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.

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.

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 crosslinking multiple target molecules, both of which could affect the original molecular dynamics. In this work, using various labeling schemes, we investigate the effects of the use of 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 crosslink 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 nm to 40 nm), which is comparable to the diffusion of dye-labeled lipids, indicating that the adverse size and crosslinking 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 crosslinker 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.

Nanoparticles have been used extensively in biology-related research and many applications require direct visualization of individual nanoparticles under optical microscopy. For long-term and high-speed measurements, scattering-based microscopy is a unique technique because of the stable and indefinite scattering signal. In scattering-based single-particle measurements, large nanoparticles are usually needed in order to generate sufficient signal for detection. However, larger nanoparticle introduces greater mass loading, experiences stronger steric hindrance, and is 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 high speed. The COBRI microscopy allows us to visualize and track single metallic and dielectric nanoparticles, as small as 10 nm, at 1,000 frames per second. A quantitative relationship between the linear scattering cross section of nanoparticle and its COBRI contrast is reported. Using COBRI microscopy, we further demonstrate tracking of 10 nm gold nanoparticles labeled to lipid molecules in supported bilayer membranes, showing that the small nanoparticle may facilitate single-molecule measurements with reduced perturbation. Furthermore, identical imaging sensitivity of COBRI and interferometric scattering (iSCAT) microscopy, the reflection counterpart of COBRI, is demonstrated under equal illumination intensity. Finally, future improvements in speed and sensitivity of scattering-based interference microscope are discussed.

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 post-processing, 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 timescales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.

Interferometric scattering (iSCAT) microscopy is currently among the most powerful techniques available for achieving high-sensitivity single-particle localization. This capability is realized through homodyne detection, where interference with a reference wave offers the promise of exceptionally precise three-dimensional (3D) localization. However, the practical application of iSCAT to 3D tracking has been hampered by rapid oscillations in the signal-to-noise ratio (SNR) as particles move along the axial direction. In this study, we introduce a novel strategy based on back pupil plane engineering, wherein a spiral phase mask is used to redistribute the phase of the scattered field of the particle uniformly across phase space, thus ensuring consistent SNR as the particle moves throughout the focal volume. Our findings demonstrate that this modified spiral phase iSCAT exhibits greatly enhanced localizability characteristics. Additionally, the uniform phase distribution enables reliable characterization of the particle’s optical properties regardless of its position. We substantiate our theoretical results with numerical and experimental demonstrations, showcasing the practical application of this approach for high-precision, ultrahigh-speed (20,000 frames per second) 3D tracking and polarizability measurement of freely diffusing nanoparticles as small as 20 nm.

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.

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.

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.

Optical interference microscopy is a powerful bioimaging technique by measuring the complex light fields associated with the specimen. Nowadays, the state-of-the-art interference microscopy makes it possible to directly visualize very small single biological nanoparticles and unlabeled macromolecules. The stable and indefinite linear scattering signal allows for continuous observation of the sample at a high speed, offering the opportunities to investigate single-molecule biophysics with the unprecedented details. Meanwhile, using interference microscopy to explore complex biological samples, such as a biological cell, emerges as an exciting research field. In this Perspective, we share our views on the impacts of optical interference microscopy on live cell imaging. Strategies for discriminating the scattering signals from different cell organelles and biological macromolecules are presented. In particular, the dynamic optical signal of live cells contains rich temporal information that is useful for enhancing the molecular specificity and functional information in label-free cell imaging. Finally, the challenges in three-dimensional imaging and turbidity suppression are discussed.

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.

Interferometric scattering (iSCAT) microscopy has been demonstrated as a powerful tool for tracking single nanoparticles at ultrahigh spatial and temporal resolutions. The unmatched performance of iSCAT single-particle tracking (SPT) relies on the fact that the scattering signal is steady and can be linearly increased under strong illumination, circumventing the problems of photobleaching and saturation of fluorescence-based approaches. However, the scattering-based imaging is complicated by the presence of nonspecific scattering background, e.g. in the complex samples of biological cells. To distinguish the signal of interest from the nonspecific scattering background, metallic nanoparticles are often used as the efficient scattering probes. In many applications, reduction of the particle size is favorable because the loading of particle may introduce labeling artifacts. To work with smaller particles of weaker signal, suppression of the heterogeneous background is necessary. In this work, we start with the characterization of dynamic iSCAT signal of living cells by calculating its spatial and temporal Fourier spectra. To reduce the influence of cell background to SPT, a common strategy of background estimation and correction by temporal average filtering is considered. The effect of residual cell background to SPT is evaluated systematically with simulated image data of various signal-to-background ratios. This work benchmarks the localization errors caused by the cell background in SPT, providing a guideline for the interpretation of single-particle diffusion data of iSCAT microscopy.

Light absorption is a common phenomenon in nature, but accurate and quantitative absorption measurement at the nanoscale remains challenging especially in the application of widefield imaging. Here, we demonstrated optical widefield interferometric photothermal microscopy that allowed us to visualize and quantify the heat generation of single nanoparticles. The working principle was to measure the scattering signal due to the refractive index change of the surrounding media induced by the dissipated heat (known as the thermal lens effect). The sensitivity of our local heat measurement was a few nanowatts—the high sensitivity made it possible to detect single gold nanoparticles, as small as 5 nm. By changing the particle sizes, we found that, for small metallic nanoparticles (gold and silver nanoparticles < 40 nm), the photothermal signal was determined by the amount of the dissipated heat, independent of the particle size. A model was established to explain our experimental results, indicating that the photothermal signal was essentially contributed by the interferometric detection of the scattered field of the thermal lens. Importantly, on the basis of this model, we further investigated the photothermal signal of large nanoparticles (40–100 nm for our setup) where the scattered light of the particle was considerable relative to the probe light. In this regime, the strong scattered field of the particle effectively served as the main reference beam that interfered with the scattered field of the thermal lens, resulting in an enhanced photothermal signal. Our work illustrates an important fact that the measured photothermal signal is fundamentally affected by the scattering property of the sample. This finding paves the way to accurate and sensitive absorption-based imaging in complex biological samples where the scattering is often spatially heterogeneous.

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 crosslinking multiple target molecules, both of which could affect the original molecular dynamics. In this work, using various labeling schemes, we investigate the effects of the use of 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 crosslink 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 nm to 40 nm), which is comparable to the diffusion of dye-labeled lipids, indicating that the adverse size and crosslinking 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 crosslinker 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.

Native cell membrane derived supported lipid bilayers (SLBs) are emerging platforms that have broad applications ranging from fundamental research to next-generation biosensors. Central to the success of the platform is proper accommodation of membrane proteins so that their dynamics and functions are preserved. Polymer cushions have been commonly employed to avoid direct contact of the bilayer membrane to the supporting substrate, and thus the mobility of 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 of distinct structures, i.e., GPI-anchored protein, single-pass transmembrane protein CD98 heavy chain, and seven-pass transmembrane protein SSTR3, were fused with green fluorescence proteins (GFPs) and their dynamics were measured by fluorescence single-molecule tracking. An automated data acquisition was implemented to study the effects of PEG polymer length to protein dynamics with large statistics. Our data showed that increasing the PEG polymer length (molecular weight from 1,000 to 5,000) enhanced the mobile fraction of the membrane proteins. Moreover, the diffusion coefficients of transmembrane proteins were raised by increasing the polymer length, whereas the diffusion coefficient of GPI-anchored protein remained almost identical with different polymer lengths. Importantly, the diffusion coefficients of the three membrane proteins became identical (2.5 μm²/s approximately) in the cushioned membrane with the longest polymer length (molecular weight of 5,000), indicating that the SLBs were fully suspended from the substrate by the polymer cushion at the microscopic length scale. Transient confinements were observed from all three proteins, and increasing the polymer length reduced the tendency of transient confinements. The measured dynamics of membrane proteins were found to be nearly unchanged after 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 future developments of membrane-based biosensors and analytical assays.

Nanoparticles have been used extensively in biology-related research and many applications require direct visualization of individual nanoparticles under optical microscopy. For long-term and high-speed measurements, scattering-based microscopy is a unique technique because of the stable and indefinite scattering signal. In scattering-based single-particle measurements, large nanoparticles are usually needed in order to generate sufficient signal for detection. However, larger nanoparticle introduces greater mass loading, experiences stronger steric hindrance, and is 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 high speed. The COBRI microscopy allows us to visualize and track single metallic and dielectric nanoparticles, as small as 10 nm, at 1,000 frames per second. A quantitative relationship between the linear scattering cross section of nanoparticle and its COBRI contrast is reported. Using COBRI microscopy, we further demonstrate tracking of 10 nm gold nanoparticles labeled to lipid molecules in supported bilayer membranes, showing that the small nanoparticle may facilitate single-molecule measurements with reduced perturbation. Furthermore, identical imaging sensitivity of COBRI and interferometric scattering (iSCAT) microscopy, the reflection counterpart of COBRI, is demonstrated under equal illumination intensity. Finally, future improvements in speed and sensitivity of scattering-based interference microscope are discussed.

Many important biological phenomena, ranging from cell signaling to viral infection, are facilitated by transportation of biological substances encapsulated in native nano-sized particles. Thermal fluctuation drives nanoparticles through cellular environments; this movement is facilitated by their small size. To understand how a specific cell function can be achieved through random collisions, it is useful to know the interactions between single particles and the local environment, as determined by measuring cell dynamics at high spatial and temporal resolutions. In this chapter, a simple yet powerful wide-field optical technique, coherent brightfield (COBRI) microscopy, is presented. COBRI microscopy detects linearly scattered light from a nanoparticle through imaging-based interferometry, which enables direct observation of small biological nanoparticles in live cells without labels. Proper image post-processing further improves the detection sensitivity of small particles by removing the scattering background of cell structures. COBRI microscopy can easily operate at a high speed due to its wide-field nature and stable, indefinite scattering signal. Using COBRI, the dynamics of single virus particles and cell vesicles in live cells can be successfully captured at a microsecond temporal resolution and nanometer spatial precision in three dimensions. The ultrahigh spatiotemporal resolution and shot-noise-limited sensitivity of COBRI microscopy provide an opportunity to study the biophysics and biochemistry of live cells at the nanoscale.

Real-time tracking of membrane proteins is essential to gain an in-depth understanding of their dynamics on cell surface. However, con-ventional 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 labelling of azide-modified membrane proteins and azide-modified antibodies of membrane proteins. The high specificity of this labelling 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.

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, as heterogeneous, 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 to 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 which 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 which aim to meet them are detailed.

Label-free microscope imaging techniques allow direct visualization of biological substances in their most native forms. This review article provides an overview of recent advancements of scattering-based, interferometric laser microscopy and their applications to ultrasensitive and ultrahigh-speed biological imaging. In particular, common-path, widefield interferometric laser microscopy, namely interferometric scattering (iSCAT) microscopy and coherent brightfield (COBRI) microscopy, are discussed in details. Using these simple yet powerful optical techniques, single proteins and individual endogenous biological nanoparticles can be imaged and tracked without any label at ultrahigh spatiotemporal resolution. The development of ultrasensitive and ultrahigh-speed scattering-based interferometric microscope imaging enables investigation of biophysical and biochemical processes with minimal perturbation and unprecedented clarity.

Localization of a single nanosized light emitter has substantial applications in bioimaging. The accuracy and precision of localization are limited by the noise and the heterogeneous background superimposed on the signal. While the effects of noise are well recognized, the influence of background is less addressed. Proper background correction not only provides more accurate localization data but also enhances the sensitivity of detection. Here, we demonstrate a new approach to background correction by estimating and removing the heterogeneous but stationary background from a series of images containing a spatially moving signal. Our approach exploits the correlated signal information encoded in the neighboring pixels governed by the point-spread function of the measurement system. This new approach makes it possible to obtain the background even when the total displacement of the signal is subdiffraction limited throughout the observation, the scenario where previous methods become invalid. We characterize our approach systematically with different types of signal motions at various signal-to-noise ratios in numerical simulations. We then verify our method experimentally by recovering the nanoscopic displacements of single gold nanoparticle moving in a specified pattern and a single virus particle randomly diffusing on a cell surface. The source code of our algorithm written in MATLAB is provided together with a sample data set. Our approach has immediate applications in high-precision optical localization measurements.

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 post-processing, 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 timescales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.

Investigation of intracellular transport at the molecular scale requires measurements at high spatial and temporal resolutions. We demonstrate label-free, direct imaging and tracking of native cell vesicles in live cells at 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. Stepwise directional motion of the vesicle on the cytoskeletal track is clearly resolved. We also observe repeated switching of transport direction of the vesicle in a continuous trajectory. Our high-resolution measurement unveils 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.

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 FND with A27(aa 21–84), a recombinant envelope protein of vaccinia virus, for glycosaminoglycans (GAGs)-specific targeting and imaging of living cells. Another recombinant protein A27(aa 33–84) that removes the GAGs-binding sequences was also used for comparison. Three types of A27-coated FNDs were generated, including A27(aa 21–84)-FND, A27(aa 33–84)-FND, and hybrid A27(aa 21–84)/A27(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.

The journey of a single molecule can be followed with nanometer spatial precision and microsecond temporal resolution by using interferometric scattering optical microscopy and an ultrahigh-speed camera.

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.

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.

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.

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.