Atomic Force Microscopy

A5 Science - Nanoscale Explorers

AFM: a different approach of microscope

This type of microscope does not rely on the passage of electrons or photons like the vast majority of conventional microscopes. Developed by G. Binnig, C. Quate, and C. Gerber in 1986, atomic force microscopy (AFM) is based on analyzing an object point by point using a scanning probe.

The AFM probe consists of a nanometer-sized tip with an very sharp geometric shape, which is positioned at the end of a microlever made of a flexible material. AFM works by measuring the attractive or repulsive interactions between the atoms that make up the nanometer-sized tip and the atoms that compose the surface of the analyzed sample. When the tip is in proximity to the surface of the sample, the interaction forces between the tip and the sample surface cause a deviation of the lever. The dynamic measurement of the lever deviation, monitored in real-time using a laser, allows both the determination of the exact path of the tip and the measurement of the interaction forces between the tip and the sample surface.

The principle of Atomic Force Microscopy is based on the physical measurement of the interaction forces between a nanometer-sized probe and the sample under analysis, and not on the observation of a beam of photons or electrons like most so-called traditional microscopies.

Illustration of the Atomic Force Microscopy principle, source: Pillet et al, 2013

Atomic Force Microscopy is a technique originally developed for fundamental physics research, such as atom observation, but its applications have since expanded to include the manipulation of atoms, the measurement of Martian soil roughness, and the development of innovative surface coatings. Today, AFM can be used to measure the properties of living surfaces and interfaces, including biological particles and objects such as cells, bacteria, microalgae, viruses, proteins and DNA, which are of great interest for research to fields such as healthcare, energy, environment and agri-food.

AFM is the only available technology that can make contact with the surface of biological samples with a resolution at the single-particle scale. However, developing manipulations and analyses in these measurement dimensions requires rare scientific expertise and know-how, in addition to significant development time.

AFM: a unique way for observing living particles.

Atomic Force Microscopy (AFM) is one of the few observation and characterization techniques of the Living samples that can be used under physiological and controlled conditions. The sample remains alive during the entire analysis, which is unique for observations at this resolution, but also presents a real technological and scientific challenge for developing measurement protocols. For instance, in the case of a living cardiac cell being studied by AFM, it must be maintained in a liquid medium at 37°C and 5% CO2 during the observation, while the AFM tip is in contact with the cell membrane surface.

Depending on the needs, acquisitions are carried out under physiological conditions, in a controlled environment, or in a culture medium. One of the critical steps in the protocols is the immobilization of the sample on a support material, which poses a significant challenge for the application of AFM to the study of biology and living particles.

For the study of a living cardiac cells by AFM, the sample is maintained in a liquid medium
at 37°C and 5% CO2 while the AFM tip is in contact with the cell surface.

Living cells are constantly exposed to mechanical stimuli from their environment and the surrounding extracellular matrix or cells. The intracellular molecular processes by which these physical signals are transformed into a biological response are collectively called mechanotransduction and are of fundamental importance in helping the cell adapt to the continuous dynamic changes in its environment and ecosystem. In the laboratory, measuring different mechanical parameters on the cell surface provides unique information on the biological body’s reaction to a stimulus or a specific environment.

The study of mechanotransduction mechanisms and indentations provides data with perspectives for applications in pharmaceuticals and health, as well as biotechnologies and nanotechnologies. Biomechanical parameters can be studied dynamically and specify the roughness, elasticity, adhesion, viscosity, hydrophobicity of surfaces, or interfaces. These data offer a better understanding of biological processes and allow for the development of innovative approaches for emerging new technologies, concepts, and products.

Key Concepts

To navigate further into the world at the nanoscale…

AFM Probe: A local probe that comes into contact with the sample surface. The nanoscale AFM probe, often pyramid-shaped, is located at the end of a lever with specific mechanical properties.

Mode Contact: With this mode, the tip is moved across the sample surface while maintaining a constant and regular applied force. The displacement is recorded by real-time tracking of the laser movement, and the measured variations are used to construct an accurate image of the topography and roughness at the surface of the sample.

Intermittent Mode: In this mode, the lever is oscillated at a predetermined amplitude at its resonance frequency (around a hundred kHz). When the tip interacts with the surface under analysis, the measured amplitude decreases and the resonance frequency is modified. This change in amplitude allows for the reconstruction of the sample topography at the nanometer scale. This mode is also known as the Tapping mode.

Force Spectroscopy Mode: With this mode, the tip is not constantly in contact with the surface under analysis, but is instead successively approached and retracted from the surface in an ordered matrix of successive points. The lever deflection is recorded during both the approach and indentation phases, as well as during the retraction phase of the AFM tip. The approach curve is used to determine the surface topography of the cell by calculating the position of the contact point, as well as the nanomechanical information. Finally, the retraction curve provides information on the adhesive properties and interaction forces between the tip and the studied surface.

Single Molecule Force Spectroscopy (SMFS) Mode: This mode is a derivative of force spectroscopy and is used to study the dynamics of interactions at the single-molecule level.

High-Speed Mode: With this mode, acquisitions are performed at very high speeds (20 frames per second). The High-Speed mode is particularly useful for observing dynamic phenomena.

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Our internal Research program is focused on life science observation and characterization at nanoscale with AFM in liquids. You can find more details about our past analysis and current projects on this page and view how we explore the biological surfaces with our technology.