9.2 Atomic force microscopy (AFM) for molecular imaging

Atomic force microscopy (AFM) is a powerful tool for molecular imaging. It uses a tiny probe to scan surfaces, revealing intricate details of molecules and structures. AFM offers various modes, from gentle non-contact to direct contact, each suited for different sample types.

AFM's versatility shines in its advanced techniques. Tip functionalization allows for selective imaging, while force spectroscopy measures molecular interactions. High-resolution imaging pushes the boundaries, enabling us to see individual atoms and molecules with incredible clarity.

AFM Modes of Operation

Contact Mode

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• Operates with the tip in close contact with the sample surface
• Cantilever deflection is used as a feedback signal to maintain constant force between tip and sample
• Provides high-resolution images of surface topography (atomic resolution)
• Can cause sample damage or tip wear due to high lateral forces
• Suitable for hard, non-deformable samples (graphite, mica)

Tapping Mode

• Oscillates the cantilever near its resonance frequency with a certain amplitude
• Tip intermittently contacts the sample surface at the bottom of each oscillation cycle
• Amplitude of oscillation is used as a feedback signal to maintain constant tip-sample interaction
• Reduces lateral forces and minimizes sample damage compared to contact mode
• Enables imaging of soft, delicate, or sticky samples (polymers, biological samples)

Non-Contact Mode

• Operates with the tip oscillating above the sample surface without making contact
• Measures changes in the resonance frequency or amplitude of the cantilever due to attractive van der Waals forces
• Provides lower resolution compared to contact and tapping modes
• Minimizes tip and sample wear as there is no direct contact
• Suitable for imaging very soft or easily deformable samples (liquid droplets, loosely bound adsorbates)

AFM Components and Forces

Cantilever and Tip

• Cantilever is a microscopic beam that acts as a force sensor, typically made of silicon or silicon nitride
• Tip is attached to the free end of the cantilever and interacts with the sample surface
• Cantilever deflection is measured using a laser beam reflected from the back of the cantilever onto a photodetector
• Tip shape and material affect the resolution and contrast of AFM images (sharp tips provide higher resolution)

Van der Waals Forces

• Weak, short-range attractive forces between atoms or molecules arising from induced dipole interactions
• Dominant forces in non-contact mode AFM, causing changes in the cantilever's resonance frequency or amplitude
• Strength of van der Waals forces depends on the distance between the tip and sample (decreases rapidly with increasing distance)
• Enables mapping of surface properties such as adhesion, hydrophobicity, or charge distribution

Force-Distance Curve

• Plots the force acting on the cantilever as a function of tip-sample distance
• Provides quantitative information about the interaction forces between the tip and sample
• Typical force-distance curve shows attractive forces (negative values) at large distances and repulsive forces (positive values) at close proximity
• Can be used to measure sample stiffness, adhesion, or elasticity by analyzing the slope and hysteresis of the curve
• Force spectroscopy techniques utilize force-distance curves to study molecular interactions or mechanical properties

Advanced AFM Techniques

Tip Functionalization

• Modifies the AFM tip by attaching specific molecules or functional groups to its apex
• Enables selective imaging or measurement of specific interactions between the tip and sample (molecular recognition)
• Common functionalization methods include self-assembled monolayers (SAMs), covalent attachment, or adsorption of molecules
• Applications include mapping of chemical functional groups, studying ligand-receptor interactions, or probing molecular forces (hydrogen bonding, hydrophobic interactions)

Force Spectroscopy

• Measures the force required to break individual molecular bonds or interactions between the tip and sample
• Involves recording force-distance curves while the tip is approached to and retracted from the sample surface
• Can provide information about the strength, kinetics, and energy landscape of molecular interactions (protein unfolding, receptor-ligand binding)
• Single-molecule force spectroscopy allows studying the mechanical properties of individual molecules or molecular complexes (DNA stretching, protein unfolding)

High-Resolution Imaging

• Achieves sub-nanometer or atomic resolution by optimizing the imaging conditions and tip geometry
• Requires sharp tips with small apex radius (few nanometers) and high aspect ratio
• Utilizes low-noise, high-stability AFM systems with precise control over tip-sample distance and force
• Enables imaging of individual atoms, molecules, or nanostructures with unprecedented detail (atomic lattices, molecular assemblies)
• Applications include studying surface reconstructions, defects, or adsorption of molecules on surfaces (self-assembled monolayers, graphene)