1) Interpreting what you see is important!2) EM Resolution!3) TEM!4) Membranes!5) Freeze Fracture!6) Metal shadowing!7) Other TEM techniques!8) SEM!!1) What you see!Things to look for to help you identify cells: state of differentiation, nuclear/cytoplasmic ratio, •heterochromatin/euchromatin, basophilic, acidophilic !!2) EM Resolution!EM resolving power > LM resolving power!•limit of resolution= 0.61x wavelength/N.A. (the quality of lens)!•theoretical limit (0.002 nm) can't be achieved for biological specimens (practical= 1nm)!•the greater the resolution, the greater the range of useful magnification-> can get it to within the •resolving power of our eyes!!3) Transmission EM= electrons pass thru specimen to form image, resolution for bio= 1nm!Electrons emitted from cathode (at top), move thru a column in a vacuum, e beam is focused by ◦magnetic coils, image is collected by screen/plate, electron dense areas of specimen scatter electrons and appear dark!Electrons scattered by electron-dense stain are lost, other electrons can get through and form ◦image on screen!Preparation of thin sections!◦Fixation: Glutaraldehyde to cross link proteins, freezing (cyro-preservation)!‣Dehydration: so you can put the specimens in vacuum!‣Embedding: in plastic resin!‣Sectioning: into ultra thin sections electrons can pass through, place on metal grid!‣Staining: add contrast to block electrons ex. immuno-gold, metal shadowing!‣!4) Membranes!Lipid molecules= amphiphilic, hydrophilic polar head, hydrophobic fatty acid tail!•Lipid bilayer!•Membrane proteins perform most of the membrane's specific tasks (transport across membrane, •transmitting info, structural links)!!5) Freeze Fracture!Technique to look at the inner surfaces of cells and membranes!•1) Infiltrate tissue with glycerol (cyroprotectant) and rapidly freeze to 160C 2) fracture frozen tissue •by hitting it with a knife edge 3) Membranes usually fracture along the lipid bilayer!Can view the internal surfaces of the cytoplasmic and extracellular monolayers!•Most proteins retained in the cytoplasmic monolayer (P-face)!•Extracellular monolayer (E-face) shows depressions where proteins used to be !•Used to reveal the protein arrangements of intercellular junctions!•!6) Metal shadowing!Use to look at surface features using a TEM!•1) coat specimen with metal sprayed at an angle 2) get a thicker layer on one side-> shadowing-> •looks 3D 3) Get a metal replica of the surface!!7) Other TEM techniques!Rapid freeze: cells can be viewed as looking as similar as possible to living tissue!•Quick freeze to prevent ice crystals, water forms vitreous ice-> replace with solvents and cont.!◦Deep etching!•Take fractured surface of a frozen specimen and remove the ice by sublimation to look at the ◦features below the surface of the fracture plane!Then metal shadow the tissue!◦Immunogold Electron Microscopy!•Technique to localize specific proteins, tag the secondary antibody with colloidal gold!◦Negative Staining!•to look at the fine details of macromolecules, incubate macromolecs with heavy metal salts- thin ◦layer of electron dense film around electron transparent molecules!EM tomography!•Tilt specimen holder to get multiple views and the computer combines them to form a 3D ◦reconstruction!!8) Scanning EM= electrons hit surface of object and are scattered to form an image, resolution= 10nm!1) Fix or freeze specimen 2) Coat with metal film 3) Electron beam scans the surface 4) Scattered •electrons are quantified to form a surface feature image 5) Number of electrons scattered depends on angle of surface relative to the beam!Electron gun is at the top of the microscope, shoots down through the beam deflector,
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