Use of the Quorum Cryo-SEM preparation system in microbial cell biology with electron cryotomography at the Jensen Laboratory

Quorum Technologies, market and technology leaders in electron microscopy coating and cryogenic preparation products, report on how their PP3010T Cryo-SEM preparation system is being used in the preparation of hydrated whole cells to be imaged using electron cryotomography in the Jensen Laboratory located at HHMI Caltech.

Image of “fission yeast” Schizosaccharomyces pombe cells prepared for TEM - by Vitrobot freezing on Quantifoil grids and Quorum PP3010T transfer for FEI Versa FIB milling.

Alasdair McDowall is the EM Center Director in the Jensen Laboratory at the Howard Hughes Medical Institute located at Caltech. Headed by Professor Grant Jensen, the Lab uses Electron Cryotomography (ECT) to study the molecular architecture of microbial cells and HIV in their native state. The focus is on the fundamentals of microbial cell biology such as cell division, movement and secretion, as well as the structure of HIV at all stages of its lifecycle.

The lab opened its doors in 2002 and continues to push the boundaries of high resolution imaging today. However, the investigation of frozen hydrated whole cells (beam and vacuum sensitive materials) in the electron microscope chamber requires new solutions. The advances in techniques for the preparation of cells by Cryo Focused Ion Beam Milling for structural characterization have recently provided a new insight of these delicate cellular architectures.

Image of “fission yeast” Schizosaccharomyces pombe cells prepared for TEM - by Vitrobot freezing on Quantifoil grids and Quorum PP3010T transfer for FEI Versa FIB milling.

Cryo Focused Ion Beam milling (cryo FIB milling) is a cutting-edge method for thinning vitrified biological samples that allows access to intracellular regions of thick specimens (> 1 um) with unprecedented ease and structural preservation. It provides the ability to move beyond imaging only small bacterial cells with electron cryotomography (ECT) and will allow the exploration of eukaryotic cells, tissues and microbial biofilms to the same molecular resolution that the group has achieved with individual bacterial cells for the past decade.

In addition, the ability to thin individual bacterial cells before imaging, without perturbing their structure, will provide higher contrast and resolution when necessary, even within already thin bacterial cells. Furthermore, the addition of a cryo-stage to the existing FIB mill at Caltech will allow for further development of much needed methods for correlating fluorescence microscopy and electron tomography for the targeting and identification of specific structures deep within eukaryotic cells, bacteria and tissues.

Image of “fission yeast” Schizosaccharomyces pombe cells prepared for TEM - by Vitrobot freezing on Quantifoil grids and Quorum PP3010T transfer for FEI Versa FIB milling.

Dr McDowall uses the Quorum PP3010T cryo sample preparation system. This is a highly automated, easy-to-use, column-mounted, gas-cooled Cryo-SEM preparation system suitable for most makes and models of SEM, FE-SEM and FIB/SEM. The Jensen group uses their prep system with an FEI Versa scanning electron microscope.

The Quorum PP3010T is ideal for our vitreous cell transfers when dedicated to maintaining the cryogenic amorphous state during SEM and DualBeam specimen preparation. The PP3010T allows easier and more practical operation for milling cryo preserved specimens for electron tomography. Its design assists the investigation of frozen hydrated cells and tissues by providing a mechanism for hydrated sample transfer, cryogenic cooling and temperature control within the SEM or Small Dual Beam during the milling process. In our operation, the Quorum PP3010T was added to an existing FEI Versa Dual Beam Scanning Electron Microscope in a climate controlled room courtesy of the Greer group, California Institute of Technology.”

Dr McDowall

To obtain full details of Cryo-SEM preparation systems and other products available from Quorum Technologies, please visit www.quorumtech.com.

Acknowledgements: Quorum Technologies thanks the following for sharing their research: Professors Grant Jensen & Julia Greer and Drs Matt Swuilius, Shrawan Mageswaran & Wei Zhao.