Conventional microscopes are limited in their resolution to 200 nm at the lowest. However, many interesting processes occur at a length scale below this limit, particularly in biological phenomena such as at the cellular level. The 2014 Nobel Prize in Chemistry was awarded for the development of super-resolved fluorescence microscopy, including STED (Stimulated Emission Depletion) microscopy, which permitted microscopy down to the 10nm-scale.
STED microscopy uses small fluorescent particles, or fluorophores, in the sample that glow (fluorescence) with the help of an excitation laser. A second deactivation laser with a donut-shaped cross-section can 'turn off' the fluorescence in a ring-shaped area, leaving only a small central spot still glowing. Scanning this beam combination across the sample creates a high-resolution image.
Traditional STED microscopy has been limited by photobleaching, where fluorophores under prolonged illumination permanently lose their fluorescence due to molecular changes caused by the lasers. This is particularly problematic for observing long-duration processes that require repeated scanning, like the changes at the molecular level inside cells.
But now, a team of researchers led by the Max Planck Institute for Polymer Research and the Okinawa Institute of Science and Technology (OIST) has addressed this issue by replacing traditional fluorophores with nanographene molecules. Their findings were recently published in Nature Communications.
Nanographenes have a robust molecular structure as well as the unique ability to recover their fluorescence. Essentially, the deactivation laser reverses the fluorescence fading process in the particles, allowing them to be reactivated without altering the sample, allowing for repeated scans of the same samples over substantially longer timeframes.
This method opens up many new research avenues using super-resolution microscopy. The ability to reactivate the nanographene molecules makes it possible to observe dynamic processes at a very high resolution over time, expanding the application of STED into new fields, like biology and materials science. "We are currently synthesizing novel functional nanographenes to observe unknown dynamic processes inside living cells using this new method," says Professor Akimitsu Narita, leader of the Organic and Carbon Nanomaterials Unit at OIST.
This press release has been adapted from the original from the Max Planck Institute for Polymer Research.
