Biomolecular imaging using atomic force microscopy
TL;DR: Atomic force microscopy will become an increasingly important tool for probing both the structural and kinetic properties of biological macromolecules, as well as studying the structure–function relationships of proteins and their functionally relevant assemblies.
About: This article is published in Trends in Biotechnology.The article was published on 2002-08-01. It has received 51 citations till now. The article focuses on the topics: Single Molecule Imaging.
Citations
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TL;DR: This review discusses single-molecule experiments (SMEs) in biological physics from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied.
Abstract: I review single-molecule experiments (SMEs) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SMEs it is possible to manipulate molecules one at a time and measure distributions describing molecular properties, characterize the kinetics of biomolecular reactions and detect molecular intermediates. SMEs provide additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SMEs it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level, emphasizing the importance of SMEs to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SMEs from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOTs), magnetic tweezers (MTs), biomembrane force probes (BFPs) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation) and proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SMEs to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.
386 citations
TL;DR: AFM is progressively becoming a usual benchtop technique and overcomes materials science applications, showing that 17 years after its invention, AFM has completely crossed the limits of its traditional areas of application.
246 citations
TL;DR: In this article, a new feedback controller and a compensator for drift in the cantilever-excitation efficiency were proposed to enable high-speed imaging of fragile biomolecular systems.
Abstract: In tapping mode atomic force microscopy, the cantilever tip intermittently taps the sample as the tip scans over the surface. This mode is suitable for imaging fragile samples such as biological macromolecules, because vertical oscillation of the cantilever reduces lateral forces between the tip and sample. However, the tapping force (vertical force) is not necessarily weak enough for delicate samples, particularly for biomolecular systems containing weak inter- or intramolecular interactions. Light tapping requires an amplitude set point (i.e., a constant cantilever amplitude to be maintained during scanning) to be set very close to its free oscillation amplitude. However, this requirement does not reconcile with fast scans, because, with such a set point, the tip may easily be removed from the surface completely. This article presents two devices to overcome this difficulty; a new feedback controller (named as “dynamic proportional-integral-differential controller”) and a compensator for drift in the cantilever-excitation efficiency. Together with other devices optimized for fast scan, these devices enable high-speed imaging of fragile samples.
184 citations
TL;DR: All three methods of aminofunctionalization were found fully satisfactory for attachment of single antibodies to AFM tips, only in a parallel macroscopic assay on silicon nitride chips a minor difference was found in that APTES appeared to yield a slightly lower surface density of amino groups.
178 citations
TL;DR: The high-resolution AFM topographs suggest that, in future studies, data revealed under various physiological conditions will provide novel insights into molecular mechanisms that drive membrane protein assembly and supply excellent boundary conditions to model protein-protein arrangements.
74 citations
References
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TL;DR: Images of mica demonstrate that atomic resolution is possible on rigid materials, thus opening the possibility of atomic-scale corrosion experiments on nonconductors and showing the potential of the AFM for revealing the structure of molecules important in biology and medicine.
Abstract: The atomic force microscope (AFM) can be used to image the surface of both conductors and nonconductors even if they are covered with water or aqueous solutions. An AFM was used that combines microfabricated cantilevers with a previously described optical lever system to monitor deflection. Images of mica demonstrate that atomic resolution is possible on rigid materials, thus opening the possibility of atomic-scale corrosion experiments on nonconductors. Images of polyalanine, an amino acid polymer, show the potential of the AFM for revealing the structure of molecules important in biology and medicine. Finally, a series of ten images of the polymerization of fibrin, the basic component of blood clots, illustrate the potential of the AFM for revealing subtle details of biological processes as they occur in real time.
965 citations
TL;DR: In this paper, a high-speed scanner, free of resonant vibrations up to 60 kHz, small cantilevers with high resonance frequencies (450-650 kHz) and small spring constants (150-280 pN/nm), and several electronic devices of wide bandwidth are presented.
Abstract: The atomic force microscope (AFM) is a powerful tool for imaging individual biological molecules attached to a substrate and placed in aqueous solution. At present, however, it is limited by the speed at which it can successively record highly resolved images. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behavior of biomolecules. For this purpose, we have developed a high-speed scanner, free of resonant vibrations up to 60 kHz, small cantilevers with high resonance frequencies (450–650 kHz) and small spring constants (150–280 pN/nm), an objective-lens type of deflection detection device, and several electronic devices of wide bandwidth. Integration of these various devices has produced an AFM that can capture a 100 × 100 pixel2 image within 80 ms and therefore can generate a movie consisting of many successive images (80-ms intervals) of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica (see http://www.s.kanazawa-u.ac.jp/phys/biophys/bmv_movie.htm).
958 citations
TL;DR: The unique capability of the AFM to directly observe single proteins in their native environments provides insights into the interactions of proteins that form functional assemblies and provides unprecedented possibilities for analyzing intramolecular and intermolecular forces.
Abstract: Progress in the application of the atomic force microscope (AFM) to imaging and manipulating biomolecules is the result of improved instrumentation, sample preparation methods and image acquisition conditions. Biological membranes can be imaged in their native state at a lateral resolution of 0.5-1 nm and a vertical resolution of 0. 1-0.2 nm. Conformational changes that are related to functions can be resolved to a similar resolution, complementing atomic structure data acquired by other methods. The unique capability of the AFM to directly observe single proteins in their native environments provides insights into the interactions of proteins that form functional assemblies. In addition, single molecule force spectroscopy combined with single molecule imaging provides unprecedented possibilities for analyzing intramolecular and intermolecular forces. This review discusses recent examples that illustrate the power of AFM.
533 citations
TL;DR: This work has imaged the ATP synthase from leaf chloroplasts by using atomic force microscopy and, surprisingly, finds that its turbine has 14 subunits, arranged in a cylindrical ring.
Abstract: ATP synthases are enzymes that can work in two directions to catalyse either the synthesis or breakdown of ATP, and they constitute the smallest rotary motors in biology The flow of protons propels the rotation1 of a membrane-spanning complex of identical protein subunits, the number of which determines the efficiency of energy conversion This proton-powered turbine is predicted to consist of 12 subunits2,3,4, based on data for Escherichia coli5 The yeast mitochondrial enzyme, however, has only 10 subunits6 We have imaged the ATP synthase from leaf chloroplasts by using atomic force microscopy and, surprisingly, find that its turbine has 14 subunits, arranged in a cylindrical ring
443 citations
TL;DR: In this article, a simple process was used to fabricate small rectangular cantilevers out of silicon nitride, with lengths of 9-50 μm, widths of 3-5 μm and thickness of 86 and 102 nm.
Abstract: We have used a simple process to fabricate small rectangular cantilevers out of silicon nitride. They have lengths of 9–50 μm, widths of 3–5 μm, and thicknesses of 86 and 102 nm. We have added metallic reflector pads to some of the cantilever ends to maximize reflectivity while minimizing sensitivity to temperature changes. We have characterized small cantilevers through their thermal spectra and show that they can measure smaller forces than larger cantilevers with the same spring constant because they have lower coefficients of viscous damping. Finally, we show that small cantilevers can be used for experiments requiring large measurement bandwidths, and have used them to unfold single titin molecules over an order of magnitude faster than previously reported with conventional cantilevers.
401 citations