Ultrafast STED nanoscopy
Nobel Laureate Stefan Hell and his team at the German Cancer Research Center in Heidelberg have achieved yet another milestone in super-resolved optical microscopy. The team have developed an ultrafast STED (stimulation emission depletion) nanoscope that now makes it possible to study molecular processes and transport processes in living cells in millisecond time steps.
At the Nobel Laureate Meeting in Lindau on 29th June 2015, Stefan Hell gave a keynote lecture on a STED nanoscope developed by the Division of Optical Nanoscopy at the German Cancer Research Center (DKFZ) in Heidelberg that can be used to take photos at millisecond intervals. The nanoscope makes it possible to examine rapid dynamic processes in the nanometre range, such as vesicle fusions or the transport of viruses and biomolecules in living cells.
In 2014, Stefan Hell was awarded the Nobel Prize in Chemistry for the development of STED microscopy, which circumvents Abbe's resolution limit. Researchers can use the STED nanoscope to study ultrafine details of living objects just a few nanometres in size with fluorescence light; such details could previously only be studied in a fixed, lifeless state with an electron microscope.
An ultrafast electro-optical scanner
After improving spatial resolution, Stefan Hell's next logical step was to take temporal resolution to its limits. However, there were enormous technical challenges to overcome. Although the fast scanners of laser scanning microscopes and the fast cameras of wide-field microscopes make it possible to increase temporal resolution to below 100 milliseconds, precise spatial resolution of subcellular nanometre motions cannot be recorded. The resulting image is blurred as it takes too long to visualise an object. STED technology cannot be combined with wide-field illumination and fast cameras because it scans, point by point, samples with two lasers of different wavelengths (one for excitation and one for extinguishing fluorescence). The scanners must ensure that beams are reflected independently from the wavelengths. In addition, the time a laser beam focuses on any of the image pixels needs to be reduced by several orders of magnitude in order to prevent excessive photobleaching.
At the heart of the ultrafast STED nanoscope recently presented in the journal "Nature Methods" is a scanner system developed by Jale Schneider as part of her doctoral thesis at the RWTH Aachen. The scanner system consists of three elements – a rapid beam deflector, an ultrafast detection unit, a control and FPGA (field programmable gate array)-based data processing system. The system has reduced pixel dwell times to 6.25 nanoseconds per frame, and thus to the order of fluorescence lifetime. Image rate increases proportionally and recorded frames are added stochastically (i.e. as temporarily ordered random processes) until the signal reaches the desired strength; rather than scanning the image field slowly and just once, Jale's new technical method scans the sample several times with a laser beam at ultrafast speed and produces a series of high-resolution images, thus reducing bleaching and blinking.
Applications in cell biology
Stefan Hell and Johann Engelhardt led the development of the world's fastest nanoscopy method in the Division of Optical Nanoscopy at the DKFZ using Jale Schneider's scanner system. The high temporal resolution – several thousand times faster than that of traditional laser scanning microscopes – enables the researchers to visualise dynamic processes in living cells and at a level of detail that has not previously been possible. In collaboration with Stephan Sigrist and his team at Freie Universität Berlin, the researchers were able to study the movements of individual neuropeptide-transporting vesicles in the nerve cells of living Drosophila larvae with a temporal resolution of 8 milliseconds (resulting from the on-line addition of single full frames); the effective frame rate was four times higher.
The researchers have also used the nanoscope to capture AIDS virus particles (HI viruses labelled with EGFP – enhanced green fluorescent protein) before and during cell penetration. These experiments were carried out in collaboration with Hans-Georg Kräusslich and his team in the Department of Virology at Heidelberg University Hospital. The virologists, who have used STED nanoscopy for AIDS research for many years, found that certain proteins of the HIV envelope had to aggregate in order for the virus to be able to infect human cells. The ultrafast STED nanoscope developed by Stefan Hell and his team will now also enable temporal analysis of the molecular processes of viral infections. The technology could therefore potentially lead to the identification of new AIDS therapy targets.