University of Groningen scientists have come up with an improved method to apply fluorescent labels in microscopy studies. They have linked a protective ‘stabilizer’ to the fluorophore resulting in increased and longer emission. Their results were published on 11 January in the journal Nature Communications.
It’s nowadays a standard method to make small objects such as cells, viruses, or even single molecules visible under an optical microscope: simply attach light emitting molecules and watch these fluorophores glow. All this can be done with great precision. In 2014, the Nobel Prize in Chemistry was awarded for the development of super-resolved fluorescence microscopy, a technique the produces the best optical images so far.
‘But all this comes at a price’, explains Thorben Cordes, Assistant Professor in Molecular Microscopy at the Zernike Institute for Advanced Materials, University of Groningen. ‘In all microscopy techniques, but especially in those with high spatial resolution, the fluorescent labels degrade in a relatively short time. This greatly reduces the time you can study the object of interest.’
How is the glowing signal generated and why does it not persist? When a blue light shines on the fluorophore, it will emit a green light. The energy of the blue photons excite the label, which causes the emission of green photons. This excitation-emission cycle is repeated millions of times to generate useful microscopy images. During those cycles, it happens occasionally that a chemically reactive form of the fluorophore is generated. ‘In that case, the light emission stops instantly and the fluorophore might never start glowing again.
To prevent this, a so-called photostabilizer can be added to the experiment that protects the fluorophore from chemical destruction. ‘But these photostabilizers are usually toxic substances, which have to be added in a very high concentration to be effective.’ While this has various practical complications, toxic photostabilizers can of course not be used for the study of cells and organisms.
Cordes came up with a more subtle solution, one that had already been tested in the 1980s. ‘The photostabilizer is simply linked directly to the fluorophore. In such close contact, a much lower concentration is required’, he explains. This works quite well, but linking photostabilizer and fluorophore first to each other and then to the target, which you want to make visible (e.g., the cell), turned out to be quite difficult. Furthermore, the technique was only developed for one type of fluorescent label and hence never really caught on.
‘By making this concept generally applicable, I saw our chance to establish a new gold standard for photostabilization. Luckily, Jasper van der Velde, a PhD student in my group and first author of the study, was similarly fascinated by this idea’, says Cordes.
With the help of colleagues from Groningen, Oxford and Göttingen, Cordes’ group developed a more or less universal and easy-to-use system. ‘We used an unnatural amino acid as a central ‘hub’, to which we could attach the photostabilizer, the fluorophore and the target molecule.’ Linking the three different compounds to the amino acid was done by simple chemistry with cheap and commercially available compounds.
Cordes: ‘The development of the precise synthesis routes of course required the help of experts. We were lucky that synthetic chemists Gerard Roelfes and Andreas Herrmann helped us. Without their enduring support this study would not have been possible. But now in principle any fluorophore can be improved via our method.’ A short movie shows single stabilized fluorophores compared to their original non-stabilized versions with a substantial increase of stability by orders of magnitudes.
In the future, interested scientists can make the stabilized fluorophores themselves using off-the-shelf chemical compounds. But Cordes is also looking at ways to produce them commercially. ‘Over the last two years we had various inquiries from colleagues, who wanted to try them out. We have sent out test samples and always got very positive feedback.’
One case was particularly noteworthy: ‘I am quite proud that Stefan Hell, one of the three winners of the 2014 Nobel Prize for super-resolved fluorescence microscopy, not only discussed the project with us but also supported it. He provided fluorophore material and access to high-resolution microscopes, which allowed us to benchmark the performance of our fluorophores in high-resolution imaging.’
Reference: van der Velde, J. H. M. et al. A simple and versatile design concept for fluorophore derivatives with intramolecular photostabilization. Nature Communications 7:10144 (2016)
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