Information
A major objective of modern structural biology is to decipher the cellular organization by elucidating the spatial arrangement of macromolecular complexes within a cell. Three-dimensional studies combined with cryo-preparation methods in electron microscopy enable the study of biological specimens in a quasi in vivo “hydrated” and “three-dimensional” state.To achieve the close-to-native state of the specimen for electron microscopy, samples have to be cryo-immobilized, either by plunge freezing, impact freezing, self-pressurized freezing or high pressure freezing (HPF). These methods vitrify the water in the sample and immobilize the cell content almost immediately. The goal is to obtain “vitreous” or amorphous ice after ultra-rapid freezing, without destroying cell structures and preserving a precise moment of their interactions, which are rapidly changing in vivo. Vitrification allows spatial and temporal resolution of cellular events.
Once the cells are immobilized by freezing, two strategies can provide the three-dimensionality of the structures of interest: Single particle analysis and electron cryo-tomography. Single particles are isolated macromolecules, molecular assemblies, small organelles, small cellular structures or small organisms not needing sectioning and the procedure carried out is the reconstruction from the different orientations of the single particle on an electron microscopy grid. Electron cryo-tomography refers to the three-dimensional reconstruction of isolated bulk structures or cryo-sections from them, acquiring images at low temperature and in as many directions of the electron beam as possible. This is achieved by tilting the holder supporting the grid containing the specimen and recording images at regular tilt intervals. Recently, the use of the dual-beam electron microscopes has been showed like a very good device for preparing samples for electron cryo-tomography.
On the other hand, freeze substitution, together with other techniques, like Tokuyasu and the news rehydration and VIS2FIX methods, can also be combined with on-section immunogold labelling for protein localization studies with reliable morphology. A special mention and discussion will be the CLEM (correlative light and electron microscopy) methods and the first results about a clonable tag for electron microscopy.
Date: 07/10/2013
Hour: 10:30h a 11:30h
Location: Aula dels CCiTUB. Edifici CCiTUB, 1ª planta. c/Lluís Solé i Sabarís, 1-3. Barcelona
Fee: Free
Speaker: Carmen López, Electron cryomicroscopy technology of the CCiTUB
|
|
