Correlated optical tweezers with fluorescence microscopy

Part of: Dynamic single-molecule analysis

One technology to provide a 360° view of biomolecular processes

Correlative optical tweezers with fluorescence (CTFM) is a dynamic 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.

With this technology it is possible to perform simultaneous manipulation, force measurements, and visualization of biomolecular complexes, such as DNA-binding proteins, while they interact with DNA.

So how does this technology work in practise? At the right we show an example of an experiment performed to study DNA-binding proteins. From this example, we can see how this technology allows –within the same experiment– to:

  • Obtain direct evidence of how the molecular mechanisms and dynamic processes of proteins on DNA work.
  • Observe the stepwise assembly of the biological complex.
  • Modulate the molecular system to test the model under different conditions.

A typical experimental setup and data output

The figure at the right shows a typical 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, widefield 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.

Below we highlight some of the different experimental use cases his technology can be used for.

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, from the kymograph at the left we can distinguish:

  • single fluorescent protein on DNA
  • single protein unbinds from DNA,
  • fast binding & unbinding event (<10ms).

Force extension, manipulation, and visualization

Polymers and filaments can be manipulated with high-resolution optical tweezers while simultaneously measuring force, extension, and microscopy data. Combining global mechanistic information with local activity provides essential insights into the dynamic function of the substrate under study. The example below shows an experiment where two fluorescent microtubules were held by two beads in a crossed pattern, and one microtubule was dragged across the other with a known force.

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. Below we show you a full length CaM protein at 10 mM Ca2+ showing equilibrium dynamics between multiple states. Data was recorded at 50 kHz (grey line) and averaged at 200 Hz (red line). The histogram quantifies the most populated states in the inset (right panel) showing two peaks at 6.5 ± 0.1 pN and 7.8 ± 0.09 pN.

Different imaging configurations to serve different needs

Let’s dive deeper into the specific imaging techniques that can be combined with optical tweezers, and more specifically, integrated into the C-Trap Optical Tweezers – Fluorescence & Label-free Microscopy system. Click on each imaging technique to learn more.

Widefield

Image biomolecular processes with low protein concentration in solution with high acquisition rates.

Confocal Fluorescence

Multicolor confocal, perfect for visualizing biological processes in solution.

STED Super Resolution

The perfect choice for performing experiments in highly crowded environments, offering unprecedented resolution (< 35 nm).

TIRF Fluorescence

Suitable for visualization on surfaces as it eliminates background fluorescence outside the focal plane.

IRM (label-free)

IRM allows you to visualize microtubules without the need for fluorescence labeling.

Our solution

The C-Trap® Optical Tweezers – Fluorescence & Label-free Microscopy is the world’s first instrument that allows simultaneous manipulation and visualization of single-molecule interactions in real time. It combines high resolution optical tweezers, fluorescence and label-free microscopy and an advanced microfluidics system in a truly integrated and correlated solution.

The C-Trap offers you a fast workflow to seamlessly catch and manipulate single molecules. The instrument measures their structural changes or interactions while you visualize them in teal time with high spatial and temporal resolution, ultimately offering you a complete and detailed picture of biomolecular properties and interactions

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