Live cell Structured Illumination Superresolution Microscopy
by Stefan Wieser of ICFO
Super-resolution imaging has emerged as a key future fluorescence imaging technology in life science research allowing to resolve cellular details below 200nm. Optical super-resolution (SR) microscopy such as STED and STORM typically uses high laser power, which compromises cell viability and long-term imaging and thus restricts these SR methods to fixed samples. On the other side, structured illumination microscopy (SIM) can be used for live cell super-resolution microscopy. STED is based on scanning the sample with an effective sub-diffraction laser spot while localization microscopy (STORM, PALM) sequentially detects a subset of single emitters for generating a high-resolution image. In comparison, SIM uses stripe patterns for sample illumination to create large-scale Moiré effects, which are used to create super-resolved images. In a typical structured illumination microscope, the stripe-patterned illumination is rotated and shifted, resulting in a series of raw diffraction-limited images. Each of the raw images encodes high frequency information (higher than the optical limit of the microscope) which is visible as low frequency features of Moiré patterns. Algebraic decoding and reconstruction of the images results in a two-fold increase in lateral (2D SIM) and axial (3D SIM) resolution. Using high numerical aperture (NA) objective lenses, a lateral resolution of 80-100nm can be achieved at 102-106 fold lower laser powers compared to STED and STORM imaging. Altogether, 2D/3D SIM allows for super-resolution imaging of living cells at low photon budget using conventional fluorophores.
Stefan Wieser did his PhD in the group of Gerhard Schütz (Vienna University of Technology) and subsequent professional research experience in the lab of Didier Marguet (Centre d’Immunologie Marseille Luminy) and post-doctoral researcher working in the group of Michael Sixt (with joint affiliation to Carl-Philipp Heisenberg) at the Institute of Science and Technology (IST) Austria.
He started his own lab in 2016 at ICFO working on immune cell migration and the development of live cell superresolution microscopy creating a team of biologists, physicist and mathematicians. His future research vision is focused on unraveling fundamental mechanisms regulating immune cell dynamics in the context of the physical micro-environment by following a multi-disciplinary research approach combining quantitative biology, super-resolution imaging, micro- and thermo-manipulation and theoretical modeling using immune cells as a model system.
The colloquium is part of the BIST Master of Research curriculum but is also open and free for anyone interested in participating.