Anastasia Gazi

Anastasia currently enjoys the following methodologies:

HT-Imaging for TEM

Development of automated acquisition methods for TEM has been boosted mainly by academic efforts (UCSF Tomography, Leginon, SerialEM) in a relatively low cost. Since 2016 Anastasia is using SerialEM as an acquisition software package to automatize the collections on Tecnai microscopes. To the time being Anastasia offers help with automated collections for:

• Serial electron tomography (dual axis and CryoET)

• High resolution mapping of entire cell profiles

• On-grid CLEM approaches

Tomographic reconstructions

In electron tomography, the tilt series acquired, by tilting the sample to different angles under the electron beam (usually every 1 or 2 degrees from about −60° to +60°), can then be computationally reconstructed into a 3D volume.

Typically, reconstruction of electron tomograms consists of five steps; pre-processing, estimation of initial image shifts, preparation of the fiducial model and tilt-series alignment, post-processing, and reconstruction. In case of dual axis tomography the tomographic volumes from each axis are build separately and combined in a final step.

Users are encouraged to reconstruct their own tomograms with IMOD, using either their own computing resources or the ones provided by UBI.  IMOD is installed in all UBI’s workstations.

Volume annotation

For volume data containing more complex structures, a direct 3D volume rendering is often difficult to analyze. Especially in CryoET where the signal to noise ratio is quite low. Their interpretation is therefore simplified by creating 3D surface models by locating objects of interest and boundaries using segmentation tools.

Anastasia prefers the use of academic software, like the IMOD segmentation tools for manual annotation and eman2 for CNN based annotation, coupled to the UCSF Chimera and ChimeraX for further 3D modeling and surface rendering.

PNAS 2021 Vol. 118 No. 32 e2105540118

Anastasia was lucky enough to be one of Prof. N. J. Panopoulos’ PhD students at University of Crete, Heraklion (2003-2007). She fall in love with the bacterial type III secretion systems (T3SS), admiring the evolution of protein nanomachines as strong as the evolution of the structural biology field itself. Thanks to Prof. M.I. Kokkinidis she visited DESY numerous times to collect data both on Small Angle X-ray Scattering and protein crystallography beamlines. In 2009 Anastasia attended what she believes is the best course for large experimental systems ever: the HERCULES course in Grenoble, where she visited ESRF, ILL and Soleil for the first time. The same year she got an EMBO short term fellowship to visit the group of Dr. Andrea Dessen @IBS, Grenoble. Still committed to T3SS, she joined the lab of Prof. P. Sansonetti end of 2010 to use Electron Microscopy (EM) as the main research methodology. A quick and dirty postdoc at a different field (biologically and methodologically -wise) then followed. In 2016 she had the pleasure to return to the historical Electron Microscopy facility of I. Pasteur, Paris thanks to a LABEX grant and to Prof. J. Krijnse-Locker. She was inspired heavily by the academic software SerialEM and decided that the EM field deserves her full attention. In 2018 she was hired by Dr. Yaser Hashem @IECB Pessac, based on his ERC grant as an EM research engineer for cryoEM data collections @TALOS Arctica. She had the pleasure to be the first to also collect cryoET data @IECB. Although she loved to work in Hashem’s team, I. Pasteur finally offered her a permanent contract the same year. Anastasia is deeply thankful to all people she met during this journey.