Travel Grant: Koen van den Dries (Albuquerque)

From the 30th of August to the 13th of September 2011 I have visited the lab of Keith Lidke at the University of New Mexico in Albuquerque to perform direct STochastic Optical Reconstruction Microscopy (dSTORM) to gain more insight into the ultrastructure of podosomes.

Podosomes are small dynamic adhesion structures that are involved in matrix degradation and invasion. They consist of a dense core of actin (500nm) surrounded by a ring of integrins and integrin-associated proteins (100-200nm). Podosomes are only formed when cells adhere to a substrate and are located at the ventral plasma membrane of cells. With an average size of 1 micron podosomes can be visualized by conventional and confocal microscopy but details concerning their nanoscale organization cannot be obtained with these techniques.

Recent progress in the field of microscopy to circumvent the diffraction limit of light resulted in a variety of novel microscopy techniques to study the nanoscale localization of cellular components. dSTORM belongs to the family of super-resolution optical techniques that exploits the sequential and stochastic readout of multiple emitters in a sample to achieve a lateral localization accuracy of typically 20 nm. dSTORM utilizes conventional stand-alone fluorophores that randomly switch between a dark and fluorescent state when illuminated at high laser power.

In total, I had 13 days to optimize the dSTORM protocol for our samples. This turned out to be just enough since many problems occurred as we tried to find the optimal settings for imaging. The first 9 days were spent to optimize the technique. Issues with sample labelling, laser intensity, blinking rate and microscope stage drift had to be solved during these days which costed a lot of time. However, with the enormous help of the people in Albuquerque and long working days we managed to get the dSTORM setup working and the last 4 days we were able to image the samples for my research. We labelled the samples for actin and specific ring components such as the integrins and integrin-associated proteins and made several images from multiple samples per specific podosome component. The acquisition of real data took a load of my mind and although the working days were still as long as before, they felt considerably shorter. After 13 days I flew back to The Netherlands with all the data we were planned to acquire and we are now writing an article on this to submit it to a journal.

I really enjoyed my stay in Albuquerque and I have greatly expanded my knowledge on super-resolution microscopy techniques in general and dSTORM microscopy in particular. I have learned that patience is always important when optimizing new protocols, even when time is short. During my two weeks visit, we systematically adjusted many conditions and parameters in order to obtain a protocol that could be used for the last days to image the samples. I could not have done this work without the excellent help of the experienced people within the lab of Keith Lidke. The atmosphere in the lab was great and very stimulating during (very) long days in the lab. I would advise everybody who has the opportunity to visit a lab inside or outside of Europe to take this opportunity. It not only helps to increase your scientific knowledge but you also learn to work with different people. Different labs have different “cultures” and it is nice to experience this. Finally, I would like to thank the VvB-BMT for providing financial support to give me the opportunity to visit the lab of Keith Lidke.