Light Microscopy

Question 1

Limit of resolution

Answer 1

how far apart adjacent objects must be in order to be distinguished as separate entities

Question 2

Resolving power:

Answer 2

ability to see fine details of structure. smaller limit of resolution = greater resolving power

Question 3

Bright Field Description

Answer 3

white light directly through specimen, background is illuminated - staining with various dyes enhances contrast

Question 4

BF Pros/Cons

Answer 4

Cheap, simple, easy  unless cell is stained, image has little contrast  specimens have to be chemically fixed (preserved), dehydrated, and stained to see transparent features  dead, so features might not be typical of living cells

Question 5

BF Image produced

Answer 5

Unstained: bright background, barely visible image  stained: bright background, dark coloured cell

Question 6

Phase Contrast - D

Answer 6

light passes through specimen, complex lenses enhance contrast in unstained cells by amplifying variations in refractive index and thickness

Question 7

PC +/-

Answer 7

especially useful for examining living and unpigmented cells  cells can be alive  better for seeing internal structures than bright field

Question 8

DIC D

Answer 8

Same as PC: exaggerate differences in refractive index, split light beam to detect phase differences

Question 9

DIC =/-

Answer 9

Same as PC: can use live cells since you don’t have to stain  more sensitive than PC because uses special prism to split light beam into two separate rays

Question 10

PC image

Answer 10

Dark background, halos around cells  highly contrasting bright and dark areas, evenly illuminated background

Question 11

DIC image

Answer 11

Dark background, has a lighter edge and a darker edge around cell  image appears 3D

Question 12

WF F - D

Answer 12

Short wavelength light excites fluorescent stain within specimen, longer wavelength light is emitted and detected

Question 13

Confocal - D

Answer 13

lasers/special optics to focus illuminating beam on single plane within specimen (same as Wide Field, but uses laser instead of broad source of light)

Question 14

WF +/-

Answer 14

Can detect specific proteins, DNA sequence  can focus on only single plane of specimen at a time  fluorescent light is emitted throughout specimen  light emitted above/below focal plane = blurring

Question 15

Confocal +/-

Answer 15

Only regions within a narrow depth of focus are imaged  regions above/below selected plane of view appear black rather than blurry  better resolution because it excludes out of focus light from the image

Question 16

WF image

Answer 16

Bright image with dark/black background, but kind of blurry

Question 17

Conofcal image

Answer 17

Like wide field, but sharper image: black instead of blurry

Question 18

DNA probes

Answer 18

Collection of single stranded DNA with high affinity for target DNA, and attached to fluorescent molecule