(By: LION/EA)
Biophysicist Rolf Harkes has developed a microscope to optically localize individual molecules in living cells. It improves monitoring of diseases like cancer and Parkinson’s at the cellular level. He successfully defended his PhD thesis on this subject today in Leiden.

Heisenberg
For some decades it has been possible to zoom in on the atomic level by using an electron microscope. However, this technique is not applicable on organisms, since the radiation is harmful to living cells. To study biological matter with ultra-high precision, scientists need a microscope that uses ordinary light. Optical microscopy faces limitations on the small scales however, because Heisenberg’s uncertainty principle tells us that we cannot measure both location and angle of an incoming photon with absolute certainty. So light originating from a point will be imaged as a hazy spot rather than a point. If molecules are stacked too closely together, which is usually the case, a regular optical microscope cannot distinguish them from each other because their images overlap.

Fluorescence
A new technique called Single-Molecule Localization Microscopy (SMLM) tackles this problem. It is based on a technology pioneered by Leiden physicist Michel Orrit. Scientists randomly make some individual molecules fluorescent. For a short time those will emit light at a distinct wavelength, making it easy to filter out their signal from the noise of the many other molecules. Because this gives an image of only a few molecules, there are no overlapping localizations. This process is then repeated, with other molecules made fluorescent, until they have all been located.

Keeping score
Harkes, supervised by Prof. Thomas Schmidt, developed an SMLM microscope to show its promise for life cell research exemplified for a protein linked to Parkinson’s disease and created new analysis methods for broader use, like a counting method. ‘You would think that you can easily keep score of the number of molecules by counting the times you localize one,’ he explains. ‘It turns out that some are localized multiple times. And this doesn’t happen on the exact same location, as there is always still some uncertainty. So I developed a mathematical analysis that keeps track of localizations nearby a previous one, and corrects for double counts.’

Study Parkinson’s
Harkes’ technology and analysis methods help visualize biological structures in ultra-high resolution, down to the molecular level. This enables medical professionals to better monitor progression of tumors and researchers to study the mechanisms behind diseases like Parkinson’s. Harkes: ‘I used SMLM for example to look at the spatial distribution of the protein α-synuclein in cells. That is a good example of SMLM’s promise to study diseases, like Parkinson’s in this case.’