The Bell Group: Single Molecule and Super-Resolution Fluorescenc

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!

Exciting News!

Same cells, same fixative, same immunostaining protocol for microtubules but one little thing is making all the difference in the amount of non-specific stain localized using dSTORM. Details to come, hopefully in our soon-to-be-submitted protocols and artefacts paper.

The first cell line we ever tried to image is back and yielding some stunning whole-cell 2D images of the actin network around the nucleus. The glutaraldehyde protocol we have specifically optimized for actin ultrastructure preservation has now been tested on three different mammalian cell lines, all with great results, and should be published as part of an upcoming paper.

This week we had the pleasure of hosting several science-savvy Year 10 work-experience students and were tasked with showing them how a working research lab operates. In between looking at the fluorescence of fingerprints and various dust samples from around the department, we also spent time checking and calibrating the pH of the stock solutions and imaging buffers in use with our favorite high-tech pH paper. Pretty cool work experience if you ask me.

$Stretched DNA labelled using YOYO-1 and imaged on the home built super resolution setup with varying success. Ongoing work by Donna and Lukas has confirmed that sub-diffraction imaging of DNA is really, really, really hard but absolutely possible....$ $Stretched DNA labelled using YOYO-1 and imaged on the home built super resolution setup with varying success. Ongoing work by Donna and Lukas has confirmed that sub-diffraction imaging of DNA is really, really, really hard but absolutely possible....$ $Stretched DNA labelled using YOYO-1 and imaged on the home built super resolution setup with varying success. Ongoing work by Donna and Lukas has confirmed that sub-diffraction imaging of DNA is really, really, really hard but absolutely possible....$

Stretched DNA labelled using YOYO-1 and imaged on the home built super resolution setup with varying success. Ongoing work by Donna and Lukas has confirmed that sub-diffraction imaging of DNA is really, really, really hard but absolutely possible. Some great images have been captured. Sometimes, as in the middle, the DNA isn’t so well stretched or labelled. But other times, as in the top and bottom images, a magnitude-improved spatial resolution is demonstrated in the double strand cross-sections.

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!

Further success on the Spheres project which is now rolling into a summer undergraduate project. Using similar protocols developed for the 100 nm spheres, Lukas has labelled a 20 nm batch with ATTO 647N dyes and grabbed some very nice single molecule images. EM once again confirms the heterogeneity of the spheres. Localization precision on the setup is exceeding expectations! [Scale bar (D) 100 nm.]

The Scope as it now stands. Ready to run three color simultaneous excitation and two channel detection. Simple as that.