Cryo-TEM and Material Science Analysis
A wealth of possibilities for the analysis of nanostructures emerged not only by the recent advances in TEM methodology and instrumentation, but also by their dissemination and adoption by various scientific disciplines[1]. TEM combines its high spatial resolution probe with the analytical techniques such as selected-area electron diffraction (SAED), scanning transmission electron microscopy (STEM), electron energy loss spectroscopy (EELS) and energy-dispersive X-ray spectroscopy (EDX), merging biological and materials science approaches to bring complete information about composition and structure of the specimen.
One of the main limitations of conventional TEM, is in the study of biological or synthetic hydrated specimens. The high vacuum of the microscope requires preparation methods that involve dehydration and chemical staining, which causes deformations and even the collapse of macromolecular architectures. Hence, by using cryogenic techniques for sample preparation, specimens are observed embedded in a thin layer of vitreous ice, which, combined with phase-contrast imaging, overcomes the need for chemical fixation and dehydration. As a result, specimens can be investigated in a state of preservation that is close to native, ensuring the preservation of the molecular structures. In combination with material science methods such as SAED, STEM and EELS, we are able to investigate in detail processes such as biomimetic mineralization, polymer structure and assemblies, etc.
Figure: Uranyl acetate map of the different stages of collagen mineralization in the presence of 10 g ml-1 of pAsp. A) CryoTEM image of stained, non-mineralized collagen. B) CryoTEM image of stained collagen, mineralized for 24 h. Calcium phosphate is associated with the fibril in a regular pattern, following the staining bands (black arrows). Peaks are labelled corresponding to their respective staining band. C) Intensity profile of A, non-mineralized collagen. D) Intensity profile of B, collagen mineralized for 24 h[3].
For the formation of CaCO3 in the presence of ammonium ions, the combination of cryo-tomography with low dose selected area electron diffraction (SAED) and dark field imaging revealed the details of the amorphous to crystalline transition[2]. Even more advanced analysis was used in the study of collagen mineralization (Figure). Here cryo-ET was employed together with electron diffraction, cryogenic positive staining, image analysis and molecular modeling to yield a nanometer detailed model of the interaction between the collagen and the mineral[3]. This study revealed that collagen functions in synergy with the inhibitors of hydroxyapatite nucleation to actively control mineralization.
References
- H. Friedrich, P.M. Frederik, G. de With, N.A.J.M. Sommerdijk, Imaging of self-assembled structures: Interpretation of TEM and Cryo-TEM images, Angewandte Chemie, International Edition, 49 [43] (2010) 7850-7858.
- E.M. Pouget, P.H.H. Bomans, A. Dey, P.M. Frederik, G. de With, N. Sommerdijk, The development of morphology and structure in hexagonal vaterite, J. American Chemical Society 132 [33] (2010) 11560-11565.
- F. Nudelman, K. Pieterse, A. George, P.H.H. Bomans, H. Friedrich, L.J. Brylka, P.A.J. Hilbers, G de With, N.A.J.M. Sommerdijk, The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors, Nature Materials 9 [12] (2010) 1004-1009.