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BIOL 331 > Unit 1-3 > Flashcards

Unit 1-3 Flashcards

1
Q

What is the “cell doctrine”?

A

The idea that all plant and animal tissues are aggregates of cells.
1p1-05

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2
Q

Bacteria and mitochondria are the smallest entities visible by light microscopy. Any attempt to increase magnification will result in fuzziness, due to what kind of effects?

A

Interference effects.

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3
Q

True or False:

2 waves out of phase will result in a bright light.

A

False.
Will result in a dim light.
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4
Q

What is the objective lens?

A

It collects a cone of light rays to create an image.

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5
Q

What is the condenser lens?

A

It focuses a cone of light rays onto each point of the specimen.
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6
Q

What is the equation for resolution?

A

0.61λ / nsinθ

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7
Q

What is nsinθ?

A 
Numerical aperture (NA).
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8
Q

What does the limit of resolution depend on?

A

Wavelength of light and the numerical aperture.

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9
Q

What does a larger NA mean?

A
  • Better resolution
  • Brighter image
  • Shorter working distance
    1p1-08
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10
Q

True or False:

Structures smaller than 0.2um cannot be observed.

A

False. They can be observed, but they’re always blurred and appear to be at least 0.2um thick.
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11
Q

In order to better visualize cells, what do we need to enhance?

A

Contrast.

1p1-09

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12
Q

True or False:

Unstained cells show very little contrast.

A

True.

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13
Q

What is “differential-interference-contrast (DIC, or Nomarski) microscopy”?

A

A more complex optical system that also exploits interference effects to impart contrast.
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14
Q

What is dark-field microscopy?

A

Light is coming in from the side - only scattered light will enter the objective.
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15
Q

True or False:

Reduction of amplitude = Reduced brightness

A

True.

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16
Q

What are the benefits of using “electronic imaging systems”?

A
  • Can overcome difficulties in detecting very dim light.
  • Detecting small differences in light intensity in a very bright background.
    1p1-12
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17
Q

What does light microscopy require?

A

Samples thin enough for light to pass through them - chromosome spreads are simply “squashed”.
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18
Q

Thicker tissues must be…

A

1) Fixed
2) Embedded
3) Sectioned
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19
Q

What are FIXED tissues?

A

Killed, immobilized, preserved.

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20
Q

What are the most common fixatives?

A

Formaldehyde and glutaraldehyde. They form covalent bonds with free amino groups of proteins.
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21
Q

What are EMBEDDED tissues?

A

Prior to sectioning, tissues are placed in supporting medium (wax or plastic resin).
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22
Q

What is a benefit of fluorescence microscopy?

A

It allows the visualization of specific molecules within cells.
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23
Q

What will happen if a fluorescent compound is illuminated at its absorbing wavelength and viewed through a filter that only allows light of the emitted wavelength to pass?

A

The object will appear to glow brightly against a dark
background.
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24
Q

True or False:

Fluorescent molecules absorb light at one wavelength and emit it at a different, shorter, wavelength.

A

False.
Longer wavelength.
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25
Q

In the fluorescence microscope, what does the first barrier filter do?

A

It lets through only blue light with a wavelength between 450 and 490 nm.
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26
Q

In the fluorescence microscope, what does the second barrier filter do?

A

It cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560 nm.
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27
Q

In the fluorescence microscope, what does the beam-splitting mirror do?

A

It reflects light below 510 nm but transmits light above 510 nm.
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28
Q

DAPI is a fluorescent stain that is specific to which compartment of the cell?

A

DNA.

1p1-17

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29
Q

Fluorescent molecules (such as fluorescein or rhodamine) are more often coupled to molecules that do what?

A

That selectively bind certain proteins within the cell.

1p1-17

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30
Q

What are antibodies and what do they bind to?

A

They’re proteins produced by the vertebrate immune system.
They bind to specific target molecules (antigen).
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31
Q

How are antibodies generated?

A

By injecting an animal with preparation of an antigen (usually purified protein) and collecting the blood serum, which is antibody rich and known as the antiserum (polyclonal).
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32
Q

True or False: Antibodies are always polyclonal.

A

False. Can be polyclonal or monoclonal.

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33
Q

How are monoclonal antibodies recovered?

A

They’re recovered from hybridoma cell lines and can be finicky since they recognize a single antigenic determinant.
1p1-20

34
Q

True or False: Monoclonal antibodies can be produced in unlimited amounts.

A

True.

1p1-20

35
Q

Antibodies can be conjugated to:

A

1) Enzymes
2) Colloidal gold
3) Fluorescent molecules
1p1-20

36
Q

What types of antibodies are used in indirect immunocytochemistry?

A

Labelled (=conjugated) “secondary” antibodies.

1p1-21

37
Q

In conventional light microscopy, what is light from above and below the plane of focus observed as?

A

Out-of-focus blur.

1p1-23

38
Q

What are the 2 different approaches for reducing the blur?

A

1) Confocal (an optical solution)
2) Image deconvolution (a computational solution)
1p1-23

39
Q

What is a Z stack?

A

A series of such optical sections taken at different depths through a specimen.
1p1-23

40
Q

What is a Z stack projection?

A

A reconstructed image of the specimen that is essentially an overlay of many individual Z stack images.
1p1-23

41
Q

How does deconvolution microscopy work?

A

Z stack sections are processed computationally to “deblur” the images.
1p1-24

42
Q

True or False: In confocal microscopy, decreasing the detector pinhole size results in a thinner optical slice.

A

True.

1p1-25

43
Q

The pinhole aperture in front of the detector is confocal with the illuminating pinhole. What does this mean?

A

This means that it’s located at exactly the point that the fluorescence coming from the specimen is focused.
1p1-26

44
Q

Which can penetrate deeper into a specimen: Confocal or deconvolution?

A

Confocal.

1p1-27

45
Q

What is multi-photon imaging?

A

Multi-photon imaging use 2 or more photons of lower energy, BUT they must be w/i ~ a femtosecond of each other.
Can penetrate deeper by using longer wavelength excitation.
1p1-28

46
Q

How is the GFP chromophore formed?

A

Proper protein folding permits formation by autocatalysis.

1p2-4

47
Q

True or False: GFP is an enzyme.

A

False.

1p2-4

48
Q

Why are specific amino acid sequences added to GFP?

A

To target specific subcellular locations.

1p2-4

49
Q

What can GFP be used for?

A
  • Report gene expression.
  • To act as a tag for specific cell types or tissues.
    1p2-5
50
Q

What does a fusion protein result from?

A

Chimeric genes that fuse a gene of interest to the GFP coding sequence.
1p2-5

51
Q

What can GFP fusion proteins be used for?

A

To observe subcellular localization of specific proteins in living cells.
1p2-5

52
Q

Why live-cell imaging?

A
  • High resolution imaging of live tissues (or cells) reveals phenomena & structures that are difficult to observe in fixed material.
  • Can interrogate a protein/organelle in real time.
  • Highlight movement/changes in morphology while cells are doing everything that need to do.
    1p2-6
53
Q

How can GFP and GFP spectral variants be used to evaluate molecular interactions?

A

Via Fluorescence Resonance Energy Transfer (FRET).

1p2-8

54
Q

Two molecules of interest are labelled with different fluorophores, with the emission spectrum of one overlapping with the excitation spectrum of the other. If the 2 fluorophores are in close proximity, what does that mean?

A

It means there can be a transfer of energy from the first fluorophore to the second.
1p2-8

55
Q

If there are no protein interactions in FRET, what colour light would you detect?

A

No excitation of GFP, therefore, blue light detected.

1p2-9

56
Q

If there ARE protein interactions in FRET, what colour light would you detect?

A

Fluorescence resonance energy transfer, therefore, green light detected.
1p2-9

57
Q

What is photoactivation used for?

A

To visualize protein trafficking. Can control exactly where/when the molecule becomes fluorescent.
1p2-10

58
Q

What can happen if a portion of a cell is exposed to the light source/laser?

A

Can lead to photobleaching of the fluorescent marker.

1p2-11

59
Q

What can we determine from the recovery of fluorescence (FRAP) within a bleached area?

A

The rate of movement of fluorescently-labelled molecules.

1p2-11

60
Q

What are 2 types of fluorescence “indicators”?

A

1) Ca2+ indicators
2) Indicators of intracellular pH
1p2-13

61
Q

What are some examples of supperresolution microscopy?

A
  • SIM
  • STED
  • PALM
  • STORM
    1p2-14
62
Q

What is the purpose of the electron microscope?

A

To resolve ultrastructure of cell.

1p2-15

63
Q

Resolution is a function of ______________?

A

Wavelength.

1p2-15

64
Q

Describe TEM (Transmission Electron Microscopy)?

A
  • The cathode emits electrons from top of column.
  • Electrons pass through a tiny hole, creating an electron beam, focused by magnetic coils.
  • Electrons may pass directly through specimen or be scattered by electron dense structures.
    1p2-16
65
Q

In TEM, why do you need a vacuum?

A

Because electrons will collide with air molecules and scatter.
1p2-16

66
Q

What are the steps towards preparing samples for TEM?

A
  • Samples must be thin.
  • Fixation
  • Embedding
  • Cutting sections
  • Staining
    1p2-17
67
Q

Why are specimen sections stained with electron-dense material to achieve contrast?

A

Tissue composed of atoms of low atomic number (C, O, N, H) = low contrast by TEM.
Make them visible by staining with salts of heavy metals (=high contrast by TEM).
1p2-17

68
Q

What does flash freezing tissue result in?

A

Non-crystalline vitreous ice (a glass) such that ice crystals do not disrupt structure.
1p2-18

69
Q

What is the method of choice for immuno EM?

A

Cryo-fixation.

1p2-18

70
Q

What is immunogold EM?

A

Secondary antibodies can be attached to very small particles prepared from colloidal gold.
1p2-19

71
Q

True or False: In EM immunolabelling, only the exposed surface of the thin section is accessible to antibody labelling.

A

True.

72
Q

What is serial reconstruction?

A

If you’re really good at sectioning, it’s possible to recover several consecutive ribbons of ultra-thin sections.
From this, a 3-D image can be projected.
1p2-20

73
Q

What is 3D EM reconstruction (tomography)?

A

Information of the third dimension can be recovered by viewing the specimen from several different angles.
1p2-21

74
Q

How does SEM work?

A

SEM detects electrons that are scattered or emitted from the surface of a specimen.
1p2-22

75
Q

What is the resolution of SEM?

A

~10 nm.
20,000X magnification.
1p2-22

76
Q

How can large macromolecules be visualized?

A

If they’re shadowed with heavy metal.

1p2-24

77
Q

What must be done to molecules in order for them to be visualized at high resolution?

A

Molecules to be visualized are biochemically purified, mixed with an aqueous solution of heavy metals, spread over a thin carbon film, and then dried.
1p2-24

78
Q

When the electron dense stain is absorbed by the background but not the molecules of interest, what do the molecules appear as (light or dark)?

A

The molecules of interest appear as light areas surrounded by dark.
1p2-24

79
Q

What is single particle reconstruction?

A

An averaging method of taking tens of thousands of images of the same molecule combined to produce an “average” image.
Will reveal structures hidden by “noise”.
1p2-25

80
Q

What is “noise”?

A

Inherent random variability that obscures the underlying image.
1p2-25

BIOL 331 (1 decks)

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