Microscopes Part 2
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What was the phase contrast microscope invented for?
to increase contrast and see better contrast in live cells
advantage of phase contrast microscopes?
increase contrast with living cells as opposed to staining, which kills
How does the PCM enhance contrast?
- enhancing differences in wavelengths that pass through the cell
- different parts of the cell don't have the same densities; so, if we have wavelengths of light, there will be different wavelengths that are seen
Disadvantage of PCM
halo effect, which is an artifact produced by the microscope
- detects movement, location, etc.
- not for internal sstructure
Differential interference Contrast microscope (Nomarski microscope)
- depth was why it was invented
- uses polarized light/ prisms, etc.
- effect is three-D
What is the resolution of the PCM, darkfield, adn DIC microscopes?
- they're all the same as the LM: 250 nm
- they change contrast but not resolution
- can be
- can see
- becomes digital file
- can be modified (take out sound, etc)
- can see things below resolution (what the eye can't perceive, but is still present)
Adv of Video microscopy
see in dim light things your eyes wouldn't see
Video microscopy is __--> can play around with to detect/ enhance more
something not present in cell, but is still produced by lenses
year of operation and place
- first operational one in North America
The PCM is widely used for examining such specimens as __. It is a type of __ that does what?
- biological tissues
- enhances contrasts of transparent and colorless objects by influencing the optical path of light
The PCM is able to show __.
components in a cell or bacteria, which would be very difficult to see in an ordinary light microscope
What does the PCM take advantage of?
the fact that the light passing through a transparent part of the speciment travels slower and, due to this, is shifted compared to the uninfluenced light
True or False:
The difference in shifting wavelengths is visible to the eye.
False: not visible
However, in PCM, the change in phase can be increased to __ by a __ in the microscope and thereby causing a difference in __. This makes the transparent object shine out in contrast to its surroundings.
- half a wavelength
- transparent phase-plate
Why is the PCM a vital instrument in biological and medical research?
- when dealing with transparent and colorless components in a cell, dyeing , although it is an alternative, also stops all processes int he cell
- The PCM allows study of living cells
What is the darkfield microscope for?
- to detect if something is there or not
- you can see movement, where they are, etc. but not for the internal structure
True or False: The darkfield microscope transmits all light that goes through the specimen
- most of the light that goes into the specimen doesn't make it through
For a brightfield microscope (compound light microscope), the light path goes from __. For maximum resolution, __.
The maximum half-angle obtainable is about __.
- condenser to the objective lens
- the half angle of the cone of light entering the objective lens should be as large as possible
- 70 degrees
Explain the principles of fluorescence.
A) Light of a certain energy is absorbed. The electrom jumps from its ground state to an excited state. It returns to the ground state by emitting a photon of lower energy and hence longer wavelength
Explain the optics of the fluorescence microscope.
- Light from the source passes through an exciter filter that transmits only UV light.
- Illumination of the specimen with UV light induces fluorescent molecules in the specimen to emit visible light.
- The barrier filter subsequently removes the UV light, while allowing passage of the visible light. The image is therefore formed exclusively by visible light emitted by fluorescent molecules in the specimen
Explain the exciter filter of the Fluorescent microscope:
screens light and transmits only UV rays; only thing that gets throguh it is the wavelength of light used
Explain the barrier filter of the Fluorescent microscope:
blocks transmission of all UV light, allowing passage of only visible light
Explain fluorescent antibody labelling.
Specific antibodies get fluorescent dyes attached to them and become labeled. They then bind to specific antigens on the surface of the cell
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