14.2 Atomic force microscopy and applications
6 min read•august 1, 2024
Atomic force microscopy (AFM) is a powerful tool for studying biological systems at the nanoscale. It uses a tiny tip to scan surfaces, creating detailed 3D maps of molecules and cells. AFM's high resolution and ability to work in various environments make it invaluable for biophysics research.
AFM shines in single-molecule studies, allowing scientists to observe and manipulate individual biomolecules. It can measure forces between molecules, track protein folding, and even watch enzymes at work. This technique bridges the gap between molecular interactions and larger-scale biological phenomena.
Atomic Force Microscopy Principles
Basic Components and Operation
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- Atomic force microscopy (AFM) is a high-resolution scanning probe microscopy technique that uses a sharp tip mounted on a to scan and map the surface topography of a sample at the nanoscale level
- The basic components of an AFM include:
- A sharp tip (typically made of silicon or silicon nitride)
- A cantilever
- A laser beam deflection system
- A photodetector
- A piezoelectric scanner
- A feedback control system
- The AFM tip interacts with the sample surface through various forces, such as van der Waals, electrostatic, and capillary forces, which cause the cantilever to deflect as it scans across the surface
- The deflection of the cantilever is measured by a laser beam that is reflected off the back of the cantilever onto a photodetector, which converts the deflection into an electrical signal
Nanoscale Imaging and Feedback Control
- The piezoelectric scanner precisely controls the movement of the sample or the tip in three dimensions (x, y, and z) to generate a high-resolution topographical map of the sample surface
- The feedback control system maintains a constant force or distance between the tip and the sample surface by adjusting the z-position of the scanner in response to changes in the cantilever deflection signal
- This feedback loop enables the AFM to maintain a consistent interaction between the tip and the sample, ensuring high-resolution imaging and minimizing sample damage
- The resulting topographical map provides detailed information about the surface features and properties of the sample at the nanoscale level ( of a few nanometers and vertical resolution of sub-nanometer)
AFM Modes and Applications in Biophysics
Imaging Modes for Biological Samples
- AFM is the most common mode of operation, where the tip is in constant contact with the sample surface, and the cantilever deflection is used to map the surface topography
- Suitable for imaging hard, stable samples but may cause damage to soft biological samples
- AFM (also known as intermittent contact mode) is a gentler imaging mode where the cantilever oscillates near its resonance frequency, and the tip intermittently contacts the sample surface
- Reduces lateral forces and minimizes sample damage, making it ideal for imaging delicate biological samples (proteins, DNA, membranes)
- Non-contact mode AFM is a mode where the tip oscillates above the sample surface without making contact, and the changes in the oscillation amplitude or frequency due to long-range forces are used to map the surface topography
- Suitable for imaging very soft or easily deformable biological samples (lipid bilayers, hydrogels)
Advanced AFM Techniques and Applications
- Force spectroscopy is an AFM technique that measures the force-distance relationship between the tip and the sample surface, providing information on the mechanical properties, adhesion forces, and molecular interactions of biological samples
- Can quantify the elasticity of cells, the unfolding of proteins, and the binding strength of receptor-ligand interactions
- is a recent development that enables the imaging of dynamic biological processes, such as conformational changes in proteins and the movement of molecular motors, with a temporal resolution of milliseconds to seconds
- Allows the real-time observation of enzymatic reactions, protein folding/unfolding, and membrane dynamics
- AFM can be combined with other techniques, such as fluorescence microscopy and Raman spectroscopy, to provide complementary information on the chemical composition and functional properties of biological samples
- Correlative AFM-fluorescence imaging can map the distribution of specific molecules or organelles within a cell
- AFM-Raman spectroscopy can identify the chemical fingerprint of individual molecules or nanostructures on a sample surface
Interpreting AFM Images of Biological Samples
Topographical Information and Quantitative Analysis
- AFM images provide high-resolution topographical maps of biological samples, with a lateral resolution of a few nanometers and a vertical resolution of sub-nanometer
- The height information in AFM images is represented by a color scale, with brighter colors indicating higher features and darker colors indicating lower features
- The shape, size, and distribution of biological structures, such as proteins, nucleic acids, and membranes, can be directly visualized and quantified from AFM images
- Measuring the dimensions of individual molecules or complexes
- Determining the packing arrangement or periodicity of molecular assemblies
- Surface roughness and texture of biological samples can be characterized by analyzing the height variations and spatial frequencies in AFM images using statistical parameters, such as root mean square (RMS) roughness and power spectral density (PSD)
- Quantifying the surface properties of cell membranes or extracellular matrices
Molecular Interactions and Artifact Identification
- Molecular-level interactions, such as the formation of protein complexes or the binding of ligands to receptors, can be studied by comparing AFM images of samples before and after the interaction
- Observing the changes in the size, shape, or arrangement of molecules upon complex formation or ligand binding
- Artifacts in AFM images, such as tip convolution effects, feedback instabilities, and sample deformation, should be carefully identified and minimized to ensure accurate interpretation of the biological features
- Tip convolution can broaden the apparent size of small features due to the finite size and shape of the AFM tip
- Feedback instabilities can cause streaks or distortions in the image due to improper tracking of the sample surface
- Sample deformation can occur when the tip applies excessive force on soft or delicate biological samples, leading to compressed or altered structures
AFM Advantages vs Limitations in Biological Studies
Advantages of AFM in Biophysics
- High spatial resolution: AFM provides nanometer-scale resolution, enabling the visualization of individual molecules and molecular complexes
- Resolving the subunit arrangement of protein assemblies or the helical structure of DNA
- Non-destructive imaging: AFM can image biological samples in their native state without the need for staining, labeling, or fixation, preserving their structural and functional integrity
- Studying the surface morphology of living cells or the conformation of membrane proteins in their native environment
- Versatile sample environment: AFM can operate in various environments, including air, liquid, and controlled temperature and humidity conditions, allowing the study of biological processes under physiologically relevant conditions
- Imaging the dynamics of proteins or cells in aqueous buffers or culture media
- Mechanical property measurement: AFM force spectroscopy can quantify the mechanical properties of biological samples, such as elasticity, adhesion, and molecular interactions, providing insights into their structure-function relationships
- Measuring the stiffness of different regions within a cell or the unbinding force of receptor-ligand pairs
Limitations of AFM in Biophysics
- Limited scan size and speed: AFM has a relatively small scan size (typically less than 100 μm) and slow imaging speed compared to other microscopy techniques, which may limit its application in studying large-scale biological structures or fast dynamic processes
- Difficulty in imaging whole cells or tissue sections with high throughput
- Sample preparation: AFM requires the sample to be immobilized on a flat substrate, which may involve complex sample preparation procedures and potentially introduce artifacts or alter the native state of the biological system
- Need for surface functionalization or chemical fixation to anchor molecules or cells onto the substrate
- Tip-sample interactions: The physical interaction between the AFM tip and the sample may cause deformation, damage, or artifacts, especially for soft or delicate biological samples, requiring careful optimization of imaging parameters and tip selection
- Soft samples like lipid bilayers or hydrogels can be easily deformed or disrupted by the tip force
- Interpretation of images: AFM images can be affected by various factors, such as tip geometry, sample properties, and imaging conditions, requiring expertise in image analysis and interpretation to extract accurate biological information
- Distinguishing between true biological features and imaging artifacts requires knowledge of the sample and the limitations of the technique
- Limited chemical specificity: AFM provides primarily topographical information and lacks inherent chemical specificity, requiring complementary techniques, such as fluorescence or spectroscopy, to obtain chemical or functional information about the biological sample
- Unable to directly identify the composition or activity of molecules or structures without additional labeling or spectroscopic methods
Key Terms to Review (17)
AFM vs. Electron Microscopy: Atomic Force Microscopy (AFM) and Electron Microscopy (EM) are two distinct techniques used to visualize samples at the nanoscale. AFM employs a cantilever with a sharp tip to scan the surface of a sample, measuring forces between the tip and the surface, allowing for topographical mapping in three dimensions. In contrast, EM utilizes a beam of electrons to illuminate a sample, providing high-resolution images by detecting secondary electrons emitted from the specimen. Each method has unique applications, strengths, and limitations that are critical for different areas of research.
Biomolecule imaging: Biomolecule imaging refers to techniques and methods used to visualize and analyze biological molecules, such as proteins, nucleic acids, and lipids, within various environments. This field is crucial for understanding the structure, function, and interactions of biomolecules in biological systems, enabling researchers to gain insights into cellular processes and disease mechanisms.
Cantilever: A cantilever is a structural element that is anchored at one end while the other end extends freely into space. This design is crucial in many applications, particularly in atomic force microscopy, where cantilevers are used to measure forces at the nanoscale by interacting with surfaces and biomolecules.
Contact mode: Contact mode is a technique used in atomic force microscopy (AFM) where the sharp tip of the probe maintains a continuous contact with the surface being studied. This method allows for high-resolution imaging of the surface topography by measuring the interaction forces between the tip and the sample, providing valuable information about the material properties at the nanoscale.
Force-Distance Curves: Force-distance curves are graphical representations that show the relationship between the force applied to a probe and the distance it moves towards a sample surface, often used in atomic force microscopy (AFM). These curves provide crucial insights into the mechanical properties of materials at the nanoscale, including adhesion, elasticity, and surface roughness.
Functionalized Tips: Functionalized tips refer to specialized probe tips used in atomic force microscopy (AFM) that have been chemically modified to enhance their interactions with specific molecules or surfaces. By altering the properties of these tips, researchers can obtain detailed information about the mechanical, electrical, and chemical characteristics of materials at the nanoscale, making them crucial for various applications in biophysics and material science.
High-speed AFM: High-speed atomic force microscopy (AFM) is an advanced imaging technique that enables real-time observation of dynamic processes at the nanoscale. By significantly increasing the scanning speed compared to traditional AFM, this technology allows scientists to capture rapid biological and physical events, making it invaluable for studying surface interactions and molecular dynamics.
Image processing algorithms: Image processing algorithms are computational methods used to enhance, analyze, or manipulate images for various purposes, including improving image quality and extracting meaningful information. These algorithms play a crucial role in refining the data obtained from microscopy techniques, allowing for clearer visualization and better interpretation of complex biological structures. They are essential for advancing technologies such as super-resolution microscopy and atomic force microscopy, where precise image analysis is vital.
Lateral resolution: Lateral resolution refers to the ability of an imaging system to distinguish between two points that are close together in a plane parallel to the imaging surface. In the context of atomic force microscopy (AFM), it is crucial for accurately imaging and measuring features on a sample surface, allowing researchers to identify structural and compositional details at a nanoscale level. Higher lateral resolution enables better visualization of fine features, which is essential for applications in nanotechnology and materials science.
Multi-modal AFM: Multi-modal atomic force microscopy (AFM) is a sophisticated imaging technique that combines traditional AFM with additional modalities, such as infrared spectroscopy or fluorescence microscopy, to provide complementary information about a sample at the nanoscale. This integration allows for the collection of varied data types, enhancing the understanding of material properties and biological structures in greater detail compared to single-mode AFM.
Probe tip: A probe tip is the small, pointed end of a scanning probe microscope (SPM), crucial for obtaining high-resolution images and measurements of surfaces at the atomic or molecular level. This component plays a vital role in interacting with the sample's surface to gather information about its topography, mechanical properties, and other characteristics through techniques such as atomic force microscopy (AFM). The design and materials used for the probe tip significantly affect the imaging resolution and quality.
Scanning Thermal Microscopy: Scanning thermal microscopy is a high-resolution imaging technique that combines scanning probe microscopy with thermal measurements to map temperature variations on a sample's surface at the nanoscale. This method enables researchers to investigate thermal properties, such as conductivity and heat capacity, providing insights into material behavior and phase transitions.
Surface topography analysis: Surface topography analysis is a technique used to characterize the physical features and structures of a surface at the microscopic or nanoscopic level. This analysis provides detailed information about surface roughness, texture, and features, which are crucial for understanding material properties and behaviors, particularly in fields like materials science, biology, and nanotechnology.
Tapping mode: Tapping mode is a specific operating mode of atomic force microscopy (AFM) where the cantilever tip intermittently contacts the sample surface while oscillating. This mode allows for higher resolution imaging compared to contact mode, as it minimizes lateral forces on the sample and reduces damage, making it suitable for soft or delicate samples while still providing topographical data.
Tip-sample interaction: Tip-sample interaction refers to the forces and effects that occur between the tip of an atomic force microscope (AFM) and the surface of a sample being analyzed. This interaction is critical in determining the resolution and quality of the images obtained, as well as the mechanical properties measured at the nanoscale. Understanding these interactions allows researchers to manipulate the tip's position and forces effectively, enabling detailed imaging and characterization of materials at the atomic level.
Topographical mapping: Topographical mapping is a technique used to represent the surface features of an object or area in a detailed and precise manner, highlighting the elevations and contours through contour lines. This mapping method provides a three-dimensional perspective on a two-dimensional plane, allowing for a better understanding of the physical characteristics of the surface being studied. It is particularly useful in visualizing the topology of surfaces at the nanoscale, such as in the context of materials science and biological samples.
Van der Waals forces: Van der Waals forces are weak, non-covalent interactions that occur between molecules or within different parts of a large molecule. These forces arise from temporary dipoles that occur when electron distribution around atoms fluctuates, leading to attractions that can significantly influence biomolecular interactions and stability.