Molecular Machines & Industries

Force Spectroscopy

What is Force Spectroscopy?

Force Spectroscopy is a term summarizing several methods to measure interactions and forces between single molecules, cells or other particles. Force spectroscopy measurements can be performed employing Optical Tweezers, Magnetic Tweezers, Atomic Force Microscopy (AFM) and Acoustic Force Spectroscopy (AFS). Biological forces are typically given in pico-Newton (pN).

What is Force Spectroscopy being used for?

Force spectroscopy methods are being used to precisely measure inter-molecular forces between single molecules, cells or particles. However, single molecule force spectroscopy technologies can also be applied to quantify intra-molecular forces to study for example protein folding and stability, as well as nucleic acid folding.

Force spectroscopy is mainly used in basic research settings to scrutinize inter- and intra-molecular forces. Molecular motors such as kinesins and dyneins walking on microtubules for example can be visualized and investigated using Atomic Force Spectroscopy (AFM Spectroscopy) providing fundamental insights into the mechanisms of the motor proteins and their function within a cell.

Another interesting example for dynamic force spectroscopy applications are protein-DNA interactions. Some proteins specifically recognize and bind to certain DNA sequences, as restriction enzymes typically do, or they identify specific structures, such as DNA repair proteins do.

Single Cell Force Spectroscopy is a technology being also applied in the pharma industry. Drug candidates for example can be tested on specific cell types to measure differences in cell stiffness. These data thus provide insights into the cells’ integrity and potential side effects of the drug candidate on a cellular level.

In basic research single cell force spectroscopy is also used to measure binding forces between two cells or between a cell and a molecule of interest. The molecule, in many cases an antibody, is coupled to a microbead which can be trapped using an optical tweezer, as single molecules such as proteins are too small to be optically trapped. The cell of interest is held with a second trap. By oscillating the microbead, the force between the antibody and the cell can be measured. For cell-cell interactions, both cells can directly be trapped in this force spectroscopy cell manipulation set up and used for force measurements.

What different forms of Force Spectroscopy are there?

Force spectroscopy experiments can be executed using Optical Tweezers, Magnetic Tweezers, Atomic Force Microscopy (AFM) and Acoustic Spectroscopy. The techniques mainly differ regarding the technology to generate forces onto the molecules and cells of interest.

What are the differences between Atomic and Acoustic Force Spectroscopy?

Atomic force spectroscopy is a technology based on atomic force microscopy (AFM). The AFM tip interacting with the sample is mounted on a cantilever which is sensed by a laser to provide highly precise position information.

AFM can be used in an imaging mode to scan the topography of a sample. Thus, from the position information of the AFM tip, a 3-dimensional image is generated. In the force spectroscopy mode however, the AFM tip acts as the force sensor. Functionalized AFM tips allow to study specific interactions of conjugated molecules as typically used for force pulling experiments.

The AFM instrument measures the forces to analyze and quantify interactions between two molecules (inter-molecular) or within a single molecule (intra-molecular). Intra-molecular forces provide insights into DNA, RNA or protein folding and thus into their 3-dimensional structure. Inter-molecular forces help to understand the interactions of two separate molecules forming a complex.

In contrast, acoustic force spectroscopy (AFS) exerts acoustic forces without any direct contact on tethered molecules, such as DNA tightropes. AFS systems typically consist of a flow cell and a piezo element. Applying voltages to the piezo element, acoustic forces are subjected to the molecules of interest and forces in the pN-range can be measured. AFS is often used to study protein-DNA interactions as well as for DNA and RNA folding analyses.

How do the cell manipulation solutions from MMI work and help me?

The MMI CellManipulator is an optical tweezer system which can hold, move, stretch and manipulate particles and cells in a contact-free way. With one tweezer level, up to 10 particles can be trapped simultaneously with one rapidly switching laser beam. A second tweezer level creates an independent laser beam which can trap another 10 particles. In addition, a setup of two tweezer levels allows to hold 2 particles at the very same time with a continuous laser beam thus allowing for very precise force spectroscopy measurements. The cell manipulator is highly flexible regarding the experimental set ups for force spectroscopy experiments. The system can optically trap particles with a size of between 200 nm and 20 µm such as single cells as well as functionalized microbeads. This allows for inter- and intra-molecular force measurements as well as for measuring forces between cells.

The MMI CellTools platform provides a very intuitive software interface to optically trap molecules and cells of interest. In addition, automated calibration of the quadrant detector is supported by the software enabling precise force spectroscopy analyses and providing reliable data.

Intriguingly, the cell manipulator can be combined with any other MMI system. Therefore, single cell force spectroscopy can seamlessly be coupled to single cell isolation and cell sorting technologies.
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