Label-Free Interference Imaging of Intracellular Trafficking.

ConspectusIntracellular 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.

Integrating magnetic tweezers and single-molecule FRET: A comprehensive approach to studying local and global conformational changes simultaneously.

This chapter presents the integration of magnetic tweezers with single-molecule FRET technology, a significant advancement in the study of nucleic acids and other biological systems. We detail the technical aspects, challenges, and current status of this hybrid technique, which combines the global manipulation and observation capabilities of magnetic tweezers with the local conformational detection of smFRET. This innovative approach enhances our ability to analyze and understand the molecular mechanics of biological systems. The chapter serves as our first formal documentation of this method, offering insights and methodologies developed in our laboratory over the past decade.

Monitoring the synthesis of neutral lipids in lipid droplets of living human cancer cells using two-color infrared photothermal microscopy.

There has been growing interest in the functions of lipid droplets (LDs) due to recent discoveries regarding their diverse roles. These functions encompass lipid metabolism, regulation of lipotoxicity, and signaling pathways that extend beyond their traditional role in energy storage. Consequently, there is a need to examine the molecular dynamics of LDs at the subcellular level. Two-color infrared photothermal microscopy (2C-IPM) has proven to be a valuable tool for elucidating the molecular dynamics occurring in LDs with sub-micrometer spatial resolution and molecular specificity. In this study, we employed the 2C-IPM to investigate the molecular dynamics of LDs in both fixed and living human cancer cells (U2OS cells) using the isotope labeling method. We investigated the synthesis of neutral lipids occurring in individual LDs over time after exposing the cells to excess saturated fatty acids while simultaneously comparing inherent lipid contents in LDs. We anticipate that these research findings will reveal new opportunities for studying lesser-known biological processes within LDs and other subcellular organelles.

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.

Two-color infrared photothermal microscopy.

Infrared photothermal microscopy is an infrared (IR) imaging technique that enables non-invasive, non-destructive, and label-free investigations at the sub-micrometer scale. It has been applied in various research areas targeting pharmaceutical and photovoltaic materials as well as biomolecules in living systems. Despite its potency in observing biomolecules in living organisms, its practical application for cytological research has been restricted by the deficiency of molecular information from the IR photothermal signal, due to the narrow spectral width of a quantum cascade laser that is one of the most preferred IR excitation light sources for current IR photothermal imaging (IPI) techniques. Here, we address this issue by bringing modulation-frequency multiplexing into IR photothermal microscopy for developing a two-color IR photothermal microscopy technique. We demonstrate that the two-color IPI technique can be used to obtain the IR microscopic images of two discrete IR absorption bands and to distinguish two different chemical species in live cells with a sub-micrometer spatial resolution. We anticipate that the more general multi-color IPI technique and its use for metabolic studies of live cells could be realized by extending the present modulation-frequency multiplexing method.

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.

Single-Molecule Methods to Study Z-DNA Mechanics and Dynamics.

Single-molecule methods are powerful in revealing physical and mechanobiological details about biological phenomena. Here, we describe the single-molecule methods applied to study mechanical properties of Z-DNA and dynamics of the B-Z transition.

Two Opposing Effects of Monovalent Cations on the Stability of i-Motif Structure.

At acidic pH, cytosine-rich single-stranded DNA can be folded into a tetraplex structure called i-motif (iM). In recent studies, the effect of monovalent cations on the stability of iM structure has been addressed, but a consensus about the issue has not been reached yet. Thus, we investigated the effects of various factors on the stability of iM structure using fluorescence resonance energy transfer (FRET)-based analysis for three types of iM derived from human telomere sequences. We confirmed that the protonated cytosine-cytosine (C:C+) base pair is destabilized as the concentration of monovalent cations (Li+, Na+, K+) increases and that Li+ has the greatest tendency of destabilization. Intriguingly, monovalent cations would play an ambivalent role in iM formation by making single-stranded DNA flexible and pliant for an iM structure. In particular, we found that Li+ has a notably greater flexibilizing effect than Na+ and K+. All taken together, we conclude that the stability of iM structure is controlled by the subtle balance of the two counteractive effects of monovalent cations: electrostatic screening and disruption of cytosine base pairing.

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.

Z-DNA as a Tool for Nuclease-Free DNA Methyltransferase Assay.

Methylcytosines in mammalian genomes are the main epigenetic molecular codes that switch off the repertoire of genes in cell-type and cell-stage dependent manners. DNA methyltransferases (DMT) are dedicated to managing the status of cytosine methylation. DNA methylation is not only critical in normal development, but it is also implicated in cancers, degeneration, and senescence. Thus, the chemicals to control DMT have been suggested as anticancer drugs by reprogramming the gene expression profile in malignant cells. Here, we report a new optical technique to characterize the activity of DMT and the effect of inhibitors, utilizing the methylation-sensitive B-Z transition of DNA without bisulfite conversion, methylation-sensing proteins, and polymerase chain reaction amplification. With the high sensitivity of single-molecule FRET, this method detects the event of DNA methylation in a single DNA molecule and circumvents the need for amplification steps, permitting direct interpretation. This method also responds to hemi-methylated DNA. Dispensing with methylation-sensitive nucleases, this method preserves the molecular integrity and methylation state of target molecules. Sparing methylation-sensing nucleases and antibodies helps to avoid errors introduced by the antibody's incomplete specificity or variable activity of nucleases. With this new method, we demonstrated the inhibitory effect of several natural bio-active compounds on DMT. All taken together, our method offers quantitative assays for DMT and DMT-related anticancer drugs.

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.

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.

Unveiling the pathway to Z-DNA in the protein-induced B-Z transition.

Left-handed Z-DNA is an extraordinary conformation of DNA, which can form by special sequences under specific biological, chemical or physical conditions. Human ADAR1, prototypic Z-DNA binding protein (ZBP), binds to Z-DNA with high affinity. Utilizing single-molecule FRET assays for Z-DNA forming sequences embedded in a long inactive DNA, we measure thermodynamic populations of ADAR1-bound DNA conformations in both GC and TG repeat sequences. Based on a statistical physics model, we determined quantitatively the affinities of ADAR1 to both Z-form and B-form of these sequences. We also reported what pathways it takes to induce the B-Z transition in those sequences. Due to the high junction energy, an intermediate B* state has to accumulate prior to the B-Z transition. Our study showing the stable B* state supports the active picture for the protein-induced B-Z transition that occurs under a physiological setting.

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.

Competition between B-Z and B-L transitions in a single DNA molecule: Computational studies.

Under negative torsion, DNA adopts left-handed helical forms, such as Z-DNA and L-DNA. Using the random copolymer model developed for a wormlike chain, we represent a single DNA molecule with structural heterogeneity as a helical chain consisting of monomers which can be characterized by different helical senses and pitches. By Monte Carlo simulation, where we take into account bending and twist fluctuations explicitly, we study sequence dependence of B-Z transitions under torsional stress and tension focusing on the interaction with B-L transitions. We consider core sequences, (GC)_{n} repeats or (TG)_{n} repeats, which can interconvert between the right-handed B form and the left-handed Z form, imbedded in a random sequence, which can convert to left-handed L form with different (tension dependent) helical pitch. We show that Z-DNA formation from the (GC)_{n} sequence is always supported by unwinding torsional stress but Z-DNA formation from the (TG)_{n} sequence, which are more costly to convert but numerous, can be strongly influenced by the quenched disorder in the surrounding random sequence.

Annealed random copolymer model of the B-Z transition in DNA: torsional responses.

Both in vivo and in vitro, specific sequences in double-stranded DNA can adopt the left-handed Z-form when underwound. Recently, the B-Z transition of DNA has been studied in detail in magnetic tweezers experiments by several groups. We present a theoretical description of this transition, based on an annealed random copolymer model. The transition of a switchable sequence is discussed as a function of energetic and geometric parameters of the B- and Z-forms, of the applied boundary conditions, and of the characteristics of the B-Z interface. We address a possible torsional softening upon the B-Z transition. The model can be also applied to other biofilaments with annealed torsional/flexural degrees of freedom.

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.