single particle analysis
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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.
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
- algoritmy MeSH
- Caenorhabditis elegans genetika růst a vývoj metabolismus MeSH
- embryo nesavčí metabolismus ultrastruktura MeSH
- fixace tkání metody MeSH
- fluorescenční barviva chemie MeSH
- genetická transkripce MeSH
- hybridizace in situ fluorescenční metody MeSH
- messenger RNA chemie genetika metabolismus MeSH
- poměr signál - šum MeSH
- vývojová regulace genové exprese MeSH
- zobrazení jednotlivé molekuly metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- Publikační typ
- časopisecké články 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).
- Publikační typ
- časopisecké články 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.
- Publikační typ
- časopisecké články 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
- algoritmy MeSH
- bakteriální proteiny chemie MeSH
- fluorescenční barviva chemie MeSH
- lidé MeSH
- luminescentní proteiny chemie MeSH
- receptory růstových faktorů chemie izolace a purifikace MeSH
- zobrazení jednotlivé molekuly metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.
- MeSH
- adenosintrifosfát metabolismus MeSH
- bodová mutace MeSH
- helikasy RecQ genetika metabolismus ultrastruktura MeSH
- homologní rekombinace * MeSH
- hydrolýza MeSH
- jednovláknová DNA metabolismus ultrastruktura MeSH
- kinetika MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- missense mutace MeSH
- molekulární motory metabolismus ultrastruktura MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- rekombinasa Rad51 genetika metabolismus MeSH
- replikační protein A metabolismus MeSH
- substrátová specifita MeSH
- zobrazení jednotlivé molekuly * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Resistive pulse sensing is a well-known and established method for counting and sizing particles in ionic solutions. Throughout its development the technique has been expanded from detection of biological cells to counting nanoparticles and viruses, and even registering individual molecules, e.g., nucleotides in nucleic acids. This technique combined with microfluidic or nanofluidic systems shows great potential for various bioanalytical applications, which were hardly possible before microfabrication gained the present broad adoption. In this review, we provide a comprehensive overview of microfluidic designs along with electrode arrangements with emphasis on applications focusing on bioanalysis and analysis of single cells that were reported within the past five years.
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
- algoritmy * MeSH
- elektronová kryomikroskopie metody MeSH
- makromolekulární látky ultrastruktura MeSH
- molekulární biologie metody MeSH
- počítačové zpracování obrazu metody MeSH
- průběh práce MeSH
- výpočetní biologie MeSH
- zobrazení jednotlivé molekuly metody MeSH
- zobrazování trojrozměrné metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Non-isothermal differential scanning calorimetry was used to study the influences of particle size and mechanically induced defects on the recrystallization kinetics of amorphous Enzalutamide. Enzalutamide prepared by hot melt extrusion and spray-drying was used as a model material. The recrystallization rate was primarily accelerated by the presence of the processing-damaged surface of the powder particles. The actual surface/volume ratio associated with decreasing particle size fulfilled only a secondary role. Interestingly, higher quench rate during the extrusion led to a formation of thermally less stable material (with the worse stability being manifested via lower activation energy of crystal growth in the amorphous matrix). This can be the consequence of the formation of looser structure more prone to rearrangements. The recrystallization kinetics of the prepared Enzalutamide amorphous materials was described by the two-parameter autocatalytic kinetic model. The modified single-curve multivariate kinetic analysis (optimized for the data obtained at heating rate 0.5 °C•min-1) was used to calculate the extrapolated kinetic predictions of long-term isothermal crystal growth. The predictions were made for the temperatures from the range of drug shelf-life and processing for each particle size fraction. By the combination of the mass-weighted predictions for the individual powder fractions it was possible to obtain a very reasonable (temperature-extrapolated) prediction of the crystallization rate for the as-prepared unsieved powdered amorphous Enzalutamide.
