Yauheni Novikau

SIM and PALM as tools to study protein structural organization, numbers, interaction and dynamics

Lately quite a plethora of concepts have been successfully developed, which take resolution beyond the classical limits of a light microscope. Among these structured illumination microscopy (SIM) and photo activated localization microscopy (PALM) hold the promise to provide biologists with unprecedented insights into sub-cellular organizations. A combination of these methods seems particularly attractive as it allows adapting to the required resolution and enables to map single molecules or molecule ensembles in the context of highly resolved structures. SIM achieves two fold resolution enhancements in both lateral and axial directions, so structures can be highly resolved in 3D. Adapting the structuring to the wavelength opens up the avenue for multi-color staining. Hence the distribution of one protein and its associated structure can be viewed in the context of others. Since all common fluorescent dyes can be used sample preparation is straightforward. Besides the classical approach to obtain highly resolved structures with up to 10 times the classical resolution, the power of PALM lies additionally in its ability to count and observe single molecules. As such clustering of molecules can be studied as well as many molecules tracked simultaneously to study their diffusion. New strategies open up the possibility to obtain resolution enhancement in the axial direction as well. These applications start already to have an impact on our view how a cell is organized and how different proteins contribute to its make-up.