Chromosome architecture needs to be investigated in relation with the chemical function of DNA. The kinetics of gene expression, DNA replication, and repair are driven by the mechanisms by which a functional nuclear protein finds its substrate in the nucleus. Single-particle tracking (SPT) is a method to quantify fluorescent molecules dynamics from the tracks of the single molecules recorded by high-resolution microscopes. SPT offers direct observation of the movement and single-molecule resolution. Usually SPT is performed on membranes because of higher contrast. Here, we introduce a novel method to record the trajectories of weakly fluorescent molecules in the nucleus of living cells. I-SPT uses some specific detection and analysis tools to enable the computation of reliable statistics on nuclear particle movement.
- MeSH
- Cell Nucleus metabolism MeSH
- Cell Line MeSH
- Humans MeSH
- Chromosomes, Human genetics ultrastructure MeSH
- Image Processing, Computer-Assisted MeSH
- DNA Replication MeSH
- Single Molecule Imaging methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The characterisation of inorganic nanoparticles (NPs) by single particle inductively coupled plasma mass spectroscopy is possible only if the spectrometer is capable of measurement with high time-signal resolution. The latest generation of spectrometers allow for measurements with dwell times (dt) shorter than the 100 μs gold standard, i.e. as low as 10 μs. The statistical behaviours of signals obtained with dt values of 10, 20, 50, and 100 μs were tested for 40, 60, and 100 nm silver NPs. Very low measured signals (units of counts) led to the occurrence of zero signal values inside the peaks corresponding to individual NPs. The probability of the occurrence of a zero signal inside the peak increased with decreasing dt and decreasing NP size. The standard approach to the bordering of the beginning and end of the peak by one zero signal point failed here and lead to the false detection of a larger number of smaller peaks. For example, in the case of 40 nm NPs a quadruple number of peaks were detected for a dt value of 10 μs compared to the 100 μs dt value; the mean peak width at 10 μs dt was approximately 220 μs, while at 100 μs dt it was 550 μs. The results tended to be less distorted when dt was longer and the NP size was larger. Low dt values also led to a distortion of the peak area distribution. For 40 nm NPs and 10 μs, the most frequent peak area and the width of the peak area distribution were not evaluated due to a non-Gaussian course; 20 μs dt caused (compared to 100 μs) a decrease in the most frequent peak area by approximately 35% (33 counts for 100 μs dt vs. 22 counts for 20 μs dt) and an increase in the width of the peak area distribution by 70% (10 counts for 100 μs dt vs. 17 counts for 20 μs dt). Therefore, new approaches to bordering peaks were tested, which consisted of searching for an uninterrupted zero signal point sequence with a total length of 50 μs or 100 μs. Only the criterion of a 100 μs delay between the two adjacent peaks resulted in values of the number of detected peaks, the most frequent peak areas, and the width of peak area distribution virtually independent of dt.
- Publication type
- Journal Article MeSH
This work demonstrates the effect of NaCl and carbon-related interferences on the analysis of arsenic and silver nanoparticles (NPs) by single-particle inductively coupled plasma mass spectrometry. Spectral interference caused by ArCl+ ions disturbing arsenic NPs analysis was eliminated using ammonia as reaction gas in a dynamic reaction cell of inductively coupled plasma mass spectrometer. In comparison to aqueous dispersions, non-spectral interferences caused by sodium lead to under-evaluation of arsenic and silver NPs diameter by about 7% and 15% at NaCl concentration of 450 mg L-1 and about 28% and 41% at NaCl concentration of 4500 mg L-1, respectively. As a consequence of lower transport efficiency, sodium non-spectral interferences also lead to about a 9% lower number of detected NPs for dispersions of both arsenic and silver NPs in 4500 mg L-1 NaCl. On the contrary, measurement of NPs in matrices containing methanol gives results where Ag and As NPs diameter is over-evaluated by about 3% and 15% at a methanol content of 1% (v/v) and about 6% and 20% at a methanol content of 2% (v/v), respectively, in comparison to aqueous dispersions. In addition, the organic carbon species behave as surfactants and increase the transport efficiency; this leads to an increase in the determined number concentration of NPs. In comparison to aqueous dispersions, this is over-evaluated by about 17% for Ag NPs and about 10% for As NPs at a methanol content of 5% (v/v).
- Publication type
- Journal Article MeSH
Ultrafast measurement using dwell times below 100 μs down to 10 μs is a relatively new feature of single particle analysis using ICP-MS. In this study, we tested the effect of shorter dwell times on the particle size detection limit (Dd.l.). Decreasing dwell times below 100 μs did not lead to a statistically significant decrease in Dd.l. The particle size detection limit (quadrupole ICP-MS) of silver nanoparticles (NP) was estimated to be approx. 10-11 nm. Ag NPs close to Dd.l. were analysed. The 14-nm NPs showed low detection yield; only 5% of number of NPs estimated from transport efficiency was detected. The 20-nm NPs showed 44% detection yield; only in the case of 30-nm NPs did the number of detected NPs correspond to transport efficiency. It is obvious that near Dd.l. estimates of NP concentrations should be made with great caution.
- Publication type
- Journal Article MeSH
Advances in fluorescent probe design and synthesis have allowed the uniform in situ labeling of individual RNA molecules. In a technique referred to as single molecule RNA FISH (smRNA FISH), the labeled RNA molecules can be imaged as diffraction-limited spots and counted using image analysis algorithms. Single RNA counting has provided valuable insights into the process of gene regulation. This microscopy-based method has often revealed a high cell-to-cell variability in expression levels, which has in turn led to a growing interest in investigating the biological significance of gene expression noise. Here we describe the application of the smRNA FISH technique to samples of Caenorhabditis elegans, a well-characterized model organism.
- MeSH
- Algorithms MeSH
- Caenorhabditis elegans genetics growth & development metabolism MeSH
- Embryo, Nonmammalian metabolism ultrastructure MeSH
- Tissue Fixation methods MeSH
- Fluorescent Dyes chemistry MeSH
- Transcription, Genetic MeSH
- In Situ Hybridization, Fluorescence methods MeSH
- RNA, Messenger chemistry genetics metabolism MeSH
- Signal-To-Noise Ratio MeSH
- Gene Expression Regulation, Developmental MeSH
- Single Molecule Imaging methods MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cryo-electron microscopy has established as a mature structural biology technique to elucidate the three-dimensional structure of biological macromolecules. The Coulomb potential of the sample is imaged by an electron beam, and fast semi-conductor detectors produce movies of the sample under study. These movies have to be further processed by a whole pipeline of image-processing algorithms that produce the final structure of the macromolecule. In this chapter, we illustrate this whole processing pipeline putting in value the strength of "meta algorithms," which are the combination of several algorithms, each one with different mathematical rationale, in order to distinguish correctly from incorrectly estimated parameters. We show how this strategy leads to superior performance of the whole pipeline as well as more confident assessments about the reconstructed structures. The "meta algorithms" strategy is common to many fields and, in particular, it has provided excellent results in bioinformatics. We illustrate this combination using the workflow engine, Scipion.
- MeSH
- Algorithms * MeSH
- Cryoelectron Microscopy methods MeSH
- Macromolecular Substances ultrastructure MeSH
- Molecular Biology methods MeSH
- Image Processing, Computer-Assisted methods MeSH
- Workflow MeSH
- Computational Biology MeSH
- Single Molecule Imaging methods MeSH
- Imaging, Three-Dimensional methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
PURPOSE: To demonstrate the utility of an amplification-free long-read sequencing method to characterize the Fuchs endothelial corneal dystrophy (FECD)-associated intronic TCF4 triplet repeat (CTG18.1). METHODS: We applied an amplification-free method, utilizing the CRISPR/Cas9 system, in combination with PacBio single-molecule real-time (SMRT) long-read sequencing, to study CTG18.1. FECD patient samples displaying a diverse range of CTG18.1 allele lengths and zygosity status (n = 11) were analyzed. A robust data analysis pipeline was developed to effectively filter, align, and interrogate CTG18.1-specific reads. All results were compared with conventional polymerase chain reaction (PCR)-based fragment analysis. RESULTS: CRISPR-guided SMRT sequencing of CTG18.1 provided accurate genotyping information for all samples and phasing was possible for 18/22 alleles sequenced. Repeat length instability was observed for all expanded (≥50 repeats) phased CTG18.1 alleles analyzed. Furthermore, higher levels of repeat instability were associated with increased CTG18.1 allele length (mode length ≥91 repeats) indicating that expanded alleles behave dynamically. CONCLUSION: CRISPR-guided SMRT sequencing of CTG18.1 has revealed novel insights into CTG18.1 length instability. Furthermore, this study provides a framework to improve the molecular diagnostic accuracy for CTG18.1-mediated FECD, which we anticipate will become increasingly important as gene-directed therapies are developed for this common age-related and sight threatening disease.
- MeSH
- Alleles MeSH
- CRISPR-Cas Systems genetics MeSH
- Adult MeSH
- Trinucleotide Repeat Expansion genetics MeSH
- Fuchs' Endothelial Dystrophy genetics pathology MeSH
- Genetic Predisposition to Disease * MeSH
- Genotype MeSH
- Introns genetics MeSH
- Middle Aged MeSH
- Humans MeSH
- Sequence Analysis, DNA MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Transcription Factor 4 genetics MeSH
- Trinucleotide Repeats genetics MeSH
- Single Molecule Imaging MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Aged, 80 and over MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We report a powerful method for capturing the time-resolved concentration profiles, liquid swelling and surface phenomena during the absorption of methane (CH4) in still liquid ethanol (C2D6O) and n-decane (n-C10D22) and at high spatial resolution (pixel size 21.07 μm) using neutron imaging. Absorption of supercritical methane was followed at two temperatures and two pressures of methane, namely 7.0, 37.8 °C and 80, 120 bar. Fick's second law, which was used in the liquid-fixed coordinates, enabled for an adequate parameterization of the observed concentration profiles and liquid levels using simple analytical expressions. For both studied liquids, anomalously slow diffusion was observed in the initial stage of the absorption experiment. This was ascribed to the slow formation of the surface excess on the interface, time constant ranged 130-275 s. The axial symmetry of the cell allowed for the tomographic reconstructions of the profiles of the menisci. Based on these profiles, contact angle and surface tension were evaluated using the Young-Laplace equation. Overall, neutron imaging made it possible to capture time- and space-resolved information from which the methane concentration, liquid level and meniscus shape under high-pressure conditions inside a cylindrical titanium vessel were quantitatively derived. Multiple characteristics of ethanol, a methane hydrate inhibitor, and n-decane, a model constituent of crude oil, were thus measured for the first time under industrially relevant conditions in a one-pot experiment.
- MeSH
- Alkanes chemistry MeSH
- Diffusion MeSH
- Ethanol chemistry MeSH
- Physical Phenomena MeSH
- Methane chemistry MeSH
- Neutrons MeSH
- Petroleum MeSH
- Models, Statistical MeSH
- Temperature MeSH
- Water MeSH
- Single Molecule Imaging instrumentation methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Background: Super-resolution single molecule localization microscopy (SMLM) is a method for achieving resolution beyond the classical limit in optical microscopes (approx. 200 nm laterally). Yellow fluorescent protein (YFP) has been used for super-resolution single molecule localization microscopy, but less frequently than other fluorescent probes. Working with YFP in SMLM is a challenge because a lower number of photons are emitted per molecule compared with organic dyes, which are more commonly used. Publically available experimental data can facilitate development of new data analysis algorithms. Findings: Four complete, freely available single molecule super-resolution microscopy datasets on YFP-tagged growth factor receptors expressed in a human cell line are presented, including both raw and analyzed data. We report methods for sample preparation, for data acquisition, and for data analysis, as well as examples of the acquired images. We also analyzed the SMLM datasets using a different method: super-resolution optical fluctuation imaging (SOFI). The 2 modes of analysis offer complementary information about the sample. A fifth single molecule super-resolution microscopy dataset acquired with the dye Alexa 532 is included for comparison purposes. Conclusions: This dataset has potential for extensive reuse. Complete raw data from SMLM experiments have typically not been published. The YFP data exhibit low signal-to-noise ratios, making data analysis a challenge. These datasets will be useful to investigators developing their own algorithms for SMLM, SOFI, and related methods. The data will also be useful for researchers investigating growth factor receptors such as ErbB3.
- MeSH
- Algorithms MeSH
- Bacterial Proteins chemistry MeSH
- Fluorescent Dyes chemistry MeSH
- Humans MeSH
- Luminescent Proteins chemistry MeSH
- Receptors, Growth Factor chemistry isolation & purification MeSH
- Single Molecule Imaging methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
