Methods in Cell Biology Flashcards
Define the total magnification of a light microscope.
The total magnification of a light microscope is the product of the individual lenses above the specimen.
Define the resolution of a microscope.
More important than magnification:
Resolution is the ability to distinguish between two closely positioned objects.
Resolution of microscope D = 0.61λ/N sinα
How is resolution improved?
Resolution is improved by using a shorter λ (wavelength) of light or gathering more light.
What is the limit of resolution of a light microscope?
Based on limitations on values of α, λ, and N, the limit of resolution of a light microscope is 200 nm (2,000 Å).
What we can see is limited by the _____,
not by the _____!
What we can see is limited by the resolution,
not by the magnification!
Light microscopes can resolve structures that
are _____ apart. The
limitation here is _____.
Light microscopes can resolve structures that
are 200nm (2,000 Å) apart. The
limitation here is the wavelength of light.
Electron microscopes can resolve structures that are _____ apart.
Electron microscopes can resolve structures that are 0.2nm (2 Å) apart.
Describe Förster resonance energy transfer (FRET)
FRET is a specialized method to look at protein-protein and protein-ligand interactions using a dye pair that consists of a donor and acceptor pair of fluorophores.
When the two proteins being studied are in close contact, exciting the donor fluorophore will cause the donor emission to excite the acceptor fluorophore, thereby releasing the emission wavelength of the acceptor fluorophore.
In FRET, why is the donor excitation wavelength shorter than the acceptor excitation wavelength?
What does this mean for the donor emission wavelength?
Excitation wavelengths are shorter than emission wavelengths. Since the acceptor must become excited when in close contact with the donor light emission, the donor emission wavelength must fall within the range of the acceptor excitation wavelength.
Describe the characteristics of a fluorescent microscope.
The fluorescent microscope:
- Exposes a specimen to ultraviolet, violet, or blue light
- Shows a bright image of the object resulting from the fluorescent light emitted by the specimen
Specimens either show autofluorescence (Plant cell wall; chloroplasts) or are stained with fluorophores that emit light that can be detected
Describe the characteristics of a confocal microscope.
- Has an extremely high resolution
- A laser beam is used to illuminate spots on the specimen
- A computer compiles images created from each point to generate a 3-dimensional image
