Cryo-TEM is a technique used to visualize the structure of frozen and fully hydrated macromolecular complexes. The sample is studied at cryogenic temperatures (generally liquid nitrogen temperatures) and no fixation or staining of the sample is required. A low dose of electron beam is also used to decrease the damage to the sample. To collect high-resolution nanoscale images with low signals from biological samples, sensitive and automated low-dose recording software is used.
Equipment and Software:
Leica Vitrobot – to prepare the sample
FEI Tecnai G2 Twin TEM – to perform Cryo-TEM
Sample Preparation:
Plunge-freeze the specimen using Leica Vitrobot and transfer the frozen grid to Tecnai TEM.
Challenges:
– Freezing conditions need to be optimized for each sample
– Aqueous solutions have surface tension which can cause changes to the structure of the specimen such as flattening artifacts (Ex. Compression of specimen)
– Choice of support film on the grid, aqueous solution and specimen concentration can affect the specimen conditions after the plunge-freeze
Publications:
Cheng, M. H. Y., Leung, J., Zhang, Y., Strong, C., Basha, G., Momeni, A., Chen, Y., Jan, E., Abdolahzadeh, A., Wang, X., Kulkarni, J. A., Witzigmann, D., & Cullis, P. R. (2023). Induction of Bleb Structures in Lipid Nanoparticle Formulations of mRNA Leads to Improved Transfection Potency. Advanced Materials, 35(31), 2303370. https://doi.org/10.1002/adma.202303370
Wadhwa, A., Bobak, T. R., Bohrmann, L., Geczy, R., Sekar, S., Sathyanarayanan, G., Kutter, J. P., Franzyk, H., Foged, C., Saatchi, K., & Häfeli, U. O. (2023). Pulmonary delivery of siRNA-loaded lipid-polymer hybrid nanoparticles: Effect of nanoparticle size. OpenNano, 13, 100180. https://doi.org/10.1016/j.onano.2023.100180