Knowing the exact distance

Delft researchers have made an important advancement in a new microscopic technique that is widely used in medical research. Their findings were published online this week in Nature Methods.

Due to the near transparency of cells, fluorescence microscopy is one of the best techniques to study the dynamics in living cells. The direct study of interactions between cellular components that are closely grouped together is problematic however since the resolution of the light microscope is about 250 nanometers, whereas interactions typically occur at the ten nanometer scale.

A trick that partially solves this problem has become very popular in recent years. It is called localisation microscopy. In their publication entitled ‘Measuring image resolution in optical nanoscopy’ researchers of the Quantitative Imaging group (AS faculty) describe how they improved this technique.

With localisation microscopy the light of single fluorescent molecules (green fluorescent proteins are often used for this) is measured in several places. This is repeated for many proteins in succession. When the data from these individual molecules is combined, the images get about ten times sharper.

But how sharp the pictures exactly got was not known. So you could for instance obtain a clear picture of two proteins in a cell but not know the exact distance between them. “Such information is crucial; without it you can’t interpret structure and deduce the function of these proteins”, says Dr. Bernd Rieger, one of the lead authors.

Rieger and his Delft colleagues Dr Sjoerd Stallinga and PhD candidate Robert Nieuwenhuizen developed a technique using a statistical-mathematical analysis known as Fourier Ring Correlation to calculate the resolution with much higher accuracy. Their technique also allows researchers to obtain the best resolution as quickly as possible. They collaborated with scientists of the Dutch Cancer institute NKI, the German Max Planck Institute, the University of New Mexico and the University of Massachusetts. The project was financially supported by STW.

Robert P J Nieuwenhuizen, Measuring image resolution in optical nanoscopy, Nature Methods, published online 28 April 2013


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