Microscopes Part 3
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Similarities of LM and EM
- both use photographic film
- some sort of radiation used
- LM: light. EM: electrons
- specimen in a different position: LM: glass slide/ EM: copper grid coated in metal
- direction of light is different
At top, what's happening? Bottom?
- tungsten filament is heated, generating electrons
- at top, cathode attracts electrons
- bottom is an anode
What occurs between cathode and anode?
- voltage, which can be changed
- aode: 0 V
- cathode: modified
The higher the voltage, __
the faster electrons move
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
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
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
Characteristics of Stains
- must have high density
- heavy metals
Consequences of Design of EM
beam of electrosn makes it required that specimen is really thin (0.05 to 0.1 um)
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
Other staining and fixatives
Resolution of TEM
- 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)
- 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)
Magnification with SEM
- not so great (larger specimen needed)
- about 10 nm
High voltage EM
- accelerating voltage
- TEM: 50,000-100,000 V
- HVEM: 200,000- 2 million (penetration power is large)
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
Some microscoopes don't use __. Instead, they use an __, such as __ adn __.
- indirect imaging methods
- scanning probe microscopes and atomic force microscopes
- 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
Similarities between Scanning and Atomic
- tip of probe is a single atom
- 3D surface view (same resolution in air and water)
- tip does not touch specimen
- near atomic resolution (1/10 Angstrom= 10x better than TEM)
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
What ddoes the microscope record?
the movements of the probe
The STPM can be used in __.
Requirement of specimen.
- in air and water
- specimen must be able to conduct electricity; limited in a sense
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
- x direction
EMs use what type of microscopes?
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
Compare the TEM and SEM
- electrons lenses
- screen (not directly seeing specimen)
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