Topics and Objectives

​

Over the three-days, participants will be taught the theoretical principles, potential applications and advantages and disadvantages of different advanced imaging methods. Lectures will be provided by experts from the participating institutes, product specialists from cutting-edge microscope manufacturers as well as international speakers. Each afternoon techniques will be illustrated during on-site and remote demonstration sessions. These demonstration sessions are designed to complement the theoretical lectures and are intended to provide insight into the theoretical principles (participants are encouraged to discuss their own data or research projects). Together they form a solid basis for further individual hands-on training at - or collaboration with the organizing core facilities. 

The first day will cover light sheet imaging, expansion microscopy and non-linear optical (NLO) imaging. Light sheet imaging is used to visualize larger (up to cm) samples while maintaining (sub)cellular resolution. To gain further insight into subcellular nanostructures, expansion microscopy can be used. This technique allows nanoscale imaging using conventional diffraction-limited light microscopes based on isotropic expansion of the sample. Non-linear imaging involves the visualization of (label-free) contrast mechanisms in which at least 2 photons are recombined into a single photon. Due to the inherent optical sectioning, greater optical range and the less invasive nature of red-shifted photons employed in NLO imaging, this enables live/three dimensional imaging of larger volumes over extended periods of time.

The second day will focus on a set of microscopy methods that can be used to measure molecular dynamics, i.e. the mobility, stability, interactions and conformational changes of molecules or particles. An introduction to single molecule Förster Resonance Energy Transfer (smFRET), Fluorescence Correlation Spectroscopy (FCS), Raster Imaging Correlation Spectroscopy (RICS) and Fluorescence Lifetime Imaging (FLIM) will be given followed by demonstration sessions of each technique/experimental set-up.

 On the third day, we will zoom in on super-resolution microscopy. Super-resolution microscopy refers to a collection of techniques that are able to circumvent the fundamental resolution limit imposed by diffraction through various means. The techniques covered include: Airyscan, SIM (SIM-TIRF), SML and STED. Lectures will explain the theory behind the different principles to break the diffraction barrier employed in each technique. Practical sessions will illustrate the possibilities, advantages and disadvantages of enhanced resolution imaging.