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:
Kulkarni, JA., Darjuan, MM., Mercer, JE., Chen, S., van der Meel, R., Thewalt, JL., Tam, YYC. and Cullis PR. (2018) On the formation and morphology of lipid nanoparticles containing ionizable cationic lipids and siRNA. ACS Nano 12 (5): 4787-4795.
Kulkarni, JA., Tam, YYC., Chen, S., Tam, YK., Zaifman, J., Cullis PR. and Biswas S. (2017) Rapid synthesis of lipid nanoparticles containing hydrophobic inorganic nanoparticles. Nanoscale 9: 13600-13609.
Kulkarni, JA., Myhre, JL., Chen, S., Tam, YYC., Danescu, A., Richman, JM. and Cullis PR. (2017) Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA. Nanomedicine 13 (4) : 1377-1387.