Introduction to dSTORM

Comparison of Microtubuli in HeLa cells stained with AF647 in normal epifluorescence (left) and dSTORM mode (right).

dSTORM [1] is a super-resolution fluorescence microscopy method, based on single molecule localisation.

In normal fluorescence microscopy, the spatial resolution of images recorded by the microscope is limited by the Abbe limit of diffraction to structures of about half the wavelength of the fluorescence light emitted by the sample.

By seperating the emission of the individual emitters (e.g. dye molecules or fluorescent proteins) in time, it is possible to localise the position of each individual emitter with a very high precision of up to 10nm. This separation in time is done by stochastic „switching“ [2] of emitters between a bright „on“ and dark „off“ state. The result is a large number of images (or video frames) showing the emission of a subset of all emitters in the structure. The origin (i.e. the centre)  of each individual emission “spot” is then localised by efficient algorithms. If this is done for a sufficiently large number of individual emitters, eventually a high resolution picture of the imaged structure can be recovered.

Project description

As a part of the CRC/TR 166 ReceptorLight (http://www.receptorlight.uni-jena.de/), we are developing an automated dSTORM microscope capable of high-throughput imaging at close-to-molecular resolution.

Through sequential labeling of cell components [3,4], multidimensional super-resolution imaging can provide a powerful method for biological and bio-medical research.

A particular focus is put on the study of membrane receptors within the frame of the ReceptorLight research cluster.

References

[1] van de Linde, S. et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nature Protocols 6, 991–1009 (2011).

[2] van de Linde, S. & Sauer, M. How to switch a fluorophore: from undesired blinking to controlled photoswitching. Chem. Soc. Rev. 43, 1076–1087 (2014).

[3] Valley, C. C., Liu, S., Lidke, D. S. & Lidke, K. A. Sequential superresolution imaging of multiple targets using a single fluorophore. PloS one 10, e0123941 (2015).

[4] Schueder, F. et al. Universal Super-Resolution Multiplexing by DNA Exchange. Angewandte Chemie International Edition 56, 4052–4055 (2017).

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