Step-by-step guide to efficient subtomogram averaging of virus-like particles with Dynamo.

Subtomogram averaging (STA) is a powerful image processing technique in electron tomography used to determine the 3D structure of macromolecular complexes in their native environments. It is a fast growing technique with increasing importance in structural biology. The computational aspect of STA is very complex and depends on a large number of variables. We noticed a lack of detailed guides for STA processing. Also, current publications in this field often lack a documentation that is practical enough to reproduce the results with reasonable effort, which is necessary for the scientific community to grow. We therefore provide a complete, detailed, and fully reproducible processing protocol that covers all aspects of particle picking and particle alignment in STA. The command line-based workflow is fully based on the popular Dynamo software for STA. Within this workflow, we also demonstrate how large parts of the processing pipeline can be streamlined and automatized for increased throughput. This protocol is aimed at users on all levels. It can be used for training purposes, or it can serve as basis to design user-specific projects by taking advantage of the flexibility of Dynamo by modifying and expanding the given pipeline. The protocol is successfully validated using the Electron Microscopy Public Image Archive (EMPIAR) database entry 10164 from immature HIV-1 virus-like particles (VLPs) that describe a geometry often seen in electron tomography.

FTLD-TDP assemblies seed neoaggregates with subtype-specific features via a prion-like cascade.

Morphologically distinct TDP-43 aggregates occur in clinically different FTLD-TDP subtypes, yet the mechanism of their emergence and contribution to clinical heterogeneity are poorly understood. Several lines of evidence suggest that pathological TDP-43 follows a prion-like cascade, but the molecular determinants of this process remain unknown. We use advanced microscopy techniques to compare the seeding properties of pathological FTLD-TDP-A and FTLD-TDP-C aggregates. Upon inoculation of patient-derived aggregates in cells, FTLD-TDP-A seeds amplify in a template-dependent fashion, triggering neoaggregation more efficiently than those extracted from FTLD-TDP-C patients, correlating with the respective disease progression rates. Neoaggregates are sequentially phosphorylated with N-to-C directionality and with subtype-specific timelines. The resulting FTLD-TDP-A neoaggregates are large and contain densely packed fibrils, reminiscent of the pure compacted fibrils present within cytoplasmic inclusions in postmortem brains. In contrast, FTLD-TDP-C dystrophic neurites show less dense fibrils mixed with cellular components, and their respective neoaggregates are small, amorphous protein accumulations. These cellular seeding models replicate aspects of the patient pathological diversity and will be a useful tool in the quest for subtype-specific therapeutics.

Magnetically Assembled SERS Substrates Composed of Iron-Silver Nanoparticles Obtained by Laser Ablation in Liquid.

The widespread application of surface-enhanced Raman scattering (SERS) would benefit from simple and scalable self-assembly procedures for the realization of plasmonic arrays with a high density of electromagnetic hot-spots. To this aim, the exploitation of iron-doped silver nanoparticles (NPs) synthesized by laser ablation of a bulk bimetallic iron-silver target immersed in ethanol is described. The use of laser ablation in liquid is key to achieving bimetallic NPs in one step with a clean surface available for functionalization with the desired thiolated molecules. These iron-silver NPs show SERS performances, a ready response to external magnetic fields and complete flexibility in surface coating. All these characteristics were used for the magnetic assembly of plasmonic arrays which served as SERS substrates for the identification of molecules of analytical interest. The magnetic assembly of NPs allowed a 28-fold increase in the SERS signal of analytes compared to not-assembled NPs. The versatility of substrate preparation and the SERS performances were investigated as a function of NPs surface coating among different thiolated ligands. These results show a simple procedure to obtain magnetically assembled regenerable plasmonic arrays for repeated SERS investigation of different samples, and it can be of inspiration for the realization of other self-assembled and reconfigurable magnetic-plasmonic devices.

Metastable alloy nanoparticles, metal-oxide nanocrescents and nanoshells generated by laser ablation in liquid solution: influence of the chemical environment on structure and composition.

Alloy nanoparticles are characterized by the combination of multiple interesting properties, which are attractive for technological and scientific purposes. A frontier topic of this field is nanoalloys with compositions not thermodynamically allowed at ordinary temperature and pressure (i.e. metastable), because they require out-of-equilibrium synthetic approaches. Recently, laser ablation synthesis in solution (LASiS) was successfully applied for the realization of metastable nanoalloys because of the fast kinetics of nanoparticle formation. However, the role played by the chemical environment on the final composition and structure of laser generated nanoalloys still has to be fully elucidated. Here, we investigated the influence of different synthetic conditions on the LASiS of metastable nanoalloys composed of Au and Fe, such as the use of water instead of ethanol, the bubbling of inert gases and the addition of a few vol% of H2O2 and H2O. The two elements showed different reactivity when LASiS was performed in water instead of ethanol, while minor effects were observed from bubbling pure gases such as N2, Ar and CO2 in the liquid solution. Moreover, the plasmonic response and the structure of the nanoalloys were sensibly modified by adding H2O2 to water. We also found that nanoparticle production is dramatically influenced just by adding 0.2% of H2O in ethanol. These results suggest that the formation of a cavitation bubble with long lifetime and large size during LASiS is useful for the preservation of the metastable alloy composition, whereas an oxidative environment hampers the formation of metastable alloy nanoparticles. Overall, by acting on the type of solvent and solutes, we were able to switch from a traditional synthetic approach for the composition of Au-Fe nanoalloys to one using a reactive environment, which gives unconventional structures such as metal@iron-oxide nanoshells and nanocrescents of oxide supported on metal nanospheres. These results expand the knowledge about the mechanism of the formation of nanoalloys using LASiS and show how to obtain multielement nanoparticles of enormous interest for nanomedicine, plasmonics, magneto-plasmonics and catalysis.

Magneto-plasmonic Au-Fe alloy nanoparticles designed for multimodal SERS-MRI-CT imaging.

Diagnostic approaches based on multimodal imaging are needed for accurate selection of the therapeutic regimens in several diseases, although the dose of administered contrast drugs must be reduced to minimize side effects. Therefore, large efforts are deployed in the development of multimodal contrast agents (MCAs) that permit the complementary visualization of the same diseased area with different sensitivity and different spatial resolution by applying multiple diagnostic techniques. Ideally, MCAs should also allow imaging of diseased tissues with high spatial resolution during surgical interventions. Here a new system based on multifunctional Au-Fe alloy nanoparticles designed to satisfy the main requirements of an ideal MCA is reported and their biocompatibility and imaging capability are described. The MCAs show easy and versatile surface conjugation with thiolated molecules, magnetic resonance imaging (MRI) and computed X-ray tomography (CT) signals for anatomical and physiological information (i.e., diagnostic and prognostic imaging), large Raman signals amplified by surface enhanced Raman scattering (SERS) for high sensitivity and high resolution intrasurgical imaging, biocompatibility, exploitability for in vivo use and capability of selective accumulation in tumors by enhanced permeability and retention effect. Taken together, these results show that Au-Fe nanoalloys are excellent candidates as multimodal MRI-CT-SERS imaging agents.

Automated fiducial-based alignment of cryo-electron tomography tilt series in Dynamo.

With the advent of modern technologies for cryo-electron tomography (cryo-ET), high-quality tilt series are more rapidly acquired than processed and analyzed. Thus, a robust and fast-automated alignment for batch processing in cryo-ET is needed. While different software packages have made available several approaches for automated marker-based alignment of tilt series, manual user intervention remains necessary for many datasets, thus preventing high-throughput tomography. We have developed a MATLAB-based framework integrated into the Dynamo software package for automatic detection of fiducial markers that generates a robust alignment model with minimal input parameters. This approach allows high-throughput, unsupervised volume reconstruction. This new module extends Dynamo with a large repertory of tools for tomographic alignment and reconstruction, as well as specific visualization browsers to rapidly assess the biological relevance of the dataset. Our approach has been successfully tested on a broad range of datasets that include diverse biological samples and cryo-ET modalities.

Architecture of the ESCPE-1 membrane coat.

Recycling of membrane proteins enables the reuse of receptors, ion channels and transporters. A key component of the recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), which rescues transmembrane proteins from the endolysosomal pathway for transport to the trans-Golgi network and the plasma membrane. This rescue entails the formation of recycling tubules through ESCPE-1 recruitment, cargo capture, coat assembly and membrane sculpting by mechanisms that remain largely unknown. Herein, we show that ESCPE-1 has a single-layer coat organization and suggest how synergistic interactions between ESCPE-1 protomers, phosphoinositides and cargo molecules result in a global arrangement of amphipathic helices to drive tubule formation. Our results thus define a key process of tubule-based endosomal sorting.

Magnetic tuning of SERS hot spots in polymer-coated magnetic-plasmonic iron-silver nanoparticles.

Plasmonic nanostructures are intensively studied for their ability to create electromagnetic hot spots, where a great variety of optical and spectroscopic processes can be amplified. Understanding how to control the formation of hot spots in a dynamic and reversible way is crucial to further expand the panorama of plasmon enhanced phenomena. In this work, we investigate the ability to modulate the hot spots in magnetic-plasmonic iron-doped silver nanoparticles dispersed in aqueous solution, by applying an external magnetic field. Evidence of magnetic field induction of hot spots was achieved by measuring the amplification of surface enhanced Raman scattering (SERS) from analytes dispersed in the solution containing Ag-Fe NPs. A polymeric shell was introduced around Ag-Fe NPs to confer colloidal stability, and it was found that the length and density of the polymer chains have a significant influence on SERS performance, and therefore on the formation of electromagnetic hot spots, under the action of the external magnetic field. These findings are expected to provide an important contribution to understanding the growing field of tuneable electromagnetic enhancement by external stimuli, such as magnetic fields applied to magnetic-plasmonic nanoparticles.

Subtomogram averaging from cryo-electron tomograms.

Cryo-electron tomography (cryo-ET) allows three-dimensional (3D) visualization of frozen-hydrated biological samples, such as protein complexes and cell organelles, in near-native environments at nanometer scale. Protein complexes that are present in multiple copies in a set of tomograms can be extracted, mutually aligned, and averaged to yield a signal-enhanced 3D structure up to sub-nanometer or even near-atomic resolution. This technique, called subtomogram averaging (StA), is powered by improvements in EM hardware and image processing software. Importantly, StA provides unique biological insights into the structure and function of cellular machinery in close-to-native contexts. In this chapter, we describe the principles and key steps of StA. We briefly cover sample preparation and data collection with an emphasis on image processing procedures related to tomographic reconstruction, subtomogram alignment, averaging, and classification. We conclude by summarizing current limitations and future directions of this technique with a focus on high-resolution StA.

Formation of alloy nanoparticles by laser ablation of Au/Fe multilayer films in liquid environment.

Laser ablation in liquids (LAL) emerged as a powerful technique for the synthesis of multielement nanoparticles (NPs) such as metal alloys with thermodynamically forbidden composition. Consequently, there is a great interest in expanding the current knowledge about NPs formation during LAL, in order to improve the control on product structure and to extend the range of compositions accessible by this technique. Here we performed a systematic investigation on alloy NPs formation by nanosecond LAL of Au/Fe/glass multilayers with different thickness and order of deposition. The experiments were carried out in ethanol and water, which have, respectively, favourable and unfavourable effects on alloy formation. Results were analyzed with optical absorption spectroscopy, transmission electron microscopy and Mie theory for simple and core-shell spheres. Since alloy NPs were obtained in all cases, our findings provide the evidence that the two metals are mixed during particles formation. Besides, our results suggest that the probability of interaction between ablated matter and solution species is higher for the topmost layer of the target, i.e. the one closer to the solid/liquid interface. This provides useful insight for the synthesis of nanoalloys with new compositions, that are of interest in several fields, from catalysis to photonics and nanomedicine.

Strong dependence of surface plasmon resonance and surface enhanced Raman scattering on the composition of Au-Fe nanoalloys.

Nanoalloys of noble metals with transition metals are crucial components for the integration of plasmonics with magnetic and catalytic properties, as well as for the production of low-cost photonic devices. However, due to synthetic challenges in the realization of nanoscale solid solutions of noble metals and transition metals, very little is known about the composition dependence of plasmonic response in nanoalloys. Here we demonstrate for the first time that the elemental composition of Au-Fe nanoalloys obtained by laser ablation in liquid solution can be tuned by varying the liquid environment. Due to surface passivation and reaction with thiolated ligands, the nanoalloys obtained by our synthetic protocol are structurally and colloidally stable. Hence, we studied the dependence of the surface plasmon resonance (SPR) on the iron fraction and, for the first time, we observed surface enhanced Raman scattering (SERS) in Au-Fe nanoalloys. SPR and SERS performances are strongly affected by the iron content and are investigated using analytical and numerical models. By demonstrating the strong modification of plasmonic properties on the composition, our results provide important insights into the exploitation of Au-Fe nanoalloys in photonics, nanomedicine, magneto-plasmonic and plasmon-enhanced catalysis. Moreover, our findings show that several other plasmonic materials exist beyond gold and silver nanostructures.