This week’s Australian Synchrotron User Meeting marked Donna’s fifth consecutive attendance and Toby’s first. Donna presented a talk debuting results from beamtime in August which combined SMLM and S-FTIR for the first time in order to examine both the structural and compositional makeup on single cells before and after fixation. The results went down well with a lot of positive feedback. The paper should be out soon! 

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. 

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. 

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! 

It’s not every day you see detail like that! We are so pleased with how our actin staining protocols are coming along and ongoing tweaks to the scope are improving every image as we go. 

A great new test sample for benchmarking the setup has been helpfully provided by Dr Georg Ramm. Single molecule imaging of LC3 reveals both a high level of homogeneous background as well as allowing visualization of autophagosomes in starved cells. Very cool.   

Some of our very first results! Epifluorescence and super resolved images of microtubules side by side!