Imaging ONE WORLD - "3D Imaging of Cells by FIBSEM with Correlation to Cryo Fluorescence Microscopy"
15 March 2021
This week will feature Harald Hess from Janelia Research Campus.
Scientific Organisers: Stefanie Reichelt, Alex Sossick, Nick Barry, Alessandro Esposito and Kirti Prakash
The meeting will begin at 13:00GMT.
As part of the 'Imaging ONE WORLD' series, the focus of these lectures is on microscopy and image analysis methods and how to apply these to your research. Almost all aspects of imaging such as sample preparation, labelling strategies, experimental workflows, ‘how-to’ image and analyse, as well as facilitating collaborations and inspiring new scientific ideas will be covered. Speakers will be available for questions and answers. The organisers, CRUK CI core facility staff, Gurdon Institute, MRC-LMB, MRC Cancer Unit and NPL will be able to continue the discussion and provide advice on your imaging projects.
Janelia Research Campus
After a PhD in Physics at Princeton in 1982, Harald Hess pursued cryogenic hydrogen atom trapping and its Bose-Einstein condensation at MIT as a postdoc and then developed various low temperature scanning probe microscopes to visualize diverse physics phenomenon at Bell Labs. After 1997 he spent 8 years in industry developing advanced equipment for hard disk drive and semiconductor inspection and production. In 2005, he and a colleague, Eric Betzig, learned about photoactivatable fluorescent proteins and invented PALM (photo-activated localization microscopy) to reveal details of cell structure beyond the diffraction limit. It was built in his La Jolla condo, tested at the National Institute of Health and perfected at Janelia Farms/Howard Hughes Medical Institute where he is extending PALM to a 3D super-resolution microscopy and exploring its application for cell biology research. He is also developing 3D electron microscopy techniques for volume imaging of cells and neural tissue as well as exploring modalities to correlate such electron microscopy with super-resolution microscopy.
3D electron microscopy data can be acquired by Focused Ion Beam Scanning Electron Microscopy FIB-SEM where fine sequence of 4-8 nm increments are ablated off of a sample surface and each such surface is imaged with the SEM. At the finest resolution and with month long stable operation, comprehensive whole cells can be acquired that transcends the limited cut section views of traditional TEM used in biology. Several examples of such data are presented along with the potential that segmentation offers to explore and formulate biological questions. Correlative microscopy can be achieved by a cryogenic protocol where samples are vitrified, imaged with PALM or SIM at low temperatures followed by EM staining and FIBSEM. A 3D registration procedure can keep most position errors between PALM and EM data at ~ 30 nm. Examples validating the approach with mitochondrial and endoplasmic reticulum labels are presented along with examples showcasing how unknown vesicle types and other structures can be identified by an associated protein.