Lecture: Testing and Pushing the Resolution of Modern Fluorescence Microscopes

18.04.2017, 11:45

Dr. Jürgen Schmied, GATTAquant GmbH, Braunschweig

Time, place:
Tuesday, 18th April 2017, 11:45h
Seminar Room A1.500, MPL, Staudtstr. 2, 91058 Erlangen


Abstract:

Modern fluorescence microscopes are capable of improved resolution and performance properties, especially taking the newly developed super-resolution systems into account. But improved resolution and more versatile systems come along with a higher complexity. What is the resolution of my system? Where are the limits? How do I interpret my results? And if the outcome is not as good as expected, what is the origin – the setup, the sample, or the settings?

The quantitative analysis of a system plays a key role in successful imaging, but many systems lack in appropriate standards to test and verify the imaging parameters, to make results comparable and to allow a statistical evaluation of the data. Therefore reliable tools are needed, enabling a quantitative treatment of resolution and imaging parameters.

Based on DNA nanotechnology so-called nanorulers and beads enable the quantitative analysis of resolution and FWHM values of fluorescence microscopes. The core technique behind these rulers is the folding of DNA into predefined shapes (DNA origami) and the precise placement of fluorophores on those DNA structures.1 Up to hundreds of equivalent rulers or beads can be imaged simultaneously in one ROI and allow for the statistical evaluation of the system based on platform-independent samples.2

DNA nanostructures are unique regarding their size properties and flexibility. Variable mark-to-mark distances can be used to approach the resolution limits of a system. The precise control of fluorophore positions makes it possible to build nano-scaled point light sources with superior brightness density. Those beads can further be modified and applied as fiducial markers in localization-based microscopy techniques like DNA-PAINT to push image resolutions by post-processed lateral drift-correction.

 

  1. Schmied, J. J. et al. Fluorescence and super-resolution standards based on DNA origami. Nat Meth 9, 1133–1134 (2012).
  2. Schmied, J. J. et al. DNA origami-based standards for quantitative fluorescence microscopy. Nat Protoc 9, 1367–1391 (2014).
  3. Schmied, J. Testing and Pushing the Limits of Super‐Resolution Microscopy. Optik & Photonik (2016). doi:10.1002/opph.201600013