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.

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.

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.

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.

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.

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.

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.

Label-free, ultrahigh-speed, 3D observation of bidirectional and correlated intracellular cargo transport by coherent brightfield microscopy.

The investigation of intracellular transport at the molecular scale requires measurements at high spatial and temporal resolutions. We demonstrate the label-free, direct imaging and tracking of native cell vesicles in live cells at an ultrahigh spatiotemporal resolution. Using coherent brightfield (COBRI) microscopy, we monitor individual cell vesicles traveling inside the cell with nanometer spatial precision in 3D at 30 000 frames per second. The stepwise directional motion of the vesicle on the cytoskeletal track is clearly resolved. We also observe the repeated switching of the transport direction of the vesicle in a continuous trajectory. Our high-resolution measurement unveils the transient pausing and subtle bidirectional motion of the vesicle, taking place over tens of nanometers in tens of milliseconds. By tracking multiple particles simultaneously, we found strong correlations between the motions of two neighboring vesicles. Our label-free ultrahigh-speed optical imaging provides the opportunity to visualize intracellular cargo transport at the nanoscale in the microsecond timescale with minimal perturbation.

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.

Coherent Brightfield Microscopy Provides the Spatiotemporal Resolution To Study Early Stage Viral Infection in Live Cells.

Viral infection starts with a virus particle landing on a cell surface followed by penetration of the plasma membrane. Due to the difficulty of measuring the rapid motion of small-sized virus particles on the membrane, little is known about how a virus particle reaches an endocytic site after landing at a random location. Here, we use coherent brightfield (COBRI) microscopy to investigate early stage viral infection with ultrahigh spatiotemporal resolution. By detecting intrinsic scattered light via imaging-based interferometry, COBRI microscopy allows us to track the motion of a single vaccinia virus particle with nanometer spatial precision (<3 nm) in 3D and microsecond temporal resolution (up to 100,000 frames per second). We explore the possibility of differentiating the virus signal from cell background based on their distinct spatial and temporal behaviors via digital image processing. Through image postprocessing, relatively stationary background scattering of cellular structures is effectively removed, generating a background-free image of the diffusive virus particle for precise localization. Using our method, we unveil single virus particles exploring cell plasma membranes after attachment. We found that immediately after attaching to the membrane (within a second), the virus particle is locally confined within hundreds of nanometers where the virus particle diffuses laterally with a very high diffusion coefficient (∼1 µm2/s) at microsecond time scales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.

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.

Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking.

Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes.

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.

Tracking single particles on supported lipid membranes: multimobility diffusion and nanoscopic confinement.

Supported lipid bilayers have been studied intensively over the past two decades. In this work, we study the diffusion of single gold nanoparticles (GNPs) with diameter of 20 nm attached to GM1 ganglioside or DOPE lipids at different concentrations in supported DOPC bilayers. The indefinite photostability of GNPs combined with the high sensitivity of interferometric scattering microscopy (iSCAT) allows us to achieve 1.9 nm spatial precision at 1 ms temporal resolution, while maintaining long recording times. Our trajectories visualize strong transient confinements within domains as small as 20 nm, and the statistical analysis of the data reveals multiple mobilities and deviations from normal diffusion. We present a detailed analysis of our findings and provide interpretations regarding the effect of the supporting substrate and GM1 clustering. We also comment on the use of high-speed iSCAT for investigating diffusion of lipids, proteins, or viruses in lipid membranes with unprecedented spatial and temporal resolution.

Two-photon microscopy of the mouse cochlea in situ for cellular diagnosis.

Sensorineural hearing loss is the most common type of hearing loss worldwide, yet the underlying cause is typically unknown because the inner ear cannot be biopsied today without destroying hearing, and intracochlear cells have not been imaged with resolution sufficient to establish diagnosis. Intracochlear imaging has been technologically challenging because of the cochlea's small size and encasement in bone. We report, for the first time, imaging of the mouse cochlea in situ without exogenous dyes, through a membranous round window, using a near-infrared femtosecond laser as the excitation and endogenous two-photon excitation fluorescence (TPEF) and second harmonic generation as the contrast mechanisms. We find that TPEF exhibits strong contrast allowing cellular, and even subcellular resolution, and detection of specific, noise-induced pathologic changes. Our results demonstrate that the round window provides a useful access to the cochlea through the middle ear, and they motivate future development of a new and efficient diagnostic tool based on two-photon micro-endoscopy.

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.

Imaging with second-harmonic radiation probes in living tissue.

We demonstrate that second-harmonic radiation imaging probes are efficient biomarkers for imaging in living tissue. We show that 100 nm and 300 nm BaTiO(3) nanoparticles used as contrast markers could be detected through 50 µm and 120 µm of mouse tail tissue in vitro or in vivo. Experimental results and Monte-Carlo simulations are in good agreement.