Cryo-tomography
In material and life sciences, electron tomography (ET) has been extensively used for 3D imaging at the nanoscale. Low temperature, Low-dose ET (cryo-electron tomography) is employed for the nanoscale imaging of soft, electron beam sensitive materials.
Figure 1: Principle of electron tomography; enables the study of non-repetitive structures with unique topologies. A set of 2D projection images is recorded while object is tilted around an axis perpendicular to the electron beam[1].
One constraint of cryoTEM is that multiple features are overlapped and cannot be distinguished when the images are obtained as 2-dimensional projections of the 3-dimensional objects. Cryo-ET technique overcomes this restriction by iteratively imaging specimens at different tilt angles and reconstructing into a 3D object [1]. Detailed information on the structure, morphology or 3D spatial organization of bio(macromolecules) and macro(molecular) assemblies then can be obtained.
Figure 2:TEM analysis of aggregates of PNOEG–PNGLF 1. a) Conventional TEM using negative staining; b) cryoTEM image of a vitrified film; c) gallery of z slices showing different cross sections of a 3D SIRT (simultaneous iterative reconstruction technique) reconstruction of a tomographic series recorded from the vitrified film in (b); d,e) visualization of the segmented volume showing d) a cross section of the aggregate and e) a view from within the hydrated channels[2].
Tomographic reconstruction of PNOEG–PNGLF aggregates
Cryo-electron tomography experiments with different degrees of complexity were used to investigate the self-assembly of macromolecular systems. With dr. S. J. Holder (University of Kent, UK) complex morphologies of comb-type block copolymers were investigated with cryo-tomography(Figure 2)[2]. Here, advanced image processing involving segmentation and skeletonization played a crucial role in the visualization of the resulting morphologies, which also revealed the temperature dependent aggregation behavior[3].
Figure 3: TEM analysis of PEO39-b-PODMA17 aggregates: (a) cryoTEM image of a vitrified film at 4 °C; (b) gallery of z slices showing different cross sections of a 3D SIRT reconstruction of a tomographic series recorded from the vitrified film in (a); (c) computer-generated 3D visualization showing only an inner section of the whole structure, where all the channels and compartments are visible; (d) skeletonization of (c);(e) cryoTEM image of a vitrified film at 45 °C; (f) slice from a 3D SIRT reconstruction of a tomographic series recorded from the vitrified film in (e); and (g) volume rendering of the whole structure in (e), showing the overall morphology.[3].
In collaboration with the group of prof. E.W. Meijer (TU/e), the formation of dynamic networks of aggregated hydrophobically modified dendrimers were investigated by cryo-electron tomography for which box-counting analysis revealed the mode of aggregation [4].
References
- F. Nudelman, G. De With, N. A.J.M. Sommerdijk, Cryo-electron tomography: 3-dimensional imaging of soft matter, Soft Matter, Advanced article, (2011), in press.
- A.L. Parry, P.H.H. Bomans, S.J. Holder, N.A.J.M. Sommerdijk, S.C.G. Biagini, Cryo-Electron Tomography reveals Confined Complex Morphologies from Tripeptide-containing Amphiphilic Double Comb Diblock Copolymers, Angew Chem. Int Ed. 47 (2008) 8859-8862.
- B.E. McKenzi, F. Nudelman, P.H.H. Bomans, S.J. Holder, N. Sommerdijk, Temperature-responsive nanospheres with bicontinuous internal structures from a semicrystalline amphiphilic block copolymer, J. American Chemical Society 132 [30] (2010) 10256-10259.
- T.M. Hermans, M.A.C. Broeren, N. Gomopoulos, , P.P.A.M. van der Schoot , M.H.P. van Genderen, N.A.J.M. Sommerdijk, G. Fytas, E.W. Meijer, Self-assembly of soft nanoparticles with tunable patchiness, Nature Nanotechnology, 4(11), (2009)721-726.