Integration of optical clearing and optical sectioning microscopy for three-dimensional imaging of natural biomaterial scaffolds in thin sections

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

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3-D reconstruction of molecular shape from microcrystal thin sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077311 ◽

1983 ◽

Vol 41 ◽

pp. 748-749

Author(s):

S. Cusack ◽

J.-C. Jésior

Keyword(s):

Three Dimensional ◽

Molecular Shape ◽

Biological Molecules ◽

Fourier Space ◽

Single Particles ◽

Thin Sections ◽

Standard Methods ◽

X Ray ◽

X Ray Crystallography ◽

Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

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Cell Fine Structure as Revealed in Dessicator-Dried Resinless Sections

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099532 ◽

1981 ◽

Vol 39 ◽

pp. 622-623

Author(s):

R. P. Becker ◽

J. J. Wolosewick ◽

J. Ross-Stanton

Keyword(s):

Fine Structure ◽

Tannic Acid ◽

Three Dimensional ◽

Albino Rats ◽

Cell Organelles ◽

Vascular Perfusion ◽

Thin Sections ◽

Carbon Coated ◽

Embedding Medium ◽

Polyethylene Glycol 6000

Methodology has been introduced recently which allows transmission and scanning electron microscopy of cell fine structure in semi-thin sections unencumbered by an embedding medium. Images obtained from these “resinless” sections show a three-dimensional lattice of microtrabeculfee contiguous with cytoskeletal structures and membrane-bounded cell organelles. Visualization of these structures, especially of the matiiDra-nous components, can be facilitated by employing tannic acid in the fixation step and dessicator drying, as reported here.Albino rats were fixed by vascular perfusion with 2% glutaraldehyde or 1.5% depolymerized paraformaldehyde plus 2.5% glutaraldehyde in 0.1M sodium cacodylate (pH 7.4). Tissues were removed and minced in the fixative and stored overnight in fixative containing 4% tannic acid. The tissues were rinsed in buffer (0.2M cacodylate), exposed to 1% buffered osmium tetroxide, dehydrated in ethyl alcohol, and embedded in pure polyethylene glycol-6000 (PEG). Sections were cut on glass knives with a Sorvall MT-1 microtome and mounted onto poly-L-lysine, formvar-carbon coated grids while submerged in a solution of 95% ethanol containing 5% PEG.

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