Microscopes Part 3

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Microscopes Part 3
2013-09-14 09:24:30

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  1. Similarities of LM and EM
    • lenses
    • both use photographic film
    • some sort of radiation used
  2. Differences
    • LM: light. EM: electrons
    • specimen in a different position: LM: glass slide/ EM: copper grid coated in metal
    • direction of light is different
  3. EM:
    At top, what's happening? Bottom?
    • tungsten filament is heated, generating electrons
    • at top, cathode attracts electrons
    • bottom is an anode
  4. What occurs between cathode and anode?
    • voltage, which can be changed 
    • aode: 0 V
    • cathode: modified
  5. The higher the voltage, __
    the faster electrons move
  6. Electrons aren't good with __; can be __. 
    WHat does the EM have?
    • penetratinv power
    • can be scattered
    • vacuum that prevents electron dispersion
    • specimen isnt alive
  7. What is the deal with viewing with an EM?
    • can't see electrons and need a viewing screen
    • the specimen can be moved around, but not seen directly 
    • can't see color
  8. When a bean of electrons go through the specimen, what happens?
    • if they go through, they hit detector, making a bright area
    • no penetration means dark, which increases contrast
  9. Characteristics of Stains
    • must have high density
    • heavy metals
  10. Consequences of Design of EM
    beam of electrosn makes it required that specimen is really thin (0.05 to 0.1 um)
  11. Process of staining
    small piece of tissue--> fixative--> dehydrated--> propylene oxide (gets rid of alcohols)--> put in plastic (epoxy) instead of paraffin (too soft)--> sectioned wtih diamond or glass knife--> copper grid in trough of water--> floats up
  12. Other staining and fixatives
    • negative
    • shadow
    • cryofixation
  13. Resolution of TEM
    - wavelength?
    - two values
    • electrons have much shorter wavelength than visible light
    • theoretical : 0.1 and 0.2 nm
    • practical: about 2 nm
    • means you can see at increased magnification (can go up to 250, 000 x)
  14. SEM
    • first part similar to TEM
    • fixative-->  dehydrate--> needs to be totally dry, but no distorition= critical point drying (replace alcohol with another liquid so the whole thing cools down adn gets converted)--> put on little Aluminum post
    • coat specimen with heavy layer of heavy metal to cover up electrons that were in excess (sputter coater does this and creates a vacuum)
  15. Magnification with SEM
    • not so great (larger specimen needed)
    • about 10 nm
  16. High voltage EM
    - accelerating voltage
    • TEM: 50,000-100,000 V
    • HVEM: 200,000- 2 million (penetration power is large)
  17. Advantage of HVEM
    • look at thicker specimen (5 um in thickness)
    • larger specimen can get 3D view of organiells
    • if specimen in isolated chamber, you can look at living cells
    • more similar to TEM
  18. Some microscoopes don't use __. Instead, they use an __, such as __ adn __.
    • lenses
    • indirect imaging methods
    • scanning probe microscopes and atomic force microscopes
  19. Atomic force
    - tip
    • tip touches specimen
    • specimin doesn't have to be an electrical conductor
    • prisms and lasers measure series of probe 
    • design very similar to scanning probe
    • does not require electricity to function
  20. Similarities between Scanning and Atomic
    • tip of probe is a single atom
    • 3D surface view (same resolution in air and water)
  21. Scanning
    - tip
    - resolution
    • tip does not touch specimen
    • near atomic resolution (1/10 Angstrom= 10x better than TEM)
  22. Scanned Tunneling Probe Microscope
    How it works
    • moves in three dimensions
    • a quantum tunneling current set up between probe and specimen
    • strength of current depends on distance from specimen
  23. What ddoes the microscope record?
    the movements of the probe
  24. The STPM can be used in __. 
    Requirement of specimen.
    • in air and water
    • specimen must be able to conduct electricity; limited in a sense
  25. The STM uses __ to scan a metallic tip across the surface of a specimen.
    The point of the tip is composed of __. An __ is produced between the tip and the specimen surface. As the tip scans the specimen in __ and __ directions, __ occurs at a rate dependent on the __ between the tip and the first layer of atoms in the surface. The instrument is designed to move the tip in the __ to maintain a constant current flow.
    • electronic methods
    • one or a few atoms
    • electrical voltage
    • x and y directions
    • electron tunneling
    • distance
    • x direction
  26. EMs use what type of microscopes?
  27. Contrast the TEM and SEM.
    • TEM: light goes through
    • SEM: indirect/ bounces off
    • TEM: beam not inhibited by anything and through vacuum
    • SEM: beam deflector
    • TEM: specimen in the middle
    • SEM: specimen on bottom
    • TEM: electromagnetic lenses
    • SEM: magnetic
  28. Compare the TEM and SEM
    • electrons lenses
    • screen (not directly seeing specimen)