The availability of the Airyscan detector in the Zeiss LSM 880 has made possible the development of a new concept in fluctuation correlation spectroscopy using super-resolution. The Airyscan unit ...acquires data simultaneously on 32 detectors arranged in a hexagonal array. This detector opens up the possibility to use fluctuation methods based on time correlation at single points or at a number of points simultaneously, as well as methods based on spatial correlation in the area covered by the detector. Given the frame rate of this detector, millions of frames can be acquired in seconds, providing a robust statistical basis for fluctuation data. We apply the comprehensive analysis to the molecular fluctuations of free GFP diffusing in live cells at different subcellular compartments to show that at the nanoscale different cell environments can be distinguished by the comprehensive fluctuation analysis.
A picosecond laser ablation approach has been developed for the synthesis of ligand-free AuAg bimetallic NPs where the relative amount of Ag is controlled in situ through a laser shielding effect. ...Various measurements, such as optical spectroscopy, transmission electron microscopy combined with energy dispersive X-ray spectroscopy and inductively coupled plasma optical emission spectrometry, revealed the generation of homogenous 15 nm average size bimetallic NPs with different compositions and tunable localized surface plasmon resonance. Furthermore, ligand-free metallic nanoparticles with respect to chemically synthesized nanoparticles display outstanding properties, i.e. featureless Raman background spectrum, which is a basic requirement in many plasmonic applications such as Surface Enhanced Raman Spectroscopy. Various molecules were chemisorbed on the nanoparticle and SERS investigations were carried out, by varying the laser wavelength. The SERS enhancement factor for AuAg bimetallic NPs shows an enhancement factor of about 5.7 × 10(5) with respect to the flat AuAg surface.
Silicon nanoparticles were prepared by ultrafast laser ablation of a silicon target in deionized water. The nanoparticles were characterized by using optical absorption, Raman spectroscopy, and ...transmission electron microscopy. The mean size is found to vary from 60 to 2.5 nm in the absence of any reducing chemical reagents, decreasing the pulse energy value. High-resolution transmission electron microscopy together with Raman spectroscopy confirms the crystalline structure of the generated silicon nanoparticles. The energy confinement of carriers which is evaluated from optical experiments varies from 90 to 550 meV when the mean nanoparticles size decreases from 60 to 2.5 nm. In particular, the evaluated nanoparticle sizes from optical analysis and the LCAO theoretical model are found in agreement with transmission electron microscopy and Raman measurements for the silicon nanoparticles with a size less than 6 nm. Finally, we present stability studies which show that the smallest nanoparticles aggregate over time.
In a previous study, the coexistence of different aggregation pathways of insulin and β-amyloid (Aβ) peptides was demonstrated by correlative stimulated emission depletion (STED) microscopy and ...atomic force microscopy (AFM). This had been explained by suboptimal proteins labeling strategies that generate heterogeneous populations of aggregating species. However, because of the limited number of proteins considered, the failure of the fluorescent labeling that occurs in a large portion of the aggregating fibrils observed for insulin and Aβ peptides, could not be considered a general phenomenon valid for all molecular systems. Here, we investigated the aggregation process of α-synuclein (α-syn), an amyloidogenic peptide involved in Parkinson's disease, which is significantly larger (MW ∼14 kDa) than insulin and Aβ, previously investigated. The results showed that an unspecific labeling procedure, such as that previously adopted for shorter proteins, reproduced the coexistence of labeled/unlabeled fibers. Therefore, a site-specific labeling method was developed to target a domain of the peptide scarcely involved in the aggregation process. Correlative STED-AFM illustrated that all fibrillar aggregates derived from the aggregation of α-syn at the dye-to-protein ratio of 1 : 22 were fluorescent. These results, demonstrated here for the specific case of α-syn, highlight that the labeling artifacts can be avoided by careful designing the labeling strategy for the molecular system under investigation. The use of a label-free correlative microscopy technique would play a crucial role in the control of the setting of these conditions.
Heavy-metal-free semiconductor material like Silicon Nanoparticle (Si-NPs) is attracting scientists because of their diverse applications in biomedical field. In this work, pulsed laser ablation of ...silicon in aqueous solution is employed to generate Si-NPs in one step avoiding use of chemical precursors. Characterization by absorption, electron and photoluminescence analysis proves the generation of luminescent Si-NPs. The productivity rate of Si-NPs is investigated by Inductively Coupled Plasma Spectrometry. Furthermore, Si-NPs quantum yield and confocal microscopy studies corroborate the potential use of these biocompatible Si-NPs for imaging applications.
Stable and biodegradable oil in water (O/W) nano-emulsions can have a huge impact on a wide range of bio-applications, from food to cosmetics and pharmaceuticals. Emulsions, however, are immiscible ...systems unstable over time; polymer coatings are known to be helpful, but an effective procedure to stabilize monodisperse and biodegradable O/W nano-emulsions is yet to be designed. Here, we coat biodegradable O/W nano-emulsions with a molecular layer of biodegradable polyelectrolytes such as polysaccharides--like chitosan--and polypeptides--like polylysine--and effectively re-disperse and densify the polymer coating at high pressure, thus obtaining monodisperse and stable systems. In particular, focusing on chitosan, our tests show that it is possible to obtain unprecedented ultra-stable O/W secondary nano-emulsions (diameter sizes tunable from ∼ 80 to 160 nm and polydispersion indices below 0.1) by combining this process with high concentrations of polymers. Depending on the polymer concentration, it is possible to control the level of coating that results in a tunable stability ranging from a few weeks to several months. The above range of concentrations has been investigated using a fluorescence-based approach with new insights into the coating evolution.
Aggregation dynamics in gold nanoparticles can be controlled to produce self similar pattern. Controlling the reaction time of the nanoparticles during aggregation to reduce the Helmholtz free ...surface energy is the key parameter in this study. The study showed that the dynamics is dominated by the Reaction Limited Aggregation Model (RLA) more than the Diffusion Limited Aggregation Model (DLA). Where the repulsion due to electrostatic force is dominant on the Van Der Walls attraction force, and the sticking probability of nanoparticles is low. The aggregation dynamics of AuNPs can be slowed down if the water evaporation of the drop casted colloidal AuNPs on a quartz substrate is slowed. Slowing down the evaporation allow the decrease in the electrostatic repulsion forces gradually, allowing the nanoparticles to sample all the possible orientation for the best thermodynamically favorable site. The lowest Helmholtz free surface energy for gold nanoparticles is corresponding to the dendrite fractal pattern. Display omitted
Self-assembly of gold nanoparticles (AuNPs) is an important growth mode for fabricating functional materials. In this work we report a dendrite structure formed by slowing down the aggregation dynamics of AuNPs self-assembly. The obtained results show that the aggregation dynamics is dominated by the Reaction Limited Aggregation Model (RLA) more than the Diffusion Limited Aggregation Model (DLA). In which the repulsion due to electrostatic forces is dominant by the Van Der Walls attraction forces, and low sticking probability of nanoparticles. The aggregation dynamics of AuNPs can be slowed down if the water evaporation of the drop casted colloidal AuNPs on a quartz substrate is slowed. Slowing down the evaporation allows electrostatic repulsion forces to decrease gradually. At certain point, the attraction forces become higher than the electrostatic repulsion and hence cluster aggregation take place slowly. The slow aggregation dynamics allows the nanoparticles to sample all possible orientation in the sticking site, searching for the lowest energy configuration. The size distribution of the nanoparticles in liquid is confirmed using dynamic light scattering based on Stokes–Einstein equation for diffusion coefficient in water. X-ray and photoluminescence (PL) spectra of the sample after aggregation showed a shift which is related to the aggregation compared with non-aggregated colloidal nanoparticles in the solution. The study shows that dendrite self similar structure can be formed by slowing down the aggregation dynamics of nanoparticles as a result of minimizing the Helmholtz free surface energy of the system.
Summary
In this paper we report stimulated emission depletion (STED) and two‐photon excitation (2PE) fluorescence microscopy with continuous wave (CW) laser beam using a new generation laser scanning ...confocal microscope equipped for STED‐CW (TCS STED‐CW, Leica Microsystems, Mannheim, Germany). We show the possibility to achieve CW‐2PE with the very same beam used for STED‐CW. This feature extends the performance of the microscope allowing multimodal imaging (CW‐2PE, STED‐CW, confocal).
α(s1)-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of ...α(s1)-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of α(s1)-casein on the fibrillogenesis of 1-40 β-amyloid peptide, involved in Alzheimer's disease.
The aggregation kinetics of β-peptide in presence and absence of α(s1)-casein was followed under shear at 37°C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of α(s1)-casein-Aβ(1-40) interaction.
α(s1)-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. α(s1)-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism.
Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.
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