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SMoLR: visualization and analysis of single-molecule localization microscopy data in R
摘要： Background: Single-molecule localization microscopy is a super-resolution microscopy technique that allows for nanoscale determination of the localization and organization of proteins in biological samples. For biological interpretation of the data it is essential to extract quantitative information from the super-resolution data sets. Due to the complexity and size of these data sets flexible and user-friendly software is required. Results: We developed SMoLR (Single Molecule Localization in R): a flexible framework that enables exploration and analysis of single-molecule localization data within the R programming environment. SMoLR is a package aimed at extracting, visualizing and analyzing quantitative information from localization data obtained by single-molecule microscopy. SMoLR is a platform not only to visualize nanoscale subcellular structures but additionally provides means to obtain statistical information about the distribution and localization of molecules within them. This can be done for individual images or SMoLR can be used to analyze a large set of super-resolution images at once. Additionally, we describe a method using SMoLR for image feature-based particle averaging, resulting in identification of common features among nanoscale structures. Conclusions: Embedded in the extensive R programming environment, SMoLR allows scientists to study the nanoscale organization of biomolecules in cells by extracting and visualizing quantitative information and hence provides insight in a wide-variety of different biological processes at the single-molecule level.
关键词： Image analysis,Image quantification,Super-resolution,Microscopy,R,Single-molecule localization
更新于2025-09-23 15:22:29
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Bio-Molecular Applications of Recent Developments in Optical Tweezers
摘要： In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT’s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.
关键词： plasmonic optical tweezers,femtosecond optical tweezers,photonic crystal optical tweezers,fluorescence,single molecule and cell studies
更新于2025-09-23 15:22:29
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Photothermally Assisted Thinning of Silicon Nitride Membranes for Ultrathin Asymmetric Nanopores
摘要： Sculpting solid-state materials at the nanoscale is an important step in manufacturing of numerous types of sensor devices, in particular solid-state nanopore sensors. Here we present mechanistic insight into laser-induced thinning of low-stress silicon nitride (SiNx) membranes and films. In a recent study, we observed that focusing a visible wavelength laser beam on a SiNx membrane results in efficient localized heating, and used this effect to control temperature at a solid-state nanopore sensor. A side-effect of the observed heating was that the pores expand/degrade under prolonged high-power illumination, prompting us to study the mechanism of this etching process. We find that SiNx can be etched under exposure to light of ~107 W/cm2 average intensity, with etch rates that are influenced by the supporting electrolyte. Combining this controlled etching with dielectric breakdown, an electrokinetic process for making pores, nanopores of arbitrary dimensions as small as 1-2 nm in diameter and thickness can easily be fabricated. Evidence gathered from biomolecule-pore interactions suggests that the pore geometries obtained using this method are more funnel-like, rather than hourglass-shaped. Refined control over pore dimensions can expand the range of applications of solid-state nanopores, for example, biopolymer sequencing and detection of specific biomarkers.
关键词： photothermal heating,single-molecule,dielectric breakdown,Nanopores,nanofabrication
更新于2025-09-23 15:21:21
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Single Nanocrystal Spectroscopy of Shortwave Infrared Emitters
摘要： Short-wave infrared (SWIR) emitters are at the center of ground-breaking applications in biomedical imaging, next-generation optoelectronic devices, and optical communications. Colloidal nanocrystals based on indium arsenide are some of the most promising SWIR emitters to date. However, the lack of single-particle spectroscopic methods accessible in the SWIR has prevented advances in both nanocrystal synthesis and fundamental characterization of emitters. Here, we demonstrate an implementation of a solution photon correlation Fourier spectroscopy (s-PCFS) experiment utilizing the SWIR sensitivity and time resolution of superconducting nanowire single-photon detectors to extract indium arsenide/cadmium selenide (InAs/CdSe) core/shell single-particle emission linewidths from colloidal nanocrystals emissive from 1.2 to 1.6 μm. We show that the average single InAs/CdSe nanocrystal ?uorescence linewidth is, remarkably, as narrow as 52 meV, similar to what has been observed in some of the most narrowband nanostructured emitters in the visible region. Additionally, the single nanocrystal ?uorescence linewidth increases with increasing shell thickness, suggesting exciton?phonon coupling as the dominant emission line-broadening mechanism in this system. The development of the SWIR s-PCFS technique has enabled measurements of spectral linewidths of colloidal SWIR-emissive NCs in solution and provides a platform to study the single NC spectral characteristics of SWIR emitters.
关键词： nanocrystals,single-molecule spectroscopy,core/shell,indium arsenide,short-wave infrared
更新于2025-09-23 15:21:21
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Single-molecule imaging of DNA gyrase activity in living <i>Escherichia coli</i>
摘要： Bacterial DNA gyrase introduces negative supercoils into chromosomal DNA and relaxes positive supercoils introduced by replication and transiently by transcription. Removal of these positive supercoils is essential for replication fork progression and for the overall unlinking of the two duplex DNA strands, as well as for ongoing transcription. To address how gyrase copes with these topological challenges, we used high-speed single-molecule fluorescence imaging in live Escherichia coli cells. We demonstrate that at least 300 gyrase molecules are stably bound to the chromosome at any time, with ～12 enzymes enriched near each replication fork. Trapping of reaction intermediates with ciprofloxacin revealed complexes undergoing catalysis. Dwell times of ～2 s were observed for the dispersed gyrase molecules, which we propose maintain steady-state levels of negative supercoiling of the chromosome. In contrast, the dwell time of replisome-proximal molecules was ～8 s, consistent with these catalyzing processive positive supercoil relaxation in front of the progressing replisome.
关键词： Escherichia coli,transcription,DNA gyrase,replication,supercoiling,single-molecule imaging
更新于2025-09-23 15:21:21
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State transition identification in multivariate time series (STIMTS) applied to rotational jump trajectories from single molecules
摘要： Time resolved data from single molecule experiments often suffer from contamination with noise due to a low signal level. Identifying a proper model to describe the data thus requires an approach with sufficient model parameters without misinterpreting the noise as relevant data. Here, we report on a generalized data evaluation process to extract states with piecewise constant signal level from simultaneously recorded multivariate data, typical for multichannel single molecule experiments. The method employs the minimum description length principle to avoid overfitting the data by using an objective function, which is based on a tradeoff between fitting accuracy and model complexity. We validate our method with synthetic data from Monte Carlo simulations modeling fluorescence resonance energy transfer and rotational jumps, respectively. The method is applied to quantify rotational jump dynamics of single terrylene diimide (TDI) molecules deposited on a solid substrate. Depending on the substitution pattern of the TDI molecules and the chosen substrate materials, we find significant differences in time scale and geometry of molecular reorientation. From an additional application of our state transition identification in multivariate time series approach, a significant correlation between shifts of emission spectra and the occurrence of rotational jumps was found.
关键词： minimum description length,rotational jump dynamics,state transition identification,multivariate time series,single molecule
更新于2025-09-23 15:21:21
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Build Your Own Microscope: Step-By-Step Guide for Building a Prism-Based TIRF Microscope
摘要： Prism-based total internal re?ection ?uorescence (pTIRF) microscopy is one of the most widely used techniques for the single molecule analysis of a vast range of samples including biomolecules, nanostructures, and cells, to name a few. It allows for excitation of surface bound molecules/particles/quantum dots via evanescent ?eld of a con?ned region of space, which is bene?cial not only for single molecule detection but also for analysis of single molecule dynamics and for acquiring kinetics data. However, there is neither a commercial microscope available for purchase nor a detailed guide dedicated for building this microscope. Thus far, pTIRF microscopes are custom-built with the use of a commercially available inverted microscope, which requires high level of expertise in selecting and handling sophisticated instrument-parts. To directly address this technology gap, here we describe a step-by-step guide on how to build and characterize a pTIRF microscope for in vitro single-molecule imaging, nanostructure analysis and other life sciences research.
关键词： single-molecule detection,single-molecule FRET,?uorescence microscope,pTIRF
更新于2025-09-23 15:21:01
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Optoplasmonic characterisation of reversible disulfide interactions at single thiol sites in the attomolar regime
摘要： Probing individual chemical reactions is key to mapping reaction pathways. Trace analysis of sub-kDa reactants and products is obfuscated by labels, however, as reaction kinetics are inevitably perturbed. The thiol-disulfide exchange reaction is of specific interest as it has many applications in nanotechnology and in nature. Redox cycling of single thiols and disulfides has been unresolvable due to a number of technological limitations, such as an inability to discriminate the leaving group. Here, we demonstrate detection of single-molecule thiol-disulfide exchange using a label-free optoplasmonic sensor. We quantify repeated reactions between sub-kDa thiolated species in real time and at concentrations down to 100’s of attomolar. A unique sensing modality is featured in our measurements, enabling the observation of single disulfide reaction kinetics and pathways on a plasmonic nanoparticle surface. Our technique paves the way towards characterising molecules in terms of their charge, oxidation state, and chirality via optoplasmonics.
关键词： Optoplasmonic,Label-free sensing,Attomolar regime,Thiol-disulfide exchange,Single-molecule detection
更新于2025-09-23 15:21:01
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Quantifying the Oligomeric States of Membrane Proteins in Cells through Super-Resolution Localizations
摘要： Transitions between different oligomeric states of membrane proteins are essential for proper cellular functions. However, the quantification of their oligomeric states in cells is technically challenging. Here we developed a new method to quantify oligomeric state(s) of highly-expressed membrane proteins using the probability density function of molecule density (PDFMD) calculated from super-resolution localizations. We provided the theoretical model of PDFMD, discussed the effects of protein concentration, cell geometry and photophysics of fluorescent proteins on PDFMD, and provided experimental criteria for proper quantification of oligomeric states. This method was further validated using simulated single-molecule fluorescent movies, and applied to two membrane proteins, UhpT and SbmA in E. coli. The study shows that PDFMD is useful in quantifying oligomeric states of membrane proteins in cells that can help in understanding cellular tasks. Potential applications to proteins with higher oligomeric states under high concentration and limitation of our methodology were also discussed.
关键词： PALM,Photophysics,Protein concentration,Cell geometry,Single molecule
更新于2025-09-23 15:21:01
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Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells
摘要： Monitoring virus assembly at the nanoscale in host cells remains a major challenge. Human immunodeficiency virus type 1 (HIV-1) components are addressed to the plasma membrane where they assemble to form spherical particles of 100 nm in diameter. Interestingly, HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble the immature virus. Here, we monitored VLP formation at the plasma membrane of host CD4+ T cells using a newly developed workflow allowing the analysis of long duration recordings of single-molecule Gag protein localisation and movement. Comparison of Gag assembling platforms in CD4+ T cells expressing wild type or assembly-defective Gag mutant proteins showed that VLP formation lasts roughly 15 minutes with an assembly time of 5 minutes. Trapping energy maps, built from membrane associated Gag protein movements, showed that one third of the assembling energy is due to direct Gag capsid-capsid interaction while the remaining two thirds require the nucleocapsid-RNA interactions. Finally, we show that the viral RNA genome does not increase the attraction of Gag at the membrane towards the assembling site but rather acts as a spatiotemporal coordinator of the membrane assembly process.
关键词： HIV-1,Gag protein,virus assembly,CD4+ T cells,single-molecule localisation microscopy
更新于2025-09-23 15:21:01