Training, Application and Support Center, TASC
E-mail: [email protected]

Superresolution microscopy, i.e. light microscopy beyond the traditional diffraction limit, is rapidly changing its face. From avant-garde method development research, it is currently being transformed into a tool for biomedical research by pioneer users and engineers.

The talk will give an introduction to the superresolution technologies that Carl Zeiss MicroImaging focuses on: Photoactivated Localization Microscopy (PAL-M) and Super Resolution Structured Illumination Microscopy (SR-SIM).

PAL-M is a single molecule imaging method based on the strategy to photoactivate (or photoswitch) only few fluorophores at a single timepoint. A mathematical process afterwards localizes precisely their position by a Gaussian fit. The effective lateral resolution down to 20nm for cells and tissues opens up completely new dimensions of quantitative analysis of complex biological specimens.

In SR-SIM the sample is illuminated with precisely modulated excitation light. This known line pattern is superimposed to the sample´s structure and creates due to interference a Moiré pattern. From this interference pattern in the raw data an algorithm computes the superresolution data that reaches up to twice the resolution in all three dimensions compared to confocal microscopes. SR-SIM is a flexible technique that can be applied on all fluorophores on fixed as well as living samples.

With the data dimensions that these novel imaging methods open up, they also bring up new questions about optimal fluorescence probes, sample preparation methods and data presentation. In this regard, each superresolution technology presents its unique advantages and challenges. Exemplary use cases and successful experiment design will be adressed in a second part of the talk.