Cryo-plasma FIB/SEM volume imaging of biological specimens.

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of subcellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM. We evaluated the choice of plasma ion source and imaging regimes to produce high-quality SEM images of a range of different biological samples. Using an automated workflow we produced three-dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20-50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.

Okapi-EM: A napari plugin for processing and analyzing cryogenic serial focused ion beam/scanning electron microscopy images.

An emergent volume electron microscopy technique called cryogenic serial plasma focused ion beam milling scanning electron microscopy (pFIB/SEM) can decipher complex biological structures by building a three-dimensional picture of biological samples at mesoscale resolution. This is achieved by collecting consecutive SEM images after successive rounds of FIB milling that expose a new surface after each milling step. Due to instrumental limitations, some image processing is necessary before 3D visualization and analysis of the data is possible. SEM images are affected by noise, drift, and charging effects, that can make precise 3D reconstruction of biological features difficult. This article presents Okapi-EM, an open-source napari plugin developed to process and analyze cryogenic serial pFIB/SEM images. Okapi-EM enables automated image registration of slices, evaluation of image quality metrics specific to pFIB-SEM imaging, and mitigation of charging artifacts. Implementation of Okapi-EM within the napari framework ensures that the tools are both user- and developer-friendly, through provision of a graphical user interface and access to Python programming.

The Effect of Glycol Side Chains on the Assembly and Microstructure of Conjugated Polymers.

Conjugated polymers with glycol-based chains, are emerging as a material class with promising applications as organic mixed ionic-electronic conductors, particularly in bioelectronics and thermoelectrics. However, little is still known about their microstructure and the role of the side chains in determining intermolecular interactions and polymer packing. Here, we use the combination of electrospray deposition and scanning tunneling microscopy to determine the microstructure of prototypical glycolated conjugated polymers (pgBTTT and p(g2T-TT)) with submonomer resolution. Molecular dynamics simulations of the same surface-adsorbed polymers exhibit an excellent agreement with the experimental images, allowing us to extend the characterization of the polymers to the atomic scale. Our results prove that, similarly to their alkylated counterparts, glycolated polymers assemble through interdigitation of their side chains, although significant differences are found in their conformation and interaction patterns. A model is proposed that identifies the driving force for the polymer assembly in the tendency of the side chains to adopt the conformation of their free analogues, i.e., polyethylene and polyethylene glycol, for alkyl or ethylene glycol side chains, respectively. For both classes of polymers, it is also demonstrated that the backbone conformation is determined to a higher degree by the interaction between the side chains rather than by the backbone torsional potential energy. The generalization of these findings from two-dimensional (2D) monolayers to three-dimensional thin films is discussed, together with the opportunity to use this type of 2D study to gain so far inaccessible, subnm-scale information on the microstructure of conjugated polymers.

Anisotropy of Charge Transport in a Uniaxially Aligned Fused Electron-Deficient Polymer Processed by Solution Shear Coating.

Precise control of the microstructure in organic semiconductors (OSCs) is essential for developing high-performance organic electronic devices. Here, a comprehensive charge transport characterization of two recently reported rigid-rod conjugated polymers that do not contain single bonds in the main chain is reported. It is demonstrated that the molecular design of the polymer makes it possible to achieve an extended linear backbone structure, which can be directly visualized by high-resolution scanning tunneling microscopy (STM). The rigid structure of the polymers allows the formation of thin films with uniaxially aligned polymer chains by using a simple one-step solution-shear/bar coating technique. These aligned films show a high optical anisotropy with a dichroic ratio of up to a factor of 6. Transport measurements performed using top-gate bottom-contact field-effect transistors exhibit a high saturation electron mobility of 0.2 cm2 V-1 s-1 along the alignment direction, which is more than six times higher than the value reported in the previous work. This work demonstrates that this new class of polymers is able to achieve mobility values comparable to state-of-the-art n-type polymers and identifies an effective processing strategy for this class of rigid-rod polymer system to optimize their charge transport properties.

The Effect of Ring Expansion in Thienobenzo[b]indacenodithiophene Polymers for Organic Field-Effect Transistors.

A fused donor, thienobenzo[b]indacenodithiophene (TBIDT), was designed and synthesized using a novel acid-promoted cascade ring closure strategy, and then copolymerized with a benzothiadiazole (BT) monomer. The backbone of TBIDT is an expansion of the well-known indacenodithiophene (IDT) unit and was expected to enhance the charge carrier mobility by improving backbone planarity and facilitating short contacts between polymer chains. However, the optimized field-effect transistors demonstrated an average saturation hole mobility of 0.9 cm2 V-1 s-1, lower than the performance of IDT-BT (∼1.5 cm2 V-1 s-1). Mobilities extracted from time-resolved microwave conductivity measurements were consistent with the trend in hole mobilities in organic field-effect transistor devices. Scanning tunneling microscopy measurements and computational modeling illustrated that TBIDT-BT exhibits a less ordered microstructure in comparison to IDT-BT. This reveals that a regular side-chain packing density, independent of conformational isomers, is critical to avoid local free volume due to irregular packing, which can host trapping impurities. DFT calculations indicated that TBIDT-BT, despite containing a larger, planar unit, showed less stabilization of planar backbone geometries in comparison to IDT-BT. This is due to the reduced electrostatic stabilizing interactions between the peripheral thiophene of the fused core and the BT unit, resulting in a reduction of the barrier to rotation around the single bond. These insights provide a greater understanding of the general structure-property relationships required for semiconducting polymer repeat units to ensure optimal backbone planarization, as illustrated with IDT-type units, guiding the design of novel semiconducting polymers with extended fused backbones for high-performance field-effect transistors.

Sequencing conjugated polymers by eye.

The solid-state microstructure of a conjugated polymer is the most important parameter determining its properties and performance in (opto)-electronic devices. A huge amount of research has been dedicated to tuning and understanding how the sequence of monomers, the nature and frequency of defects, the exact backbone conformation, and the assembly and crystallinity of conjugated polymers affect their basic photophysics and charge transporting properties. However, because of the lack of reliable high-resolution analytical techniques, all the structure-property relations proposed in the literature are based either on molecular modeling or on indirect experimental data averaged on polydisperse samples. We show that a combination of electrospray vacuum deposition and high-resolution scanning tunneling microscopy allows the imaging of individual conjugated polymers with unprecedented detail, thereby unraveling structural and self-assembly characteristics that have so far been impossible to determine.

Monolayer-to-thin-film transition in supramolecular assemblies: the role of topological protection.

The ability to control the transition from a two-dimensional (2D) monolayer to the three-dimensional (3D) molecular structure in the growth of organic layers on surfaces is essential for the production of functional thin films and devices. This has, however, proved to be extremely challenging, starting from the currently limited ability to attain a molecular scale characterization of this transition. Here, through innovative application of low-dose electron diffraction and aberration-corrected transmission electron microscopy (acTEM), combined with scanning tunneling microscopy (STM), we reveal the structural changes occurring as film thickness is increased from monolayer to tens of nanometers for supramolecular assembly of two prototypical benzenecarboxylic acids - terephthalic acid (TPA) and trimesic acid (TMA) - on graphene. The intermolecular hydrogen bonding in these molecules is similar and both form well-ordered monolayers on graphene, but their structural transitions with film thickness are very different. While the structure of TPA thin films varies continuously towards the 3D lattice, TMA retains its planar monolayer structure up to a critical thickness, after which a transition to a polycrystalline film occurs. These distinctive structural evolutions can be rationalized in terms of the topological differences in the 3D crystallography of the two molecules. The templated 2D structure of TPA can smoothly map to its 3D structure through continuous molecular tilting within the unit cell, whilst the 3D structure of TMA is topologically distinct from its 2D form, so that only an abrupt transition is possible. The concept of topological protection of the 2D structure gives a new tool for the molecular design of nanostructured films.

Synthesis and controlled growth of osmium nanoparticles by electron irradiation.

We have synthesised osmium nanoparticles of defined size (1.5-50 nm) on a B- and S-doped turbostratic graphitic structure by electron-beam irradiation of an organometallic osmium complex encapsulated in self-spreading polymer micelles, and characterised them by transmission electron microscopy (TEM), high-resolution TEM (HRTEM), and atomic force microscopy (AFM) on the same grid. Oxidation of the osmium nanoparticles after exposure to air was detected by X-ray photoelectron spectroscopy (XPS).

Supramolecular nesting of cyclic polymers.

Advances in template-directed synthesis make it possible to create artificial molecules with protein-like dimensions, directly from simple components. These synthetic macromolecules have a proclivity for self-organization that is reminiscent of biopolymers. Here, we report the synthesis of monodisperse cyclic porphyrin polymers, with diameters of up to 21 nm (750 C­C bonds). The ratio of the intrinsic viscosities for cyclic and linear topologies is 0.72, indicating that these polymers behave as almost ideal flexible chains in solution. When deposited on gold surfaces, the cyclic polymers display a new mode of two-dimensional supramolecular organization, combining encapsulation and nesting; one nanoring adopts a near-circular conformation, thus allowing a second nanoring to be captured within its perimeter, in a tightly folded conformation. Scanning tunnelling microscopy reveals that nesting occurs in combination with stacking when nanorings are deposited under vacuum, whereas when they are deposited directly from solution under ambient conditions there is stacking or nesting, but not a combination of both.

Surface-based supramolecular chemistry using hydrogen bonds.

CONSPECTUS: The arrangement of molecular species into extended structures remains the focus of much current chemical science. The organization of molecules on surfaces using intermolecular interactions has been studied to a lesser degree than solution or solid-state systems, and unanticipated observations still lie in store. Intermolecular hydrogen bonds are an attractive tool that can be used to facilitate the self-assembly of an extended structure through the careful design of target building blocks. Our studies have focused on the use of 3,4,9,10-perylene tetracarboxylic acid diimides (PTCDIs), and related functionalized analogues, to prepare extended arrays on surfaces. These molecules are ideal for such studies because they are specifically designed to interact with appropriate diaminopyridine-functionalized molecules, and related species, through complementary hydrogen bonds. Additionally, PTCDI species can be functionalized in the bay region of the molecule, facilitating modification of the self-assembled structures that can be prepared. Through a combination of PTCDI derivatives, sometimes in combination with melamine, porous two-dimensional arrays can be formed that can entrap guest molecules. The factors that govern the self-assembly processes of PTCDI derivatives are discussed, and the ability to construct suitable target arrays and host-specific molecular species, including fullerenes and transition metal clusters, is demonstrated.

Vernier-templated synthesis, crystal structure, and supramolecular chemistry of a 12-porphyrin nanoring.

Vernier templating exploits a mismatch between the number of binding sites in a template and a reactant to direct the formation of a product that is large enough to bind several template units. Here, we present a detailed study of the Vernier-templated synthesis of a 12-porphyrin nanoring. NMR and small-angle X-ray scattering (SAXS) analyses show that Vernier complexes are formed as intermediates in the cyclo-oligomerization reaction. UV/Vis/NIR titrations show that the three-component assembly of the 12-porphyrin nanoring figure-of-eight template complex displays high allosteric cooperativity and chelate cooperativity. This nanoring-template 1:2 complex is among the largest synthetic molecules to have been characterized by single-crystal analysis. It crystallizes as a racemate, with an angle of 27° between the planes of the two template units. The crystal structure reveals many unexpected intramolecular C-H⋅⋅⋅N contacts involving the tert-butyl side chains. Scanning tunneling microscopy (STM) experiments show that molecules of the 12-porphyrin template complex can remain intact on the gold surface, although the majority of the material unfolds into the free nanoring during electrospray deposition.

Height dependent molecular trapping in stacked cyclic porphyrin nanorings.

Stacked layers of cyclic porphyrin nanorings constitute nanoscale receptacles with variable height and diameter which preferentially adsorb sublimed molecules. Using scanning tunnelling microscopy we determine the filling capacity of these nanoring traps, and the dependence of adsorbate capture on stack height and diameter.

Mechanical stiffening of porphyrin nanorings through supramolecular columnar stacking.

Solvent-induced aggregates of nanoring cyclic polymers may be transferred by electrospray deposition to a surface where they adsorb as three-dimensional columnar stacks. The observed stack height varies from single rings to four stacked rings with a layer spacing of 0.32 ± 0.04 nm as measured using scanning tunneling microscopy. The flexibility of the nanorings results in distortions from a circular shape, and we show, through a comparison with Monte Carlo simulations, that the bending stiffness increases linearly with the stack height. Our results show that noncovalent interactions may be used to control the shape and mechanical properties of artificial macromolecular aggregates offering a new route to solvent-induced control of two-dimensional supramolecular organization.

Haptic-STM: a human-in-the-loop interface to a scanning tunneling microscope.

The operation of a haptic device interfaced with a scanning tunneling microscope (STM) is presented here. The user moves the STM tip in three dimensions by means of a stylus attached to the haptic instrument. The tunneling current measured by the STM is converted to a vertical force, applied to the stylus and felt by the user, with the user being incorporated into the feedback loop that controls the tip-surface distance. A haptic-STM interface of this nature allows the user to feel atomic features on the surface and facilitates the tactile manipulation of the adsorbate/substrate system. The operation of this device is demonstrated via the room temperature STM imaging of C(60) molecules adsorbed on an Au(111) surface in ultra-high vacuum.

Self-assembled aggregates formed by single-molecule magnets on a gold surface.

The spontaneous ordering of molecules into two-dimensional self-assembled arrays is commonly stabilized by directional intermolecular interactions that may be promoted by the addition of specific chemical side groups to a molecule. In this paper, we show that self-assembly may also be driven by anisotropic interactions that arise from the three-dimensional shape of a complex molecule. We study the molecule Mn(12)O(12)(O(2)CCH(3))(16)(H(2)O)(4) (Mn(12)(acetate)(16)), which is transferred from solution onto a Au(111) substrate held in ultrahigh vacuum using electrospray deposition (UHV-ESD). The deposited Mn(12)(acetate)(16) molecules form filamentary aggregates because of the anisotropic nature of the molecule-molecule and molecule-substrate interactions, as confirmed by molecular dynamics calculations. The fragile Mn(12)O(12) core of the Mn(12)(acetate)(16) molecule is compatible with the UHV-ESD process, which we demonstrate using near-edge X-ray adsorption fine-structure spectroscopy. UHV-ESD of Mn(12)(acetate)(16) onto a surface that has been prepatterned with a hydrogen-bonded supramolecular network provides additional control of lateral organization.

Tailoring pores for guest entrapment in a unimolecular surface self-assembled hydrogen bonded network.

A unimolecular hydrogen-bonded network is formed by a perylene-diimide derivative following surface self-assembly leading to the formation of pores of appropriate dimensions to accommodate regularly spaced guest C(60) molecules.

Entrapment of decanethiol in a hydrogen-bonded bimolecular template.

We have used scanning tunneling microscopy to investigate the deposition of 1-decanethiol onto a bimolecular self-assembled network composed of PTCDI (perylene tetracarboxylic diimide) and melamine on a Au(111) surface. A new laterally organized phase in which the pores of a parallelogram bimolecular arrangement trap two decanethiol molecules is identified. Disruption of the hexagonal PTCDI-melamine network arrangement after decanethiol deposition is also observed, providing insights about the interplay between supramolecular and substrate-adsorbate interactions.

Functionalized supramolecular nanoporous arrays for surface templating.

Controlled self-assembly and chemical tailoring of bimolecular networks on surfaces is demonstrated using structural derivatives of 3,4:9,10-perylenetetracarboxylic diimide (PTCDI) combined with melamine (1,3,5-triazine-2,4,6-triamine). Two functionalised PTCDI derivatives have been synthesised, Br(2)-PTCDI and di(propylthio)-PTCDI, through attachment of chemical side groups to the perylene core. Self-assembled structures formed by these molecules on a Ag-Si(111)sqrt3 x sqrt3R30 degrees surface were studied with a room-temperature scanning tunneling microscope under ultrahigh vacuum conditions. It is shown that the introduction of side groups can have a significant effect upon both the structures formed, notably in the case of di(propylthio)-PTCDI which forms a previously unreported unimolecular hexagonal arrangement, and their entrapment behaviour. These results demonstrate a new route of functionalisation for network pores, opening up the possibility of designing nanostructured surface structures with chemical selectivity and applications in nanostructure templating.