Search results for "single molecule"
showing 10 items of 27 documents
Localization microscopy of DNA in situ using Vybrant(®) DyeCycle™ Violet fluorescent probe: A new approach to study nuclear nanostructure at single m…
2016
Higher order chromatin structure is not only required to compact and spatially arrange long chromatids within a nucleus, but have also important functional roles, including control of gene expression and DNA processing. However, studies of chromatin nanostructures cannot be performed using conventional widefield and confocal microscopy because of the limited optical resolution. Various methods of superresolution microscopy have been described to overcome this difficulty, like structured illumination and single molecule localization microscopy. We report here that the standard DNA dye Vybrant(®) DyeCycle™ Violet can be used to provide single molecule localization microscopy (SMLM) images of …
Quantitative super-resolution localization microscopy of DNA in situ using Vybrant® DyeCycle™ Violet fluorescent probe.
2016
Single Molecule Localization Microscopy (SMLM) is a recently emerged optical imaging method that was shown to achieve a resolution in the order of tens of nanometers in intact cells. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of DNA and proteins, is arranged in the eukaryotic cell nucleus. Such an approach utilizing switching of a fluorescent, DNA-binding dye Vybrant® DyeCycle™ Violet has been previously demonstrated by us (Żurek-Biesiada et al., 2015) [1]. Here we provide quantitative information on the influence of the chemical environment on the behavior of the dye, discuss the variability in the DNA-associated signal density,…
Conformational dynamics of a single protein monitored for 24 hours at video rate
2018
We use plasmon rulers to follow the conformational dynamics of a single protein for up to 24 h at a video rate. The plasmon ruler consists of two gold nanospheres connected by a single protein linker. In our experiment, we follow the dynamics of the molecular chaperone heat shock protein 90 (Hsp90), which is known to show “open” and “closed” conformations. Our measurements confirm the previously known conformational dynamics with transition times in the second to minute time scale and reveals new dynamics on the time scale of minutes to hours. Plasmon rulers thus extend the observation bandwidth 3–4 orders of magnitude with respect to single-molecule fluorescence resonance energy transfer a…
High-throughput sequencing (HTS) for the analysis of viral populations
2020
The development of High-Throughput Sequencing (HTS) technologies is having a major impact on the genomic analysis of viral populations. Current HTS platforms can capture nucleic acid variation across millions of genes for both selected amplicons and full viral genomes. HTS has already facilitated the discovery of new viruses, hinted new taxonomic classifications and provided a deeper and broader understanding of their diversity, population and genetic structure. Hence, HTS has already replaced standard Sanger sequencing in basic and applied research fields, but the next step is its implementation as a routine technology for the analysis of viruses in clinical settings. The most likely appli…
Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers
2018
Influenza hemagglutinin (HA) is the canonical type I viral envelope glycoprotein and provides a template for the membrane-fusion mechanisms of numerous viruses. The current model of HA-mediated membrane fusion describes a static "spring-loaded" fusion domain (HA2) at neutral pH. Acidic pH triggers a singular irreversible conformational rearrangement in HA2 that fuses viral and cellular membranes. Here, using single-molecule Förster resonance energy transfer (smFRET)-imaging, we directly visualized pH-triggered conformational changes of HA trimers on the viral surface. Our analyses reveal reversible exchange between the pre-fusion and two intermediate conformations of HA2. Acidification of p…
Super-resolution binding activated localization microscopy through reversible change of DNA conformation
2018
ABSTRACT Methods of super-resolving light microscopy (SRM) have found an exponentially growing range of applications in cell biology, including nuclear structure analyses. Recent developments have proven that Single Molecule Localization Microscopy (SMLM), a type of SRM, is particularly useful for enhanced spatial analysis of the cell nucleus due to its highest resolving capability combined with very specific fluorescent labeling. In this commentary we offer a brief review of the latest methodological development in the field of SMLM of chromatin designated DNA Structure Fluctuation Assisted Binding Activated Localization Microscopy (abbreviated as fBALM) as well as its potential future app…
FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.
2021
International audience; 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 d…
Phase Separation Comes of Age: From Phenomenology to Single Molecules
2019
Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA processing enzymes, termed processing bodies (PBs). Here, we track the dynamic localization of individual miRNAs, mRNAs and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and 3` versus 5` placement of miRNA target sites significantly impact PB-localization dynamics of mRNAs. Using comp…
Anilato-based multifunctional molecular materials
2018
En esta Tesis presentamos varias series de compuestos moleculares basados en el ligando anilato (anilato = C6O4X22- = 2,5-disustituida-3,6-dihidroxi-1,4-benzoquinona) y diferentes átomos metálicos incluyendo metales de los bloques s, p, d y f. En el capítulo uno presentamos la síntesis y caracterización de nuevas sales precursoras, basadas en el ligando anilato, con fórmula general A3[M(C6O4X2)3]; (MIII = Fe, Cr y Ga; X = Cl, Br and NO2). Hemos usado diferentes cationes A+ (A = K+, [PPh3Et]+, [PPh3Pr]+, [PBu4]+, [NBu4]+, [NHep4]+, [PPh4]+ y K+, compuestos 1-20) para estudiar el papel que juega la forma y el tamaño del catión en la estructura y simetría de las correspondientes sales. Este es…
Investigation of molecular dimers by ensemble and single molecule spectroscopy
2002
International audience; We have investigated molecular dimers with different electronic coupling strengths by bulk and single molecule spectroscopy. In one of the dimers the two monomers (perylene-monoimide) are directly connected via a single bond while in the other one they are separated by the benzil motif. The close proximity of the monomers in the first case gives rise to excitonic band splitting which is clearly observable in the bulk absorption spectra. For the benzil structure the electronic interactions are governed by Förster-type energy hopping between the monomers. Fluorescence intensity trajectories at the single molecule level show one-step and two-step bleaching behaviour whi…