...
For STED at the LMCB, use the Leica STED.
Single Molecule Localisation Microscopy (SMLM)
SMLM techniques like Photoactivation Localisation Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) rely on sparse visualisation of a small subset of fluorescent emitters. This is achieved in different ways depending on the technique. For example, in PALM, photo-activatable or photo-switchable fluorophores are used and only a few of them are converted to the emitting state at any one time, whereas in STORM the fluorophores are driven into a dark state and stochastically return to an emitting state. In both cases the images are made up of spots that blink on a frame-by-frame basis. Each spot represents an individual emitter. Capturing many thousands of these images and localising the centre of each spot allows one to reconstruct an image made up of each localised molecule. The precision of localisation depends upon the specimen and how well the experiment has been optimised, but under ideal conditions it can be less than 10 nm.
Once molecules have been localised their clustering can be analysed using such programs as SR-Tesseler. For SMLM at the LMCB, use the Carl Zeiss Elyra PS.1. In addition the ONI Nanoimager can be used for PALM/STORM and also single molecule tracking and smFRET.