AFM imaging of RGD presenting synthetic extracellular matrix using gold nanoparticles

Several high-resolution imaging techniques such as FESEM, TEM and AFM are compared with respect to their application on alginate hydrogels, a widely used polysaccharide biomaterial. A new AFM method applicable to RGD peptides covalently conjugated to alginate hydrogels is described. High-resolution images of RGD adhesion ligand distribution were obtained by labeling biotinylated RGD peptides with streptavidin-labeled gold nanoparticles. This method may broadly provide a useful tool for sECM characterization and design for tissue regeneration strategies.     

Cryogenic Correlative Single-Particle Photoluminescence Spectroscopy and Electron Tomography for Investigation of Nanomaterials

Cryogenic single-particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single-particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in-plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in-plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.     

Quantitative chemical phase imaging by means of energy filtering transmission electron microscopy

Electron spectroscopic imaging (ESI) in the transmission electron microscope (TEM) is a powerful method to produce 2-dimensional elemental distribution maps. These maps show in a clear way the chemical situation of a small specimen region. In this work we used a Gatan Imaging Filter (GIF) attached to a 200 kV TEM to investigate a Ba-Nd-titanate ceramic. The three phases occuring in this material could be visualized using inner-shell ionization edges (Ba M₄₅, Nd M₄₅ and Ti L₂₃). We applied different image correlation techniques to the ESI elemental maps for direct visualization of the chemical phases. First we simply overlaid the elemental maps assigning each element one colour to form an RGB image. Secondly we used the technique of scatter diagrams to classify the different phases. Finally we quantified the elemental maps by dividing them and multiplying them by the appropriate inner-shell ionization cross-sections which gave atomic ratio images. By using these methods we could clearly identify and quantify the various phases in the Ba-Nd-titanate specimen.Dedicated to Professor Dr. rer. nat. Dr. h.c. Hubertus Nickel on the occasion of his 65th birthday     

Cryogenic Correlative Single‐Particle Photoluminescence Spectroscopy and Electron Tomography for Investigation of Nanomaterials

Cryogenic single‐particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single‐particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in‐plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in‐plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.     

Correlating polymer structure,dynamics, and function with atomic force microscopy

Since its development, atomic force microscopy (AFM) has become an indispensable tool for investigating fundamental and technological applications of polymer materials. The versatility of AFM imaging modes and operating conditions allows for nanoscale characterization of a range of dynamic processes, such as crystallization, phase separation, self assembly, and electronic transport. Advances in AFM technology, particularly high-speed and high-resolution imaging, enable investigation of polymer structure, function, and dynamics in real world conditions and across a range of relevant spatial and temporal scales. In this perspective, we highlight a collection of recent polymer studies that utilize AFM to correlate the function and structure of polymer films, with focus on its multiparametric imaging capabilities. As the complexity of polymer materials and morphologies continues to increase, AFM is well poised to meet the accompanying demand for nanoscale imaging and characterization.     

Atomic force microscopy and transmission electron microscopy of cellulose from Micrasterias denticulata; evidence for a chiral helical microfibril twist

Atomic force microscopy (AFM), tapping mode atomic force microscopy (TM-AFM) and transmission electron microscopy (TEM) have been used to image the cell wall, ultrathin sections of whole cells and cellulose microfibrils prepared from the green alga Micrasterias denticulata. Measurements of the microfibril dimensions are in agreement with earlier observations carried out by electron microscopy. Images at the molecular level of the surface of the microfibrils were obtained with AFM and show regular periodicities along the microfibril axis that correspond to the fibre and glucose repeat distances of cellulose. Twisted regions visible at intervals along the microfibrils dried down onto substrates were noted to be right-handed in over 100 observations by TEM, AFM and TM-AFM. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microscopic features of methacrylate polymers with zwitterionic pendant groups determined by various techniques

The objective of this work was to determine microscopic structural features of three methacrylate polymers with different numbers of diethylene glycol residues and zwitterionic pendant groups. X‐ray diffraction, electron microscopy, and scanning probe microscopy techniques were employed. X‐ray data led to the adoption of a model made up of molecular aggregates forming lamellar domains, establishing in this way ordered characteristics of these kinds of polymers. Scanning electron microscopy images provided evidence of the occurrence of a lamellar structure forming the morphology of the polymers. This was corroborated by atomic force microscopy experiments. Transmission electron microscopy revealed that the lamellar aggregates conformed into clusters immersed in a polymeric matrix. From phase‐contrast images, information on the homogeneity of the composition at a molecular surface level was obtained. Then, the techniques provided evidence of the lamellar domain characteristics of the studied sulfobetaines. The crystallinity was a function of the number of ethoxy groups because as this number increased, the crystallinity became lower © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 1152–1160, 2005     

Surlyn®/silicate nanocomposite materials via a polymer in situ sol–gel process: Morphology

Surlyn®/silicate hybrid materials were produced via diffusion‐controlled polymer in situ sol–gel reactions for tetraethylorthosilicate. The heterogeneous morphologies of these materials were inspected with transmission electron (TEM), atomic force (AFM), and environmental scanning electron microscopic methods. The silicate uptake was highly dependent on the water affinity of the particular Surlyn® form (acid or ionic) rather than on the affinity of the solvent. The morphology consisted of silicate particles with diameters that were on the order of tens of nanometers. Hence, these materials can be classified as nanocomposites. The particle size distributions in both the TEM and AFM images for all composites appeared to be narrow, with un‐neutralized Surlyn® exhibiting a broader distribution. Larger particles were found near the film surfaces, and the silicon elemental distribution across the film thickness indicated higher concentrations near the surfaces, which is most likely due to the fact that the sol–gel reaction is diffusion controlled in these polymeric media. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 1563–1571, 2003     

Biohybrid fluorescent nanodiamonds as dual‐contrast markers for light and electron microscopies

Cathodoluminescence (CL) and correlative light‐electron microscopy (CLEM) are two useful analytical tools in diverse research areas. Recently, fluorescent nanodiamonds (FNDs) have emerged as promising imaging agents for both CL and CLEM owing to their exceptional photophysical and chemical properties. However, to realize their practical applications in the life sciences, surface modification and functionalization of the nanomaterials with bioactive molecules are critical and essential. Here we provide a comprehensive review on the methods of synthesizing biohybrid FNDs as well as recent advances of CL and CLEM imaging of cells with these carbon nanoparticles as dual‐contrast markers.     

Study of hydration layers near a hydrophilic surface in water through AFM imaging

In this work, a hydrophilic silica plate exposed in air, and immersed in an aqueous solution was studied through atomic force microscopy (AFM) imaging in contact‐ and tapping‐mode operations. It was experimentally found that the tapping‐mode AFM images of the silica surface were different when it was immersed in an aqueous solution from those when it was exposed in air. The former showed fewer topographic features. However, the contact‐mode AFM images of the silica surface were almost uninfluenced by the medium in which the surface was placed. This phenomenon might be attributed to the existence of hydration layers near the silica surface in the aqueous solution. The layers are like a large sheet on the surface that hides the details, so that an AFM tip in the tapping mode can read only the hydration layer and therefore image only the rough outline of the surface. This result might suggest the existence of hydration layers near a hydrophilic surface immersed in water. Copyright © 2006 John Wiley & Sons, Ltd.     

Photochemically initiated single polymer immobilization

This Concept article surveys methods for attaching single polymer molecules on solid substrates. A general approach to single polymer immobilization based on the photochemistry of perfluorophenylazides is elaborated.     

Characterization of Collagen/Lipid Nanoparticle–Curcumin Cryostructurates for Wound Healing Applications

Curcumin‐loaded collagen cryostructurates have been devised for wound healing applications. Curcumin displays strong antioxidant, antiseptic, and anti‐inflammatory properties, while collagen is acknowledged for promoting cell adhesion, migration and differentiation. However, when curcumin is loaded directly into collagen hydrogels, it forms large molecular aggregates and clogs the matrix pores. A double‐encapsulation strategy is therefore developed by loading curcumin into lipid nanoparticles (LNP), and embedding these particles inside collagen scaffolds. The resulting collagen/LNP cryostructurates have an optimal fibrous structure with ≈100 µm average pore size for sustaining cell migration. Results show that collagen is structurally unaltered and that nanoparticles are homogeneously distributed amidst collagen fibers. Hydrogels soaked in saline buffer release about 20 to 30% of their nanoparticles content within 24 h, while achieved 100% release after 25 days. When exposed to NIH 3T3 fibroblasts, these hydrogels provide a satisfactory scaffold for cell interaction as early as 4 h after seeding, with no cytotoxic counter effect. These positive features make the collagen/lipid cryostructurates a promising material for further use in wound healing.     

Nanoprobes for super-resolution fluorescence imaging at the nanoscale

Compared with other imaging techniques,fluorescence microscopy has become an essential tool to study cell biology due to its high compatibility with living cells.Owing to the resolution limit set by the diffraction of light,fluorescence microscopy could not resolve the nanostructures in the range of<200 nm.Recently,many techniques have been emerged to overcome the diffraction barrier,providing nanometer spatial resolution.In the course of development,the progress in fluorescent probes has helped to promote the development of the high-resolution fluorescence nanoscopy.Here,we describe the contributions of the fluorescent probes to far-field super resolution imaging,focusing on concepts of the existing super-resolution nanoscopy based on the photophysics of fluorescent nanoprobes,like photoswitching,bleaching and blinking.Fluorescent probe technology is crucial in the design and implementation of super-resolution imaging methods.     

Molecular imaging of membrane proteins and microfilaments using atomic force microscopy

Atomic force microscopy (AFM) is an emerging technique for a variety of uses involving the analysis of cells. AFM is widely applied to obtain information about both cellular structural and subcellular events. In particular, a variety of investigations into membrane proteins and microfilaments were performed with AFM. Here, we introduce applications of AFM to molecular imaging of membrane proteins, and various approaches for observation and identification of intracellular microfilaments at the molecular level. These approaches can contribute to many applications of AFM in cell imaging.     

When tethered chains meet free ones; the stability of polymer wetting films on polymer brushes

We present a combined experimental and theoretical self-consistent field (SCF) investigation of the wetting behavior of a polystyrene melt (composed of chains with degree of polymerization P) on top of a polystyrene brush (composed of chains with length N) grafted onto a silica surface. The control variables are the grafting density σ of the brush chains and the length of mobile chains P. Experiments show in agreement with the theory that there is a window of complete wetting. Both at very low and at high grafting densities the system remains partial wet. At large degree of polymerization P, there is a difference between the experimental and theoretical results. Theory predicts partial wetting only, whereas the window of complete wetting persists in the experiments even when P >> N. This difference is attributed to the double-well structure of the disjoining pressure as revealed by the SCF theory. With this type of disjoining pressure it is conceivable that a metastable zero contact angle remains present for very long times.     

Model experiments for a molecular understanding of polymer crystallization

Time‐resolved real‐space observations of morphology and pattern formation resulting from crystallization of ultrathin films of low‐molecular‐weight poly(ethylene oxide) (PEO) or diblock copolymers containing PEO shed light on the mechanisms of how polymer crystals are formed. We used simple but restricted geometries like thin films of controlled thickness or confinement resulting from block copolymer mesotructures. Under such conditions, we were able to relate the observed morphology and its temporal evolution directly to molecular processes and the kinetics of crystal growth. We demonstrate that changes in the morphology with time are due to different thermal histories and are the consequence of the mestable nature of polymer crystals. Information about the nucleation process was obtained by examining crystal formation in 12‐nm small spherical cells of a block copolymer mesostructure. We discuss the advantages of thin‐film studies for a better understanding of polymer crystallization. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 1869–1877, 2003     

Spectroscopic imaging techniques for characterization of polymer morphologies: a combination of infrared and electron microscopy

The morphological characterization of polymer blends consisting of polyamide and poly(tetrafluoroethylene) using FT-IR spectroscopy and electron microscopy is described. To enhance the lateral resolution - one of the main limits in infrared spectroscopy - a combination with scanning electron microscopy and analytical electron microscopic methods of a transmission electron microscope was made. The possibilities of electron energy loss spectroscopy and energy filtered transmission electron microscopy (EFTEM) in the area of polymer characterization are outlined.     

Simultaneous imaging of the structure and fluorescence of a supramolecular nanostructure formed by the coupling of π‐conjugated polymer chains in the intermolecular interaction

We fabricated a micrometer‐long supramolecular chain in which π‐conjugated polyrotaxane was coupled. A new experimental setup was designed and constructed, and the simultaneous direct imaging of the structure and fluorescent function was achieved. Furthermore, we identified the formation of a polymer intertwined network and observed novel fluorescence due to a long‐range interaction via this intertwined network over a distance of 5 μm or more without quenching over 15 min in the near field. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 801–809, 2006