoprobes for the High-Resolution Tracking of a Single-Membrane Molecule.

Single-molecule tracking is a powerful method to study molecular dynamics in living systems including biological membranes. High-resolution single-molecule tracking requires a bright and stable signal, which has typically been facilitated by nanoparticles due to their superb optical properties. However, there are concerns about using a nanoparticle to label a single molecule because of its relatively large size and the possibility of cross-linking multiple target molecules, both of which could affect the original molecular dynamics. In this work, using various labeling schemes, we investigate the effects using nanoparticles to measure the diffusion of single-membrane molecules. By conjugating a low density of streptavidin (sAv) to gold nanoparticles (AuNPs) of different sizes (10, 15, 20, 30, and 40 nm), we isolate and quantify the effect of the particle size on the diffusion of biotinylated lipids in supported lipid bilayers (SLBs). We find that single sAv tends to cross-link two biotinylated lipids, leading to a much slower diffusion in SLBs. We further demonstrate a simple and robust strategy for the monovalent and oriented labeling of a single lipid molecule with a AuNP by using naturally dimeric rhizavidin (rAv) as a bridge, thus connecting the biotinylated nanoparticle surface and biotinylated target molecule. The rAv-AuNP conjugate demonstrates fast and free diffusion in SLBs (2-3 µm2/s for rAv-AuNP sizes of 10-40 nm), which is comparable to the diffusion of dye-labeled lipids, indicating that the adverse size and cross-linking effects are successfully avoided. We also note that the diffusion of dye-labeled lipids critically depends on the choice of dye, which could report different diffusion coefficients by about 20% (2.2 µm2/s of ATTO647N and 2.6 µm2/s of ATTO532). By comparing the diffusion of the uniformly and randomly oriented labeling of a single lipid molecule with a AuNP, we conclude that oriented labeling is favorable for measuring the diffusion of single-membrane molecules. Our work shows that the measured diffusion of the membrane molecule is highly sensitive to the molecular design of the cross-linker for labeling. The demonstrated approach of monovalent and oriented AuNP labeling provides the opportunity to study single-molecule membrane dynamics at much higher spatiotemporal resolutions and, most importantly, without labeling artifacts.

Bioorthogonal Fluorescent Nanodiamonds for Continuous Long-Term Imaging and Tracking of Membrane Proteins.

Real-time tracking of membrane proteins is essential to gain an in-depth understanding of their dynamics on the cell surface. However, conventional fluorescence imaging with molecular probes like organic dyes and fluorescent proteins often suffers from photobleaching of the fluorophores, thus hindering their use for continuous long-term observations. With the availability of fluorescent nanodiamonds (FNDs), which have superb biocompatibility and excellent photostability, it is now possible to conduct the imaging in both short and long terms with high temporal and spatial resolution. To realize the concept, we have developed a facile method (e.g., one-pot preparation) to produce alkyne-functionalized hyperbranched-polyglycerol-coated FNDs for bioorthogonal labeling of azide-modified membrane proteins and azide-modified antibodies of membrane proteins. The high specificity of this labeling method has allowed us to continuously monitor the movements of the proteins of interest (such as integrin α5) on/in living cells over 2 h. The results open a new horizon for live cell imaging with functional nanoparticles and fluorescence microscopy.