10.3 Atomic force microscopy and single-molecule techniques
4 min read•august 1, 2024
() lets us peek at tiny biological structures up close. It uses a super-sharp tip to scan surfaces, giving us detailed images and info about biomolecules like proteins and DNA. It's like having a microscopic finger that can touch and feel individual molecules!
take it a step further, letting us watch and manipulate one molecule at a time. This gives us unique insights into how molecules behave, interact, and change shape. It's like studying a single fish instead of the whole school, revealing secrets hidden in the crowd.
Atomic Force Microscopy for Biomolecules
Principles and Applications
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- Atomic force microscopy (AFM) is a high- scanning probe microscopy technique that uses a sharp tip mounted on a cantilever to scan the surface of a sample and measure the interaction forces between the tip and the sample
- AFM can provide topographical images of biological samples with nanometer-scale resolution, allowing for the visualization of individual molecules (proteins), molecular complexes (lipid membranes), and cellular structures
- The AFM tip interacts with the sample surface through various forces, including van der Waals, electrostatic, and capillary forces, which can be measured and used to characterize the surface properties and interactions of biological molecules
- AFM can be used to study the morphology, mechanical properties, and interactions of proteins, nucleic acids, lipid membranes, and other biomolecules under physiological conditions
- The high sensitivity and spatial resolution of AFM enable the investigation of molecular recognition events at the single-molecule level
- Ligand-receptor binding
- Protein-protein interactions
- Enzyme-substrate interactions
Imaging and Force Measurements
- AFM provides high-resolution imaging and force measurements of biological samples under near-physiological conditions, without the need for sample labeling or extensive sample preparation
- AFM can provide quantitative information on the mechanical properties, kinetics, and thermodynamics of biomolecular interactions and conformational changes
- However, AFM experiments often require extensive sample preparation, such as surface immobilization and functionalization, which may affect the native behavior of the molecules
- The temporal resolution of AFM may be limited compared to spectroscopic methods (fluorescence, NMR), which can probe molecular dynamics on faster timescales
- The interpretation of AFM data can be challenging due to the complex nature of the interactions involved and the potential artifacts arising from surface effects and experimental conditions
AFM Operation Modes
Contact and Tapping Modes
- involves maintaining a constant force between the tip and the sample surface while scanning, allowing for high-resolution imaging of hard surfaces but potentially causing damage to soft biological samples
- , also known as intermittent contact mode, oscillates the cantilever near its resonance frequency and maintains a constant oscillation amplitude while scanning
- Minimizes the lateral forces applied to the sample
- Reduces the risk of sample damage
- combines tapping mode imaging with , allowing for the simultaneous acquisition of topographical and mechanical property maps of the sample surface
Force Spectroscopy
- Force spectroscopy is an AFM technique that measures the force-distance relationship between the tip and the sample by approaching and retracting the tip from the surface
- Provides information on the mechanical properties, adhesion forces, and unbinding events of individual molecules
- involves measuring the force-distance curves at different loading rates to study the kinetics and energy landscape of molecular interactions
- Receptor-ligand binding
- Protein unfolding
Single-Molecule Techniques
Manipulation and Observation
- Single-molecule techniques involve the manipulation and observation of individual molecules, allowing for the study of their properties, dynamics, and interactions without the averaging effects inherent in ensemble measurements
- use focused laser beams to trap and manipulate individual molecules, enabling the measurement of forces and displacements in the piconewton and nanometer range, respectively
- use magnetic fields to apply torque and stretching forces to individual molecules tethered between a surface and a magnetic bead
- Allows for the study of DNA topology, protein-DNA interactions, and molecular motors
- Fluorescence-based single-molecule techniques enable the study of conformational dynamics, protein folding, and molecular interactions with high temporal and spatial resolution
- Single-molecule FRET (Förster resonance energy transfer)
- Super-resolution microscopy
Applications
- Atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) can be used to measure the mechanical properties of individual proteins
- Unfolding and refolding forces
- Strength and kinetics of intermolecular interactions
- Single-molecule techniques allow for the direct observation of the heterogeneity and stochastic behavior of individual molecules, revealing rare events and transient intermediates that may be obscured in ensemble measurements
AFM vs Other Biophysical Methods
Advantages
- AFM and single-molecule techniques can provide unique insights into the properties and interactions of individual molecules
- They offer high-resolution imaging, force measurements, and the ability to study biomolecules under near-physiological conditions
- These techniques enable the direct observation of molecular heterogeneity, stochastic behavior, and rare events that may be obscured in ensemble measurements
Limitations
- AFM and single-molecule experiments often require extensive sample preparation, which may affect the native behavior of the molecules
- The temporal resolution of AFM and some single-molecule techniques may be limited compared to spectroscopic methods
- The interpretation of AFM and single-molecule data can be challenging due to the complex nature of the interactions involved and potential artifacts
- These techniques may not capture the collective behavior and emergent properties of biomolecular systems, which can be studied using complementary ensemble-averaged techniques
- X-ray crystallography
- NMR
- Bulk spectroscopy
Key Terms to Review (24)
Afm: Atomic force microscopy (AFM) is a powerful imaging technique used to visualize surfaces at the nanoscale by measuring the interaction forces between a sharp probe and the sample surface. AFM allows researchers to obtain high-resolution images of materials and biological samples, making it essential for studying single molecules and their interactions, as well as contributing to the development of new biophysical techniques.
Atomic Force Microscopy: Atomic Force Microscopy (AFM) is a powerful imaging technique that enables the visualization and manipulation of surfaces at the atomic level using a sharp tip attached to a cantilever. This technique allows researchers to study materials, biological samples, and nanoscale structures with high resolution, contributing significantly to understanding various molecular interactions and topological features.
Conductive AFM: Conductive atomic force microscopy (AFM) is a variation of atomic force microscopy that enables the measurement of electrical properties at the nanoscale. This technique allows researchers to obtain both topographical and electrical information simultaneously, making it particularly valuable for studying conductive materials, organic semiconductors, and nanoscale electronic devices.
Contact mode AFM: Contact mode AFM, or atomic force microscopy, is a scanning probe technique where the probe tip makes continuous contact with the sample surface to generate high-resolution images. This mode allows for detailed topographic mapping of surfaces at the nanoscale, providing information about surface roughness and mechanical properties while exerting a constant force on the sample.
Dna molecules: DNA molecules, or deoxyribonucleic acid, are long strands of nucleotides that carry genetic information essential for the development, functioning, growth, and reproduction of all known living organisms and many viruses. Their structure features a double helix formed by two complementary strands running in opposite directions, held together by base pairs of adenine-thymine and cytosine-guanine. The study of DNA at the single-molecule level using techniques like atomic force microscopy offers insights into its physical properties, dynamics, and interactions with proteins.
Dynamic force spectroscopy: Dynamic force spectroscopy is a technique used to study the mechanical properties and interactions of biomolecules at the single-molecule level by applying a controlled force and measuring the resulting changes in molecular behavior. This method enables researchers to investigate how molecules respond to external forces, providing insights into molecular stability, binding affinities, and conformational changes under tension. It often utilizes atomic force microscopy (AFM) to manipulate and measure forces on individual molecules, making it a powerful tool in the realm of biophysical chemistry.
Force Spectroscopy: Force spectroscopy is a powerful technique used to measure the forces between molecules at the single-molecule level. It allows researchers to explore molecular interactions, binding affinities, and mechanical properties by applying controlled forces to individual molecules. This technique is especially significant for understanding biological processes at a molecular level, such as protein folding, enzyme activity, and receptor-ligand interactions.
Force-volume imaging: Force-volume imaging is a technique used in atomic force microscopy (AFM) to measure the mechanical properties of materials at the nanoscale by combining force spectroscopy with topographical mapping. This method allows researchers to obtain quantitative data on the elasticity and stiffness of samples while also visualizing their surface structure, providing insights into material properties at the single-molecule level.
High-speed afm: High-speed atomic force microscopy (AFM) is a cutting-edge imaging technique that allows for the rapid acquisition of high-resolution images of surfaces at the nanoscale, making it invaluable for observing dynamic processes in real time. This technology enhances traditional AFM by employing advanced scanning mechanisms and faster cantilevers, which significantly reduce the time required to capture images while maintaining high resolution. By enabling researchers to visualize biological and material samples under near-physiological conditions, high-speed AFM bridges the gap between static imaging and dynamic observation.
Hooke's Law: Hooke's Law states that the force exerted by a spring is directly proportional to the displacement of the spring from its equilibrium position, as long as the elastic limit is not exceeded. This principle is crucial in various applications, especially in understanding the mechanical properties of materials and systems, including the behavior of molecules under force in techniques like atomic force microscopy.
Lennard-Jones Potential: The Lennard-Jones potential is a mathematical model that describes the interaction between a pair of neutral atoms or molecules. It captures the balance between attractive and repulsive forces, represented as a function of the distance between the particles. This potential is essential for understanding molecular interactions and is widely used in simulations to explore systems at the atomic level, especially in techniques that involve measuring forces at nanoscale distances.
Magnetic tweezers: Magnetic tweezers are a powerful single-molecule manipulation tool that uses magnetic fields to exert forces on biomolecules, allowing for the study of their mechanical properties and interactions. By attaching magnetic beads to molecules, researchers can apply precise tension and measure responses under varying conditions, making this technique essential for investigating molecular dynamics at the single-molecule level.
Optical Tweezers: Optical tweezers are a powerful technique that uses laser light to trap and manipulate microscopic particles, such as biological molecules or cells, with high precision. This method is essential for studying molecular interactions, providing insights into mechanical properties and dynamic processes in single-molecule biophysics and mechanochemical coupling.
Phase imaging: Phase imaging is a technique used in microscopy that captures the phase shift of light waves as they pass through a sample, revealing information about the sample's structure and composition without needing stains or dyes. This method takes advantage of the interference patterns created by variations in the refractive index of the sample, providing detailed insights into biological samples, including single molecules.
Protein complexes: Protein complexes are assemblies of multiple protein molecules that interact to perform specific biological functions. These structures can vary in size and complexity, often forming through non-covalent interactions such as hydrogen bonds, ionic bonds, and hydrophobic interactions. They play critical roles in cellular processes, including signaling, catalysis, and structural support, making their study essential in understanding molecular biology.
Protein folding studies: Protein folding studies involve the investigation of how proteins acquire their functional three-dimensional structures from linear chains of amino acids. Understanding protein folding is crucial because the structure of a protein determines its function, and misfolded proteins can lead to various diseases. Techniques used in these studies, such as atomic force microscopy and single-molecule biophysics, allow researchers to observe and manipulate the folding process at a molecular level.
Resolution: Resolution refers to the smallest distinguishable detail in an imaging system, crucial for determining the quality and clarity of the images produced. It is a key factor in various analytical techniques, as higher resolution allows for more precise measurements and better visualization of structures at the molecular level, enabling insights into protein conformations, mass-to-charge ratios of biomolecules, and interactions at a nanoscale.
Scanning tunneling microscopy: Scanning tunneling microscopy (STM) is a powerful technique used to visualize surfaces at the atomic level by measuring the tunneling current that occurs between a sharp conducting tip and the surface being examined. This method allows for the observation of individual atoms and the mapping of electronic states, providing insights into surface structures and properties. STM is particularly significant in studying conductive materials and has paved the way for advancements in nanotechnology and single-molecule techniques.
Single-molecule fluorescence: Single-molecule fluorescence is a powerful technique used to study individual molecules by detecting their emitted light when excited by a specific wavelength. This method allows scientists to gain insights into molecular interactions, conformational changes, and dynamics at the single-molecule level, providing a more detailed understanding of biological processes and molecular functions.
Single-molecule techniques: Single-molecule techniques are advanced methods used to study individual molecules, enabling researchers to observe molecular behavior and interactions at an unprecedented resolution. These techniques provide insights into the dynamics of biological processes, such as protein folding and DNA interactions, and have become essential tools in fields like biophysics, molecular biology, and nanotechnology. The ability to analyze single molecules has transformed our understanding of complex biochemical systems and their functionalities.
Surface characterization: Surface characterization refers to a set of analytical techniques used to study and understand the physical and chemical properties of surfaces at the microscopic level. This process is crucial in materials science, biology, and nanotechnology, as it helps determine how surfaces interact with their environment and affects properties like adhesion, catalysis, and biocompatibility.
Tapping mode AFM: Tapping mode AFM, or tapping mode atomic force microscopy, is a scanning technique that allows for high-resolution imaging of surfaces by intermittently tapping the surface with a cantilever tip. This method minimizes the contact between the tip and the sample, reducing damage and enabling the observation of soft materials or biological samples without significant distortion. Tapping mode enhances the resolution of imaging while also providing topographical information, making it particularly useful in biophysical chemistry applications.
Tip-sample interaction: Tip-sample interaction refers to the forces that occur between the sharp tip of a scanning probe microscope and the sample surface during imaging or manipulation. This interaction is crucial for obtaining high-resolution images and understanding molecular behavior at the nanoscale, as it dictates how the tip moves over the surface and how it detects features on the sample.
Topography Mapping: Topography mapping is a technique used to visualize the surface features of a sample at the nanoscale, providing detailed information about the physical structure and morphology. This method is essential in techniques like atomic force microscopy (AFM), where it allows researchers to obtain high-resolution images and understand the properties of materials at the atomic level.