Recent developments in cell imaging techniques, such as live cell imaging and super resolution microscopy, offer an increased insight into cellular and molecular dynamics. However, while the methods are now in place to allow researchers to utilise these techniques to ask more questions and understand more answers regarding life science the reagents needed to pursue this have not been.
Live-cell imaging allows researchers to track key biological processes, such as cell division, by lighting up the molecular components of DNA and proteins involved with fluorescent tags. Yet the tools need to stain DNA and fluorescently tag proteins have limited the use of this application. Labelling proteins with specific antibodies is impossible in live cells, as the antibodies fall apart within the reducing intracellular environment, so a more stable affinity reagent is required. Equally, current DNA stains are themselves toxic or require types of light (e.g. blue) that can damage the cells. Ideally, a safe DNA fluorescent stain would be activated in the safer far-red spectrum of light.
Labelling with fluorescent antibodies is not compatible with newer super-resolution microscope applications (link is external), due to the sheer size of the antibody tag, which reduces the achievable resolution. Similarly, many DNA stains are incompatible with super resolution microscopy. So, the bioimaging community have been waiting for solutions to these molecular labelling issues, to allow them to better engage with the advanced microscopy techniques.
We have developed Affimer® reagents that lack disulphide bonds, and so are stable within the intracellular environment (link is external) to specifically label proteins in live cell imaging. Additionally, as Affimer molecules are a mere tenth the size of an antibody the potentially achievable resolution in super-resolution microscopy is increased, opening up this technique to biological researchers.
The lab of Kai Johnsson at EPFL have developed a DNA stain that shows low toxicity, works with far-red light and can be used in super-resolution microscopy. The new stain incorporates two molecules. The first is a fluorescent molecule (silicon rhodamine or SiR) that works in the far-red spectrum and was previously developed in Johnsson’s lab. The second one is a well-known DNA stain Hoechst (the chemical name is bisbenzimide). As a result, the team named the new DNA stain ‘SiR-Hoechst (link is external)’.
The new stain works by binding to the minor groove of the DNA helix. Once bound, it turns on and emits a bright fluorescent red light, but if unbound it remains ‘off’ and so produces very little noise. More importantly, SiR-Hoechst can bind to DNA without affecting its biological function in the cell.
Because SiR-Hoechst works with far-red light, there is little risk of cellular damage. In addition, the light that it emits can be easily distinguished from any background fluorescence of living cells. Unlike other DNA stains, it can safely maintain high-quality staining in live cells for over 24 hours, allowing biologists to identify or track delicate processes, such as cell division, in real time. What’s more, the stain can be used in live-cell super-resolution microscopy, paving the way for DNA imaging in cells and biological tissues with exquisite resolution.
The team is now preparing to commercialize SiR-Hoechst through their EPFL startup company, Spirochrome, so in combination with Affimer technology the toolkit for bioimaging applications is becoming more comprehensive.