EXAM 1: LO 6

Question 1

Microscopy

Answer 1

the use of light or electrons to magnify objects

Question 2

Wavelength

Answer 2

the distance between two corresponding parts of a wave

Question 3

Magnification

Answer 3

the apparent increase in the size of an object

Question 4

Resolution

Answer 4

the ability to distinguish between two objects that are close together

Question 5

Contrast

Answer 5

the difference in intensity between two objects, or between an object and its background

Question 6

Optical sectioning

Answer 6

the process of creating a 3D reconstruction of a specimen from images captured at different focal planes.

Question 7

Explain Wavelength of radiation

Answer 7

• (NON IONIZING) Radiation = traveling energetic particles of energy
• Disruption of wavelength is what allows us to see an image
• The smaller the wavelength, the smaller the object can be that disrupts it
• Smaller the wavelength, the higher the resolving power (ability to distinguish between two objects)

Question 8

Explain magnification

Answer 8

• Enlargement of appearance
• Includes the refractive index: a measure of how greatly a substance slows the velocity of light

Question 9

What is refractive index?

Answer 9

a measure of how greatly a substance slows the velocity of light

Question 10

What is resolution?
What is the trend that’s associated with it?

Answer 10

• Distinguishing between objects/points
• The shortest distance between two point that can still be distinguished by observer as separate entities
• Increasing resolution = increasing the amount of information into lens = better image & ability to distinguish between 2 objects

Question 11

What is contrast?

Answer 11

• Important in determining resolution
• Staining increases contrast
• Use of light that is in phase increases contrast
• Difference in intensity between two objects, or between an object and background

Question 12

Explain how the use of oil affects microscopy

Answer 12

• Oil is there to reduce change in density as light travels through the air (keeps resolution high and produces sharper image as magnification increases
• Oil immersion lens increases resolution & numerical aperture

Question 13

Bright field microscopy

Answer 13

• Light passes through the specimen into the objective lens
• Can have 1 or 2 ocular lenses
• Most have a condenser lens (direct light through the specimen → increases the intensity of light)

Question 14

Dark field microscopy

Answer 14

• Best for observing pale objects (light is inverted) → the specimen appears light against dark background
• Only light rays scattered by the specimen enter the objective lens
• Increases contrast and enables observation of more details
• For a pale specimen, you would have to kill the organism to stain it to see it with a bright field.
• For a dark field, you wouldn’t need to kill the organism

Question 15

Phase contrast microscopes

Answer 15

• Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining
• Light rays “in phase” produce a brighter image. Light rays “out of phase” produce a darker image
• Contrast is created because light waves are out of phase

Question 16

Fluorescent Microscope

Answer 16

• Direct UV light source at the specimen
• Specimen radiates energy back as a longer, visible wavelength
• UV light increases resolution and contrast
• Some cells are naturally fluorescent others must be stained
• Used in immunofluorescence to identify pathogens and to make visible a variety of proteins

Question 17

Confocal microscopy

Answer 17

• Uses fluorescent dyes
• Uses UV lasers to illuminate fluorescent chemicals in a single plane
• Resolution increased because emitted light passes through pinhole aperture
• Allows for optical sectioning
• Computer constructs 3D composite images from digitized images

Question 18

Discuss the similarities and differences between light microscopy, confocal microscopy, and electron microscopy

Answer 18

• Dependent on the wavelength and resolving power
• Electron microscopes differ from light microscopes in that they produce an image of a specimen by using a beam of electrons rather than a beam of light.

Question 19

What makes electron microscopy special?

Answer 19

• Electrons have much a shorter wavelength than visible light, and this allows electron microscopes to produce higher-resolution images than standard light microscopes.

Question 20

What are the 2 types of electron microscopy?

Answer 20

Transmission Electron Microscope (TEM)
Scanning Electron Microscope (SEM)

Question 21

Transmission Electron Microscope (TEM)

Answer 21

• Can not be used to study living organisms
• Preparation can cause artifacts
• Electrons scatter when they pass through thin sections of a specimen
• Transmitted electrons are under vacuum which reduces scatter and are used to produce a clear image
• Denser regions in the specimen, scatter more electrons and appear darker

Question 22

Scanning Electron Microscope (SEM)

Answer 22

• Uses electron beam to scatter electrons from the surface of a specimen to create detailed image → Produces a realistic 3D image of a specimen’s surface features
• No sectioning required
• The whole organism can be viewed
• No internal structures

Question 23

Staining for Electron Microscopy

Answer 23

• Chemicals containing heavy metals used for TEM
• Stains may bind molecules in specimens or the background

Question 24

List the general principles of microscopy

Answer 24

1) Wavelength of radiation
2) Magnification
3) Resolution
4) Contrast

Question 25

gram stain procedure

Answer 25

1) crystal violet (primary stain) for 1-minute → water rinse
2) iodine (mordant) for 1-minute → water rinse
3) alcohol (decolorizer)for 30s → water rinse
4) safranin (counterstain) for 1-minute → water rinse → blow dry

Question 26

in the third step of gram staining, how do you tell if its gram + or gram -? What about the 4th step?

Answer 26

3. Gram + = purple / Gram - = colorless
4. Gram + = purple / Gram - = pink