Atomic force microscopy of macromolecular interactions.

The introduction of functional imaging tools and techniques that operate at molecular-length scales has provided investigators with unique approaches to characterizing biomolecular structure and function relationships. Recent advances in the field of scanning probe techniques and, in particular, atomic force microscopy have yielded tantalizing insights into the dynamics of protein self-assembly and the mechanics of protein unfolding.     

Fabrication and characterization of patterned immobilization of quantum dots on metallic nano-gratings

Surfaces featuring nano-structures and biochemical patterns are increasingly developed as novel and superior substrates for biosensors and assays. Metallic periodic nano-structures have been studied for their unique optical properties and in particular their ability to support surface plasmon waves. Here we present a new nano-structuring approach based on gentle metal lift-off process coupled with self-assembled surface chemistry for the fabrication of a zeroth-order 400nm period metallic grating with differentiated surface chemistries on the mesas and troughs. The approach, using terminated self-assembled monolayers, creates versatile functionalized substrates allowing the precise deposition of complex biomolecular structures. We use this technique to perform the guided deposition of a three-dimensional polyelectrolyte multilayer structure and the patterned adsorption of quantum dots. Finally, we demonstrate that scanning near-field optical microscopy, used in conjuncture with atomic force microscopy and scanning electron microscopy, is an ideal tool for the characterization of this nano-structured surface as it provides a complete chemical, topographical and optical image of the surface. This ability to pattern and locally measure the surface properties is likely to have an important impact on the design of novel and optimized biointerfaces and transducers for biosensors.     

Quantification of protein-protein interactions using fluorescence polarization.

Quantitative determinations of the dissociation constants of biomolecular interactions, in particular protein-protein interactions, are essential for a detailed understanding of the molecular basis of their specificities. Fluorescence spectroscopy is particularly well suited for such studies. This article highlights the theoretical and practical aspects of fluorescence polarization and its application to the study of protein-protein interactions. Consideration is given to the nature of the different types of fluorescence probes available and the probe characteristics appropriate for the system under investigation. Several examples from the literature are discussed that illustrate different practical aspects of the technique applied to diverse systems.     

Coordinate-based colocalization analysis of single-molecule localization microscopy data

Colocalization of differently labeled biomolecules is a valuable tool in fluorescence microscopy and can provide information on biomolecular interactions. With the advent of super-resolution microscopy, colocalization analysis is getting closer to molecular resolution, bridging the gap to other technologies such as fluorescence resonance energy transfer. Among these novel microscopic techniques, single-molecule localization-based super-resolution methods offer the advantage of providing single-molecule coordinates that, rather than intensity information, can be used for colocalization analysis. This requires adapting the existing mathematical algorithms for localization microscopy data. Here, we introduce an algorithm for coordinate-based colocalization analysis which is suited for single-molecule super-resolution data. In addition, we present an experimental configuration for simultaneous dual-color imaging together with a robust approach to correct for optical aberrations with an accuracy of a few nanometers. We demonstrate the potential of our approach for cellular structures and for two proteins binding actin filaments.     

Antenna-Attached Parabolic Probe for Excitation and Collection Modes in SNOM

A novel optical fiber probe with a parabola-like shape and a nano-antenna mounted on the center of its endface is proposed for simultaneous excitation and collection modes in scanning near-field optical microscopy. The working principles of the probe are demonstrated, and its optical properties are theoretically investigated and compared with the conventional tip-on-aperture probe. It shows that the probe can greatly boost both the enhancement factor for the excitation mode and the collection efficiency for the collection mode. The proposed probe is a promising tool to realize low-cost and high resolution for a wide variety of near-field measurements in biology, physics, and chemistry.     

Factors affecting protein interaction at sorbent interfaces

Interactions between surfaces and macromolecules are the fundamentals in separation and detection of diverse solutes. In this very brief review the central aspects of protein-surface interactions are discussed with the intention of identifying the important factors influencing such processes and placing them in relation to the established knowledge in this field. Some perspectives of new techniques related to scanning probe microscopy for studying interactions at the nanometer level are also discussed.     

Quantum dots for multiplexed detection and characterisation of prostate cancer cells using a scanning near-field optical microscope

In this study scanning near-field optical microscopy (SNOM) has been utilised in conjunction with quantum dot labelling to interrogate the biomolecular composition of cell membranes. The technique overcomes the limits of optical diffraction found in standard fluorescence microscopy and also yields vital topographic information. The technique has been applied to investigate cell-cell adhesion in human epithelial cells. This has been realised through immunofluorescence labelling of the cell-cell adhesion protein E-cadherin. Moreover, a dual labelling protocol has been optimised to facilitate a comparative study of the adhesion mechanisms and the effect of aberrant adhesion protein expression in both healthy and cancerous epithelial cells. This study reports clear differences in the morphology and phenotype of healthy and cancerous cells. In healthy prostate epithelial cells (PNT2), E-cadherin was predominantly located around the cell periphery and within filopodial extensions. The presence of E-cadherin appeared to be enhanced when cell-cell contact was established. In contrast, examination of metastatic prostate adenocarcinoma cells (PC-3) revealed no E-cadherin labelling around the periphery of the cells. This lack of functional E-cadherin in PC-3 cells coincided with a markedly different morphology and PC-3 cells were not found to form close cell-cell associations with their neighbours. We have demonstrated that with a fully optimised sample preparation methodology, multiplexed quantum dot labelling in conjunction with SNOM imaging can be successfully applied to interrogate biomolecular localisation within delicate cellular membranes.     

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.     

Near-field scanning optical microscopy in cell biology

Near-field optics has produced the highest optical resolution that has ever been achieved. The methods involved lie at the interface of far-field optical microscopy and scanned probe microscopy. This article describes the principles behind near-field scanning optical microscopy (NSOM) and highlights its potential in cell biology.     

A regenerable flow-through affinity sensor for label-free detection of proteins and DNA

Label-free monitoring of biomolecular reactions in real-time is of great interest since it can provide valuable information about binding kinetics and equilibrium constants. In this report, a sensor based on White Light Reflectance Spectroscopy (WLRS) is presented that is capable of real-time monitoring of biomolecular reactions taking place on top of a polymer covered silicon dioxide reflective surface. The optical set-up consists of a visible–near infrared light source, a bifurcated optical fiber and a spectrometer. The outer part of the optical fiber guides the light vertically onto the surface where the biomolecular reactions occur, whereas the reflected light is driven from the central part of the fiber to the spectrometer. A microfluidic module in combination with a pump supplies the reagents at a constant rate. The biomolecular interactions are monitored as shifts of the wavelength of the interference minimum. The proposed methodology was applied for real-time and label-free monitoring mouse gamma-globulins binding onto immobilized anti-mouse IgG antibody. Mouse gamma-globulins at concentrations down to 150 pM were detected in reaction times of 1-min. Regeneration of immobilized antibody was accomplished up to seven times without loss of its activity. In addition, real-time monitoring of hybridization reaction between complementary oligonucleotides was accomplished. The proposed sensor provides a simple, fast, low cost approach for label-free monitoring of biomolecular interactions and therefore it should by suitable for a wide range of analytical applications.     

Exploring biomolecular recognition using optical biosensors.

Understanding the basic forces that determine molecular recognition helps to elucidate mechanisms of biological processes and facilitates discovery of innovative biotechnological methods and materials for therapeutics, diagnostics, and separation science. The ability to measure interaction properties of biological macromolecules quantitatively across a wide range of affinity, size, and purity is a growing need of studies aimed at characterizing biomolecular interactions and the structural elements that drive them. Optical biosensors have provided an increasingly impactful technology for such biomolecular interaction analyses. These biosensors record the binding and dissociation of macromolecules in real time by transducing the accumulation of mass of an analyte molecule at the sensor surface coated with ligand molecule into an optical signal. Interactions of analytes and ligands can be analyzed at a microscale and without the need to label either interactant. Sensors enable the detection of bimolecular interaction as well as multimolecular assembly. Most notably, the method is quantitative and kinetic, enabling determination of both steady-state and dynamic parameters of interaction. This article describes the basic methodology of optical biosensors and presents several examples of its use to investigate such biomolecular systems as cytokine growth factor-receptor recognition, coagulation factor assembly, and virus-cell docking.     

High-speed atomic force microscopy and its future prospects

Various techniques have been developed and used to investigate how proteins produce complex biological architectures and phenomena. Among these techniques, high-speed atomic force microscopy (HS-AFM) holds a unique position. It is only HS-AFM that allows the simultaneous assessment of structure and dynamics of single protein molecules in action. This new microscopy tool has been successfully applied to a variety of proteins, from motor proteins to membrane proteins, antibodies, enzymes, and even to intrinsically disordered proteins. And yet there still remain many biomolecular phenomena that cannot be addressed by HS-AFM in its current form. Here, I present a brief history of HS-AFM development, describe the current state of HS-AFM, and then discuss which new biological scanning probe microscopy techniques will be coming up next.     

Imaging today's infectious animalcules

The study of pathogens and their interactions with host cells has advanced hand-in-hand with developments in optical microscopy. Whereas microbiology benefits enormously from modern imaging technologies, for example, digital imaging and confocal microscopy, it also presents unique challenges. To overcome these, microbiologists are adept at customising imaging methods, and recently there have been studies using state-of-the-art quantitative imaging methods to probe host-pathogen interactions at the single-cell level. Of particular interest are the studies using combined light and electron microscopy methods, bi-arsenical tetra-cysteine tag labelling and automated image-acquisition and analysis for high-throughput/high-content experimentation. These applications demonstrate how imaging methodologies, adapted for microbiology, continue to open avenues for studies that previously have proven inaccessible.     

Electrospray ionization mass spectrometry in the study of biomolecular non-covalent interactions

In the past mass spectrometry has been limited to the study of small, stable molecules, however, with the emergence of electrospray ionization mass spectrometry (ESI-MS) large biomolecules as well as non-covalent biomolecular complexes can be studied. ESI-MS has been used to study non-covalent interactions involving proteins with metals, ligands, peptides, oligonucleotides, as well as other proteins. Although complementary to other well-established techniques such as circular dichroism and fluorescence spectroscopy, ESI-MS offers some advantages in speed, sensitivity, and directness particularly in the determination of the stoichiometry of the complex. One major advantage is the ability of ESI-MS to provide multiple signals each arising from a distinct population within the sample. In this review I will discuss some of the different types of non-covalent biomolecular interactions that have been studied using ESI-MS, highlighting examples which show the efficacy of using ESI-MS to probe the structure of biomolecular complexes.     

Observations on the internal and surface morphology of malaria infected blood cells using optical and atomic force microscopy

We describe a simple and fast method to probe the morphological changes on the exterior and interior of a malaria infected erythrocyte at different stages of parasite development. This involves the imaging and scanning of Giemsa stained malaria infected erythrocytes using optical microscopy and atomic force microscopy, respectively.     

Data-driven docking for the study of biomolecular complexes

With the amount of genetic information available, a lot of attention has focused on systems biology, in particular biomolecular interactions. Considering the huge number of such interactions, and their often weak and transient nature, conventional experimental methods such as X-ray crystallography and NMR spectroscopy are not sufficient to gain structural insight into these. A wealth of biochemical and/or biophysical data can, however, readily be obtained for biomolecular complexes. Combining these data with docking (the process of modeling the 3D structure of a complex from its known constituents) should provide valuable structural information and complement the classical structural methods. In this review we discuss and illustrate the various sources of data that can be used to map interactions and their combination with docking methods to generate structural models of the complexes. Finally a perspective on the future of this kind of approach is given.     

Principle and Application of Tip-enhanced Raman Scattering

Plasmonics - Tip-enhanced Raman scattering (TERS), as a combination of scanning probe microscopy (SPM) and surface-enhanced Raman spectroscopy (SERS) makes a huge progress in high sensitive optical...     

Self-assembled monolayer coating of biological probes to avoid protein adhesion

Probes designed to locally illuminate structures within plant cells are described. The probes studied are etch-sharpened single mode optical fibers, coated with aluminum, similar to probes used for near-field scanning optical microscopy. We find that cellular material adheres to the probes that are not coated with a self-assembled monolayer octadecyltrichlorosilane. The hydrophobic monolayer coating enabled these probes to be inserted into and removed from plant cells with no protein adhesion to the probes. This allows probe reinsertion and it causes less damage to the target cell, greatly facilitating in vivo optical study of cells.