microscopy

Question 1

types of microscopy

Answer 1

• light microscopy
• epifluorescence microscopy
• electron microscopy (scanning, transition, cryo)

Question 2

light microscopy: bright field

Answer 2

• Sample illumination is via transmitted white light from below and observed from above

+ cheap, quick and easy to use.

• magnification power is limited, image contrast, requires sample staining.

Question 3

light microscopy: dark field

Answer 3

• A technique for improving the contrast of unstained, transparent specimens
  + can dramatically improve image contrast (especially of transparent objects) while requiring little equipment setup or sample preparation
• does suffer from low light intensity in final image of many biological samples and continues to be affected by low apparent resolution

Question 4

light microscopy: phase contrast

Answer 4

• Shows differences in refractive index as a difference in contrast
• Circular annulus in the condenser produces a cone of light
• Cone is superimposed on a similar sized ring within the phase- objective
• Every objective has a different size ring, so for every objective another condenser setting has to be chosen

+ Staining not required
- Halos can form which obscures detail

Question 5

resolution

Answer 5

• Resolution = ability to distinguish two very small and closely-spaced objects as separate entities.
• Resolution is best when the distance separating the two objects is short.
• Resolution is determined by certain physical parameters that include the wavelength of light, and the light-gathering power of the objective and condenser lenses.

Question 6

microscopy adaptation: resolution

Answer 6

• The human eye is best adapted for green light and our ability to see detail may be compromised with the use of blue or violet.
• Most manufacturers of microscopes correct their simplest lenses for green light.

Question 7

magnification and imaging