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Unformatted text preview: tory-hair cell examined by scattering of electrons off the sample If you want to “sense” the surface of a cell or subcellular compartment or particle… use Atomic Force Microscopy (AFM) A needle makes physical contact with the submerged (or dry) biological sample and senses its topology by tapping the surrounding areas. AFM images of the cytoplasmic (A) and nucleoplasmic (B) sides of the nuclear envelope, revealing dense packing of NPCs. AFM is also used to manipulate single molecules titan: an elastic muscle protein Useful when you want to study structural changes in a biological sample. If using a flow chamber, one can add a protein/chemical to the sample being examined to monitor structural responses that occur in real -time at high resolution (~5 nm). If you want to look at the fine ultra-structure of a cell… use transmission electron microscopy (TEM) Serial sectioning of epoxy-embedded cells allows a 3-D reconstruction of a cell Cells are fixed with gluteraldehyde, stained with osmium tetroxide and embedded in resin prior to visualization. Not useful for visualization of ‘live’ cells. Electrons pass through the sample and you can see its shadow. Visualizing macromolecules at high resolution by TEM analysis:
Negative-staining (with uranyl acetate, a heavy metal) actin filaments Cryo-electron microscopy allows 3-D reconstruction of homogeneous particles by averaging:
Hepatitis B viruses
Virus constructed by averaging Fit of protein fold into particle contour In cryo-EM cells/samples are not fixed; they are just stained, frozen & visualized under the TEM Useful to determine structure of large cellular particles by averaging images If you want to look at a macromolecule inside a cell, you can also use autoradiography
A pulse chase experiment: Incubate cells momentarily with radioactive molecule,then chase with ‘cold’ molecules and follow fate of radioactivity 3H-thymidine Examples of radioactive molecules incorporation marks cells replicating DNA If you want to look at the specific location of a macromolecule within a cell… use immuno-gold labeling and transmission electron microscopy (TEM)
Useful to localize a protein to a precise location within the cell (to within 10 nm of its location). Before visualization of the gold particles, the cells need to be fixed and processed for EM; thus, this is not appropriate to study cellular processes in ‘real-time’ Read for next class (this week): Read Chapter 8 and Chapter 9 from Alberts et al Also this weekend review Chapter 1, Chapter 2 (first section) & Chapter 3; these contain necessary background that you should remember from Biol20A, Biol20B, Biol100 or BMB100A There are a few Alberts et al text books on reserve in the library...
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