How does the technology work?
Optical Tweezers and Fluorescence Microscopy
How does the technology work?
Optical Tweezers and Fluorescence Microscopy
”To decipher complex molecular interactions, scientists need the ability to observe the same biological process from multiple points of view. The combination of optical tweezers with fluorescence microscopy enables the simultaneous and in real-time visualization of individual molecules and measurement of the mechanical properties of biomolecular complexes to reveal greater detail.
LUMICKSC-Trap® and m-Trap®
Working Principle
Correlative Optical Tweezers – Fluorescence Microscopy (CTFM) is a single-molecule technique that combines optical tweezers, fluorescence microscopy, and microfluidics into a fully integrated platform. It can be used to apply and measure forces while simultaneously visualizing individual molecules in real-time. CTFM enables scientists to correlate mechanical properties to the number, location and conformational state biomolecules
With this technology it is possible to perform simultaneous manipulation, force measurements and visualization of these complexes — for example proteins interacting with DNA. This means that scientists can now correlate mechanical properties to the number, location and conformational state of the proteins bound to DNA. This revolutionary single-molecule visualization technology enables the understanding of life to the smallest detail, which is critical for life science research and drug development.
Optical Tweezers – Fluorescence Microscopy
The figure shows a typical CTFM experimental setup, where optical tweezers are used to trap beads and catch a biomolecule such as DNA in between. Fluorescently labeled proteins are then visualized with confocal or STED fluorescence microscopy. Simultaneous force and extension measurements allow correlating the protein activity and binding kinetics with the mechanical properties of the DNA.
The importance of this technique is it provides the ability to observe the same biological process from multiple points of view. With this new ability to perform simultaneous manipulation, force measurements, and visualization
of these complexes—for example proteins interacting with DNA— scientists can correlate mechanical properties to the number, location and conformational state of the proteins bound to DNA.
Force extension, manipulation, and visualization
Polymers and filaments can be manipulated with the high-resolution optical tweezers while simultaneously measuring force, extension and fluorescence microscopy data. Combining global mechanistic information with local activity provides essential insights into the dynamic function of the substrate under study.
Constant force measurements
Equilibrium dynamics of biomolecular states can be measured by performing constant force measurements. By keeping the traps in a fixed position while measuring tension fluctuations caused by intramolecular conformational transitions with ultra-high sensitivity it is possible to detect the smallest, rarest and most transient states.
Real-time single-molecule visualization
The kymograph gives unique insights into the dynamic interactions between proteins and filament substrates, such as DNA, and protein–protein interactions. Simultaneous force and extension measurements allow for correlating the protein activity and binding kinetics with the mechanical properties of the protein substrate complex.
As an example, in the kymograph below we can distinguish:
- single fluorescent protein on DNA
- single protein unbinds from DNA,
- fast binding & unbinding event (<10ms).
