1,720,996 research outputs found
Upgrading interferometric scattering microscopy with ensemble statistical analysis
No full textInterferometric scattering (iSCAT) microscopy, label-free high-speed (up to ~1000 frames per second) imaging and tracking technique, has proven to be a versatile tool by measuring the mass and 3D position of nanoparticles and biomolecules as well as visualizing real-time dynamics of nanoscale events in complex cellular environments. However, the quantification of iSCAT signals has not been straightforwardly defined in practical terms. We delve into several issues associated with signal processing in iSCAT: error-prone post-processing routine and lack of statistical reliability in the convention of iSCAT contrast. After providing a brief account of concepts and principles of correlation spectroscopy, we here discuss an alternative ensemble (higher number density of scatterers) statistical analysis that can be used to extract the dynamic information of scattering particles from fluctuating iSCAT signals. Finally, our perspective on the correlation approach toward time-correlated iSCAT technique will be presented.11Nsciescopuskc
Two Opposing Effects of Monovalent Cations on the Stability of i-Motif Structure
No full textAt 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. (GRAPHICS)11Nsciescopu
Competition between B-Z and B-L transitions in a single DNA molecule: Computational studies
Full text linkUnder 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. © 2016 American Physical Society1221sciescopu
Two-color infrared photothermal microscopy
No full textInfrared 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. © 2023 The Royal Society of Chemistry.11Nsciescopu
Monitoring the synthesis of neutral lipids in lipid droplets of living human cancer cells using two-color infrared photothermal microscopy
No full textThere 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. © 2024 The Royal Society of Chemistry.11Ysciescopu
Long-term cargo tracking reveals intricate trafficking through active cytoskeletal networks in the crowded cellular environment
No full textA 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. © 2023, The Author(s).11Nsciescopu
Axial profiling of interferometric scattering enables an accurate determination of nanoparticle size
No full textInterferometric 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. © 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.11Nsciescopu
Sequence-dependent cost for Z-form shapes the torsion-driven B-Z transition via close interplay of Z-DNA and DNA bubble
No full textDespite 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.11Nsciescopu
Three-dimensional interferometric scattering microscopy via remote focusing technique
No full text© 2020 Optical Society of America.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 study11sci
Fluorescence-Combined Interferometric Scattering Imaging Reveals Nanoscale Dynamic Events of Single Nascent Adhesions in Living Cells
No full textFocal 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.11Nsciescopu
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