New Limit for Optical Microscopy

Many different measurements, each containing a few light-emitting points, are combined to form a single image with a high resolution. (Source: B. Rieger, TU Delft)

The field of optical micro­scopy research has developed rapidly in recent years. Thanks to the invention of the super-reso­lution fluores­cence micro­scopy, it has recently become possible to view even the smaller parts of a living cell. Now, by making a smart refinement to that technique, researchers at TU Delft have pushed its boundaries even further. Where previously objects measuring up to 10 to 20 nano­meters could be observed, their method makes it possible to focus on structures of as tiny as three nano­meters across.

Delft cloth trader and scientist Antoni van Leeuwen­hoek’s home-made micro­scopes had a resolution of less than a micro­meter, which enabled him to observe structures like bacteria and sperm cells. But even in the seven­teenth century, Van Leeuwen­hoek was already approaching the diffrac­tion limit. According to the theory, the maximum size of the object you can image using a conven­tional micro­scope is half that wavelength. The diffrac­tion limit was long thought to a hard boundary, determined by the laws of nature. But by applying clever tricks, physicists eventually succeeded in crossing it.

Not so long ago, in 2014, the Nobel Prize for Chemistry was awarded to the three researchers who invented the work­around: super-resolution fluores­cence microscopy. In this technique, certain proteins or molecules are made fluores­cent by genetic modi­fication. The weak light signal they emit can then be captured with the help of an optical microscope. “In practice, though,” says researcher Bernd Rieger, “the problem with making proteins fluores­cent is that you can’t label all those of a particular type. Only 30 to 50 percent of them, at most. When you then start taking measure­ments, you see only a number of indi­vidual luminous points, not the complete structure you are trying to view.”

To solve the problem, the Delft researchers have devised an adap­tation to super-reso­lution micro­scopy. This is comparable with what is known in photo­graphy as compositing: stacking multiple images to create a single compound picture. “Averaging the information from different measurem­ents was already being done in electron micro­scopy,” explains researcher Sjoerd Stallinga. “But that’s a completely different tech­nology. It took our doctoral candidate Hami­dreza Heydarian two years to convert the technique for use in optical micro­scopy.”

One problem was that combining hundreds, if not thousands, of snapshots requires huge amounts of processing power. With a normal computer, it took several days to construct a clear image from all the data. “For­tunately,” says Rieger, “thanks to the computer games industry, we have access to graphics cards able to calculate extremely well in parallel.” A programmer from the Nether­lands eScience Center in Amsterdam joined the project and converted an existing algorithm for normal PCs into one the researchers could run on such a graphics card. As a result, the measure­ments can now be combined into a single image within a few hours.

This research is narrowing the gap between electron and optical micro­scopy, which is important because the two techniques deliver different results and so are comple­mentary, but are still a long way apart in terms of their possibilities. “The best electron micro­scopes are 30 to 50 times more powerful than the best optical ones,” says Stallinga. “Bringing the two worlds closer together could lead to new bio­logical insights.” According to the researchers, their technique – which is already achieving reso­lutions at the three-nano­meter level – should eventually make it possible to view structures measuring just one nanometer. Below that threshold, the dimensions of the fluores­cent labels become a limiting factor. (Source: TU Delft)

Reference: H. Heydarian et al.: Template-free 2D particle fusion in localization microscopy, Nat. Meth., online 17 September 2018; DOI: 10.1038/s41592-018-0136-6

Link: Dept. of Imaging Physics, Delft University of Technology, Delft, The Netherlands

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