Sometimes all it takes is a couple of minutes and a tweak of the setup to get the very best blinking! The red laser and the Q-TIRF x-y stage were both used extensively today for ongoing experiments in collaboration with Dr Gregory Moseley into the effects of the Rabies P protein on microtubule architecture. 

More mitochondria images using an inclined illumination and the exquisite resulting rendering of a sample cross section. 

Every so often someone hip-checks the microscope mid-measurement. Luckily, rapidSTORM lets us see the drift this causes straight away. 

Progress on the DNA project with Lukas. These YOYO-1 stained double stranded DNA molecules are finally all stretching out nicely using our own variation on shear combing. Low power 488 nm excitation seems to be doing the trick well. Further optimization will hopefully allow better isolation of single molecules and improved localization precision. 

Optimization of fixation protocols and characterization of fixation artefacts for single molecule super resolution microscopy continues! Among the many images gathered was this one containing one of the most confused microtubules we’ve ever seen… Or maybe we’ve just been looking at microtubules for too long.

A new single molecule super resolution project has been thought up to run alongside FCS measurements of Nile Red stained liposomes. As shown in 2006 by Hochstrasser, the diffusion of Nile Red in and out of lipid bilayers allows for single molecule localizations as the diffusing molecule only fluoresces from within the non-polar environment of the lipid tails. Preliminary data of sub-diffraction liposomes immobilised on glass shows the potential of such a method. Future work will optimize liposomes and lipid bilayer preparation for SMLM imaging, as well as move the technique into cell samples. 

Link.

Link.

Our hard worked-for first super resolution image of DNA stretched on glass. Not the greatest elongation but the binding/unbinding detection of YOYO fluorophores seems to be working well and some areas look very promising. 

More zoomed in microtubule images. These ones were fixed using methanol and once again some interesting looking kinks in the structure manifested. Still not perfect but we’re getting there!