Microscopes SEM and Atomic Force Microscopy
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a. primarily to examine surfaces of objects sizing from a virus to an animal head
goal of specimen prep
a. goal of specimen prep: produce an object with same shape and surface properties as the living state, but devoid of fluids since water constitutes high percentage of the weight of living cells and is present in almost every macromolecule
What is the problem with removingwater?
How do we go around this?
- air dryà destroyed by surface tension
- a. they are fixed, passed through alcohols, and then go through critical-point drying
What does critical-point drying take advantage of? How?
- i. the fact that a critical temp and pressure exist for each solvent at which the density fo the vapor is equal to the density of the liquidà surface tension between gas and liquid
- i. solvent of cells replaced with liquid transitional fluid, which is vaporized under pressure to prevent surface tension (usually CO2)
Once dried, it is __ for __.
- coated in metal
- electron beam
Difference of electron use between TEM and SEM
- i. TEM: electron beam focused by condenser lenses to illuminate entire field
- ii. SEM: electrons are accelerated as a fine beam that scans the specimen
Differences in image between SEM and TEM
TEM: 1. electrons pass through the specimen to form image
1. image formed by electrns that are reflected back from the specimen or by secondary electrons given off by specimen after being struck by primary electron beamà strike detector
Image formation is __. Why?
a. indirect; another electron beam scans the surface of the specimen, another scans the face of a cathode-ray tube
a. electrons that bounce off specimen and reach detector control __ in the __ i. the more electrons collected, the __ and the __--> image of __
- strength of the beam
- cathode-ray tube
- stronger the signal to the tube
- greater the intensity of the beam on the screemà
- surface topography
a. provide great range of __
b. resolving power related to __
c. great depth of __ (about 500x that of LM)à __quality
- magnification (about 15-15,000x)
- diameter of the electron beam
a. Atomic Force Microscopy
i. not an __, but is a __ that is important in __and __
ii. operates by __
- high-resolution scanning instrument
- molecular biology
- scanning a sharp, microsized tip (probe) over the surface of the specimen
1. in one type, the probe is attached to __, whose __ changes as tip encounters changes in __-->converted to __ image of the surface of the specimen
- oscillating beam (cantilever)
- frequency of scillations
- 3D topographic
i. provides image of each individual molecule as it is oriented in field
ii. anotherrlimitation of EM and X-Ray crystallographic technologies is __
they are only able to provide static images or “snapshots”
i. the development of high-speed AFM allows __
ii. probe of AFM can be used as more than a __ device; can be employed as a __ to __ in attempt to measure various __properties
- rapid sequence imaging
- push/ pull on the specimen
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