In situ scanning probe microscopy studies of tetanus toxin-membrane interactions

Despite the considerable information available with regards to the structure of the clostridial neurotoxins, and their inherent threat as biological warfare agents, the mechanisms underpinning their interactions with and translocation through the cell membrane remain poorly understood. We report herein the results of an in situ scanning probe microscopy study of the interaction of tetanus toxin C-fragment (Tet C) with supported planar lipid bilayers containing the ganglioside receptor G(T1b). Our results show that Tet C preferentially binds to the surface of fluid phase domains within biphasic membranes containing G(T1b) and that with an extended incubation period these interactions lead to dramatic changes in the morphology of the lipid bilayer, including the formation of 40-80 nm diameter circular cavities. Combined atomic force microscopy/total internal reflection fluorescence microscopy experiments confirmed the presence of Tet C in the membrane after extended incubation. These morphological changes were found to be dependent upon the presence of G(T1b) and the solution pH.     

Imaging molecular interactions in living cells by FRET microscopy

F?rster resonance energy transfer (FRET) is applied extensively in all fields of biological research and technology, generally as a 'nanoruler' with a dynamic range corresponding to the intramolecular and intermolecular distances characterizing the molecular structures that regulate cellular function. The complex underlying network of interactions reflects elementary reactions operating under strict spatio-temporal control: binding, conformational transition, covalent modification and transport. FRET imaging provides information about all these molecular processes with high specificity and sensitivity via probes expressed by or introduced from the external medium into the cell, tissue or organism. Current approaches and developments in the field are discussed with emphasis on formalism, probes and technical implementation.     

The importance of molecular structure and conformation: learning with scanning probe microscopy

Molecular structure holds a key to understanding Nature's intricate design mechanisms and blueprints. If we can understand her blueprints and basic materials, perhaps we can begin to mimic her beautiful products more cost effectively and with less detrimental environmental consequences. Higher resolution instrumentation has allowed us to study single molecules. Indeed, many stellar contributions to the field have come forth in the last couple of years. We can measure the forces required to unravel individual domains of biological molecules such as titin or DNA to a few picoNewtons resolution. This review will attempt to provide a general overview of the field of single molecule analysis using scanning force microscopy.     

Potentiostatic deposition of DNA for scanning probe microscopy.

We describe a procedure for reversible adsorption of DNA onto a gold electrode maintained under potential control. The adsorbate can be imaged by scanning probe microscopy in situ. Quantitative control of a molecular adsorbate for microscopy is now possible. We found a potential window (between 0 and 180 mV versus a silver wire quasi reference) over which a gold (111) surface under phosphate buffer is positively charged, but is not covered with a dense adsorbate. When DNA is present in these conditions, molecules adsorb onto the electrode and remain stable under repeated scanning with a scanning tunneling microscope (STM). They become removed when the surface is brought to a negative charge. When operated at tunnel currents below approximately 0.4 nA, the STM yields a resolution of approximately 1 nm, which is better than can be obtained with atomic force microscopy (AFM) at present. We illustrate this procedure by imaging a series of DNA molecules made by ligating a 21 base-pair oligonucleotide. We observed the expected series of fragment lengths but small fragments are adsorbed preferentially.     

Three-dimensional FRET reconstruction microscopy for analysis of dynamic molecular interactions in live cells

Analysis of cellular pathways requires concentration measurements of dynamically interacting molecules within the three-dimensional (3D) space of single living cells. Förster resonance energy transfer (FRET) microscopy from widefield, from confocal, and potentially from superresolution microscopes can access this information; however, these measurements are distorted by the inherent 3D blurring of optical imaging, spectral overlap of fluorophores, and detection noise. We propose a mathematical model of these processes and demonstrate, through simulation, how these distortions limit the dynamic range and sensitivity of conventional FRET microscopy. Using this model, we devise and validate a new approach (called 3D-FRET stoichiometry reconstruction, 3DFSR) for reconstructing 3D distributions of bound and free fluorescent molecules. Previous attempts to reconstruct 3D-FRET data relied on sequential spectral unmixing and deconvolution, a process that corrupts the detection statistics. We demonstrate that 3DFSR is superior to these approaches since it simultaneously models spectral mixing, optical blurring, and detection noise. To achieve the full potential of this technique, we developed an instrument capable of acquiring 3D-FRET data rapidly and sensitively from single living cells. Compared with conventional FRET microscopy, our 3D-FRET reconstruction technique and new instrumentation provides orders of magnitude gains in both sensitivity and accuracy wherein sustained high-resolution four-dimensional (x,y,z,t) imaging of molecular interactions inside living cells was achieved. These results verify previous observations that Cdc42 signaling is localized to the advancing margins of forming phagosomes in macrophages.     

Electromechanical imaging of biomaterials by scanning probe microscopy

The majority of calcified and connective tissues possess complex hierarchical structure spanning the length scales from nanometers to millimeters. Understanding the biological functionality of these materials requires reliable methods for structural imaging on the nanoscale. Here, we demonstrate an approach for electromechanical imaging of the structure of biological samples on the length scales from tens of microns to nanometers using piezoresponse force microscopy (PFM), which utilizes the intrinsic piezoelectricity of biopolymers such as proteins and polysaccharides as the basis for high-resolution imaging. Nanostructural imaging of a variety of protein-based materials, including tooth, antler, and cartilage, is demonstrated. Visualization of protein fibrils with sub-10nm spatial resolution in a human tooth is achieved. Given the near-ubiquitous presence of piezoelectricity in biological systems, PFM is suggested as a versatile tool for micro- and nanostructural imaging in both connective and calcified tissues.     

Combined scanning probe and total internal reflection fluorescence microscopy

Combining scanning probe and optical microscopy represents a powerful approach for investigating structure-function relationships and dynamics of biomolecules and biomolecular assemblies, often in situ and in real-time. This platform technology allows us to obtain three-dimensional images of individual molecules with nanometer resolution, while simultaneously characterizing their structure and interactions though complementary techniques such as optical microscopy and spectroscopy. We describe herein the practical strategies for the coupling of scanning probe and total internal reflection fluorescence microscopy along with challenges and the potential applications of such platforms, with a particular focus on their application to the study of biomolecular interactions at membrane surfaces.     

Plant cell wall characterization using scanning probe microscopy techniques

Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy.     

Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy

Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.     

High-resolution imaging of proteins in human teeth by scanning probe microscopy

High-resolution studies of dental tissues are of considerable interest for biomedical engineering and clinical applications. In this paper, we demonstrate the application of piezoresponse force microscopy (PFM) to nanoscale imaging of internal structure of human teeth by monitoring the local mechanical response to an electrical bias applied via a conductive tip. It is shown that PFM is capable of detecting dissimilar components of dental tissues, namely, proteins and calcified matrix, which have resembling morphology but different piezoelectric properties. It is demonstrated that collagen fibrils revealed in chemically treated intertubular dentin exhibit high piezoelectric activity and can be visualized in PFM with spatial resolution of 10 nm. Evidence of the presence of protein inclusions of 100-200 nm wide and several micrometers long in tooth enamel has been obtained. Furthermore, it is found that the peritubular dentin and intertubular dentin exhibit different piezoelectric behavior suggesting different concentration of collagen fibrils. The obtained results demonstrate a high potential of PFM in providing an additional insight into the structure of dental tissues. It is suggested that the PFM approach can be used to study the structure of a wide range of biological materials by monitoring their electromechanical behavior at the nanoscale.     

A review of scanning probe microscopy investigations of liposome-DNA complexes

Liposome-DNA complexes are one of the most promising systems for the protection and delivery of nucleic acids to combat neoplastic, viral, and genetic diseases. In addition, they are being used as models in the elucidation of many biological phenomena such as viral infection and transduction. In order to understand these phenomena and to realize the mechanism of nucleic acid transfer by liposome-DNA complexes, studies at the molecular level are required. To this end, scanning probe microscopy (SPM) is increasingly being used in the characterization of lipid layers, lipid aggregates, liposomes, and their complexes with nucleic acid molecules. The most attractive attributes of SPM are the potential to image samples with subnanometer spatial resolution under physiological conditions and provide information on their physical and mechanical properties. This review describes the application of scanning tunneling microscopy and atomic force microscopy, the two most commonly applied SPM techniques, in the characterisation of liposome-DNA complexes.