Towards high-resolution three-dimensional imaging of native mammalian tissue: electron tomography of frozen-hydrated rat liver sections.
Cryo-electron tomography of frozen-hydrated specimens holds considerable promise for high-resolution three-dimensional imaging of organelles and macromolecular complexes in their native cellular environment. While the technique has been successfully used with small, plunge-frozen cells and organelles, application to bulk mammalian tissue has proven to be difficult. We report progress with cryo-electron tomography of frozen-hydrated sections of rat liver prepared by high-pressure freezing and cryo-ultramicrotomy. Improvements include identification of suitable grids for mounting sections for tomography, reduction of surface artifacts on the sections, improved image quality by the use of energy filtering, and more rapid tissue excision using a biopsy needle. Tomographic reconstructions of frozen-hydrated liver sections reveal the native structure of such cellular components as mitochondria, endoplasmic reticulum, and ribosomes, without the selective attenuation or enhancement of ultrastructural details associated with the osmication and post-staining used with freeze-substitution.
Hsieh
Chyong-Ere
CE
Resource for Visualization of Biological Complexity, Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, 12201, USA.
Leith
ArDean
A
Mannella
Carmen A
CA
Frank
Joachim
J
Marko
Michael
M
D000818:Animals; D004721:Endoplasmic Reticulum / Q000648:ultrastructure; D005629:Frozen Sections / Q000379:methods / Q000592:standards; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008099:Liver / Q000648:ultrastructure*; D046032:Microscopy, Energy-Filtering Transmission Electron / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D008928:Mitochondria / Q000648:ultrastructure; D051381:Rats; D012270:Ribosomes / Q000648:ultrastructure; D014057:Tomography, X-Ray Computed / Q000295:instrumentation / Q000379:methods* / Q000592:standards