Label-Free Interference Imaging of Intracellular Trafficking.

Intracellular cargo trafficking is a highly regulated process responsible for transporting vital cellular components to their designated destinations. This intricate journey has been a central focus of cellular biology for many years. Early investigations leaned heavily on biochemical and genetic approaches, offering valuable insight into molecular mechanisms of cellular trafficking. However, while informative, these methods lack the capacity to capture the dynamic nature of intracellular trafficking. The advent of fluorescent protein tagging techniques transformed our ability to monitor the complete lifecycle of intracellular cargos, advancing our understanding. Yet, a central question remains: How do these cargos manage to navigate through traffic challenges, such as congestion, within the crowded cellular environment? Fluorescence-based imaging, though valuable, has inherent limitations when it comes to addressing the aforementioned question. It is prone to photobleaching, making long-term live-cell imaging challenging. Furthermore, they render unlabeled cellular constituents invisible, thereby missing critical environmental information. Notably, the unlabeled majority likely exerts a significant influence on the observed behavior of labeled molecules. These considerations underscore the necessity of developing complementary label-free imaging methods to overcome the limitations of fluorescence imaging or to integrate them synergistically.In this Account, we outline how label-free interference-based imaging has the potential to revolutionize the study of intracellular traffic by offering unprecedented levels of detail. We begin with a brief introduction to our previous findings in live-cell research enabled by interferometric scattering (iSCAT) microscopy, showcasing its aptitude and adeptness in elucidating intricate nanoscale intracellular structures. As we delved deeper into our exploration, we succeeded in the label-free visualization of the entire lifespan of nanoscale protein complexes known as nascent adhesions (NAs) and the dynamic events associated with adhesions within living cells. Our continuous efforts have led to the development of Dynamic Scattering-particle Localization Interference Microscopy (DySLIM), a generalized concept of cargo-localization iSCAT (CL-iSCAT). This label-free, high-speed imaging method, armed with iSCAT detection sensitivity, empowers us to capture quantitative and biophysical insights into cargo transport, providing a realistic view of the intricate nanoscale logistics occurring within living cells. Our in vivo studies demonstrate that intracellular cargos regularly contend with substantial traffic within the crowded cellular environment. Simultaneously, they employ inherent strategies for efficient cargo transport, such as collective migration and hitchhiking, to enhance overall transport rates─intriguingly paralleling the principle and practice of urban traffic management. We also highlight the synergistic benefits of combining DySLIM with chemical-selective fluorescent methods. This Account concludes with a "Conclusions and Outlook" section, outlining promising directions for future research and developments, with a particular emphasis on the functional application of iSCAT live-cell imaging. We aim to inspire further investigation into the efficient transport strategies employed by cells to surmount transportation challenges, shedding light on their significance in cellular phenomena.

Long-term cargo tracking reveals intricate trafficking through active cytoskeletal networks in the crowded cellular environment.

A eukaryotic cell is a microscopic world within which efficient material transport is essential. Yet, how a cell manages to deliver cellular cargos efficiently in a crowded environment remains poorly understood. Here, we used interferometric scattering microscopy to track unlabeled cargos in directional motion in a massively parallel fashion. Our label-free, cargo-tracing method revealed not only the dynamics of cargo transportation but also the fine architecture of the actively used cytoskeletal highways and the long-term evolution of the associated traffic at sub-diffraction resolution. Cargos frequently run into a blocked road or experience a traffic jam. Still, they have effective strategies to circumvent those problems: opting for an alternative mode of transport and moving together in tandem or migrating collectively. All taken together, a cell is an incredibly complex and busy space where the principle and practice of transportation intriguingly parallel those of our macroscopic world.

Axial profiling of interferometric scattering enables an accurate determination of nanoparticle size.

Interferometric scattering (iSCAT) microscopy has undergone significant development in recent years. It is a promising technique for imaging and tracking nanoscopic label-free objects with nanometer localization precision. The current iSCAT-based photometry technique allows quantitative estimation for the size of a nanoparticle by measuring iSCAT contrast and has been successfully applied to nano-objects smaller than the Rayleigh scattering limit. Here we provide an alternative method that overcomes such size limitations. We take into account the axial variation of iSCAT contrast and utilize a vectorial point spread function model to uncover the position of a scattering dipole and, consequently, the size of the scatterer, which is not limited to the Rayleigh limit. We found that our technique accurately measures the size of spherical dielectric nanoparticles in a purely optical and non-contact way. We also tested fluorescent nanodiamonds (fND) and obtained a reasonable estimate for the size of fND particles. Together with fluorescence measurement from fND, we observed a correlation between the fluorescent signal and the size of fND. Our results showed that the axial pattern of iSCAT contrast provides sufficient information for the size of spherical particles. Our method enables us to measure the size of nanoparticles from tens of nanometers and beyond the Rayleigh limit with nanometer precision, making a versatile all-optical nanometric technique.

Cisplatin fastens chromatin irreversibly even at a high chloride concentration.

Cisplatin is one of the most potent anti-cancer drugs developed so far. Recent studies highlighted several intriguing roles of histones in cisplatin's anti-cancer effect. Thus, the effect of nucleosome formation should be considered to give a better account of the anti-cancer effect of cisplatin. Here we investigated this important issue via single-molecule measurements. Surprisingly, the reduced activity of cisplatin under [NaCl] = 180 mM, corresponding to the total concentration of cellular ionic species, is still sufficient to impair the integrity of a nucleosome by retaining its condensed structure firmly, even against severe mechanical and chemical disturbances. Our finding suggests that such cisplatin-induced fastening of chromatin can inhibit nucleosome remodelling required for normal biological functions. The in vitro chromatin transcription assay indeed revealed that the transcription activity was effectively suppressed in the presence of cisplatin. Our direct physical measurements on cisplatin-nucleosome adducts suggest that the formation of such adducts be the key to the anti-cancer effect by cisplatin.

Sequence-dependent cost for Z-form shapes the torsion-driven B-Z transition via close interplay of Z-DNA and DNA bubble.

Despite recent genome-wide investigations of functional DNA elements, the mechanistic details about their actions remain elusive. One intriguing possibility is that DNA sequences with special patterns play biological roles, adopting non-B-DNA conformations. Here we investigated dynamics of thymine-guanine (TG) repeats, microsatellite sequences and recurrently found in promoters, as well as cytosine-guanine (CG) repeats, best-known Z-DNA forming sequence, in the aspect of Z-DNA formation. We measured the energy barriers of the B-Z transition with those repeats and discovered the sequence-dependent penalty for Z-DNA generates distinctive thermodynamic and kinetic features in the torque-induced transition. Due to the higher torsional stress required for Z-form in TG repeats, a bubble could be induced more easily, suppressing Z-DNA induction, but facilitate the B-Z interconversion kinetically at the transition midpoint. Thus, the Z-form by TG repeats has advantages as a torsion buffer and bubble selector while the Z-form by CG repeats likely behaves as torsion absorber. Our statistical physics model supports quantitatively the populations of Z-DNA and reveals the pivotal roles of bubbles in state dynamics. All taken together, a quantitative picture for the transition was deduced within the close interplay among bubbles, plectonemes and Z-DNA.

Fluorescence-Combined Interferometric Scattering Imaging Reveals Nanoscale Dynamic Events of Single Nascent Adhesions in Living Cells.

Focal adhesions (FAs) are dynamic protein nanostructures that form mechanical links between cytoskeletal actin fibers and the extracellular matrix. Here, we demonstrate that interferometric scattering (iSCAT) microscopy, a high-speed and time-unlimited imaging technique, can uncover the real-time dynamics of nanoscopic nascent adhesions (NAs). The high sensitivity and stability of the iSCAT signal enabled us to trace the whole life span of each NA spontaneously nucleated under a lamellipodium. Such high-throughput and long-term image data provide a unique opportunity for statistical analysis of adhesion dynamics. Moreover, we directly revealed that FAs play critical roles in both the extrusion of filopodia as nucleation sites on the leading edge and the one-dimensional transport of cargos along cytoskeletal fibers as fiber docking sites. These experimental results show that iSCAT is a sensitive tool for tracking real-time dynamics of nanoscopic objects involved in endogenous and exogenous biological processes in living cells.

Three-dimensional interferometric scattering microscopy via remote focusing technique.

Interferometric scattering (iSCAT) microscopy enables us to track nm-sized objects with high spatial and temporal resolutions and permits label-free imaging of biomolecules. Its superb sensitivity, however, comes at a cost by several downsides, such as slow three-dimensional imaging and limited vertical tracking. Here, we propose a new method, Remote Focusing-iSCAT (RF-iSCAT) microscopy, to visualize a volume specimen by imaging sections at different depths without translation of either the objective lens or sample stage. We demonstrate the principle of RF-iSCAT by determining the z-position of submicrometer beads by translating the reference mirror instead. RF-iSCAT features an unprecedentedly long range of vertical tracking and permits fast but vibration-free vertical scanning. We anticipate that RF-iSCAT would enhance the utility of iSCAT for dynamics study.

Label-free and live cell imaging by interferometric scattering microscopy.

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli, and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g., focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

Decoding Single Molecule Time Traces with Dynamic Disorder.

Single molecule time trajectories of biomolecules provide glimpses into complex folding landscapes that are difficult to visualize using conventional ensemble measurements. Recent experiments and theoretical analyses have highlighted dynamic disorder in certain classes of biomolecules, whose dynamic pattern of conformational transitions is affected by slower transition dynamics of internal state hidden in a low dimensional projection. A systematic means to analyze such data is, however, currently not well developed. Here we report a new algorithm-Variational Bayes-double chain Markov model (VB-DCMM)-to analyze single molecule time trajectories that display dynamic disorder. The proposed analysis employing VB-DCMM allows us to detect the presence of dynamic disorder, if any, in each trajectory, identify the number of internal states, and estimate transition rates between the internal states as well as the rates of conformational transition within each internal state. Applying VB-DCMM algorithm to single molecule FRET data of H-DNA in 100 mM-Na+ solution, followed by data clustering, we show that at least 6 kinetic paths linking 4 distinct internal states are required to correctly interpret the duplex-triplex transitions of H-DNA.

Deciphering Kinetic Information from Single-Molecule FRET Data That Show Slow Transitions.

Single-molecule FRET is one of the most powerful and widely used biophysical techniques in biological sciences. It, however, often suffers from limitations such as weak signal and limited measurement time intrinsic to single-molecule fluorescence measurements. Despite several ameliorative measures taken to increase measurement time, it is nearly impossible to acquire meaningful kinetic information on a molecule if conformational transitions of the molecule are ultraslow such that transition times (⟨τ⟩orig) are comparable to or longer than measurement times (δt) limited by the finite lifetime of fluorescent dye. Here, to extract a reliable and accurate mean transition time from a series of short time traces with ultraslow kinetics, we suggest a scheme called sHaRPer (serialized Handshaking Repeated Permutation with end removal) that concatenates multiple time traces. Because data acquisition frequency f and measurement time (δt) affect the estimation of mean transition time (⟨τ⟩), we provide mathematical criteria that f, δt, and ⟨τ⟩ should satisfy to make ⟨τ⟩ close enough to ⟨τ⟩orig. Although application of the sHaRPer method has a potential risk of distorting the time constants of individual kinetic phases if the data are described with kinetic partitioning, we also provide criteria to avoid such distortion. Our sHaRPer method is a useful way to handle single-molecule data with slow transition kinetics. This study provides a practical guide to use sHaRPer.

Destabilization of i-motif by submolar concentrations of a monovalent cation.

Counterions are crucial for self-assembly of nucleic acids. Submolar monovalent cations are generally deemed to stabilize various types of base pairs in nucleic acids such as Watson-Crick and Hoogsteen base pairs via screening of electrostatic repulsion. Besides monovalent cations, acidic pH is required for i-motif formation because protons facilitate pairing between cytosines. Here we report that Li(+) ions destabilize i-motif, whereas other monovalent cations, Na(+) and K(+), have the usual stabilizing effect. The thermodynamics data alone, however, cannot reveal which mechanism, enhanced unfolding or suppressed folding or both, is responsible for the Li(+)-induced destabilization. To gain further insight, we examined the kinetics of i-motif. To deal with slow kinetics of i-motif, we developed a method dubbed HaRP to construct a long FRET time trace to observe a sufficient number of transitions. Our kinetics analysis shows clearly that Li(+) ions promote unfolding of i-motif but do not hinder its folding, lending strong support for our hypothesis on the origin of this unusual effect of Li(+). Although the subangstrom size of Li(+) ions allows them to infiltrate the space between cytosines in competition with protons, they cannot adequately fulfill the role of protons in mediating the hydrogen bonding of cytosine pairs.

DNA triplex folding: moderate versus high salt conditions.

Some specific sequences in duplex DNA can give rise to local formation of a triple helical DNA called triplex together with a separate strand. Recent single-molecule FRET experiments, performed on DNA strands designed to fold into a triplex, allow us to measure the folding and unfolding time distributions under neutral p H conditions. The average times of both processes are of the order of 1 s. The folding time is moderately sensitive to salt concentration. The average unfolding time is found to be nearly constant. Interestingly, the distributions of the unfolding time revealed heterogeneous kinetics at moderate salt concentration (∼ 10 mM), but not at high salt (∼ 100 mM). We relate this salt dependence to different folding paths and folded states, which are governed by the (salt-dependent) stiffness of the third single-stranded donor sequence. Finally we comment on the formation of intramolecular triplex named H-DNA in a torsionally constrained duplex under physiological salt conditions, which mimics the in vivo situation of triplex folding.

Kinetics of the triplex-duplex transition in DNA.

The kinetics of triplex folding/unfolding is investigated by the single-molecule fluorescence resonance energy transfer (FRET) technique. In neutral pH conditions, the average dwell times in both high-FRET (folded) and low-FRET (unfolded) states are comparable, meaning that the triplex is marginally stable. The dwell-time distributions are qualitatively different: while the dwell-time distribution of the high-FRET state should be fit with at least a double-exponential function, the dwell-time distribution of the low-FRET state can be fit with a single-exponential function. We propose a model where the folding can be trapped in metastable states, which is consistent with the FRET data. Our model also accounts for the fact that the relevant timescales of triplex folding/unfolding are macroscopic.