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UVic BIOL 360 - Fall 2019 > Visualizing Cells + Manipulation of Cells (ch 8, 9) > Flashcards

Visualizing Cells + Manipulation of Cells (ch 8, 9) Flashcards

1
Q

What is the limit of resolution for the naked eye? What about light microscopes? Electron microscopes?

A

Eye: ~0.5 mm
Light microscope: ~100nm
Electron microscope: ~0.1 nm

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2
Q

Who coined the term “microscope”?

A

Giovanni Faber.

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3
Q

What is meant by “optical diffraction”?

A

Two light waves out of phase can limit the resolution of a microscope because of interference effects.

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4
Q

Using light microscopy, when do we reach the limit of resolution for two points close together?

A

When the points are less than 200 nm apart.

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5
Q

Resolution is dependent on 2 factors. What are they?

A
  1. Wavelength

2. Numerical aperture

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6
Q

How can we calculate the resolution of a microscope?

A 
= 0.61λ / nsinθ
Where:
n is the index of refraction
θ is the angle of incidence
λ is the wavelength
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7
Q

What is the purpose of the objective lens in a light microscope?

A

Collects a cone of light rays to form an image.

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8
Q

What is the purpose of the condenser in a light microscope?

A

Focuses a cone of light rays onto each point of the specimen.

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9
Q

What differentiates resolution and detection?

A

Resolution: discern details about the specimen
Detection: discern the presence of a subject

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10
Q

What is the resolution given n=1, λ=530nm, θ=20?

A

~950 nm.

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11
Q

What are the 4 basic types of light microscope?

A
  1. Bright field
  2. Dark field
  3. Phase contrast
  4. Differential-interference contrast
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12
Q

What is the main problem with bright field microscopy? How can we fix this? What drawback exists?

A

Specimens are grey-on-grey with no contrast. Can use dyes or stains but have to fix (kill) cells to add colour.

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13
Q

How does dark field microscopy work?

A

Prevent direct light from entering objective, only allow scattered light.

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14
Q

How does phase contrast microscopy work?

A

Waves out of phase generate contrast when combined. The phase shift causes the change in brightness.

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15
Q

How does differential-interference contrast microscopy work?

A

Like phase contrast microscopy, but uses polarized light to give the appearance of a 3D image.

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16
Q

How can we detect small objects that are below the resolution limit of light microscopes?

A
  1. Electron microscopy

2. Fluorescence microscopy (labelling)

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17
Q

In fluorescence microscopy, electrons are _____ at a shorter wavelength and ______ at a longer wavelength.

A

Excited at shorter wavelength, emitted at longer wavelength.

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18
Q

Which fluorescent dye is used to stain all DNA?

A

DAPI.

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19
Q

Which fluorescent dye is used to stain all proteins?

A

FITC.

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20
Q

Which 4 fluorescently labelled antibodies were shown in lecture?

A
  1. Rhodamine B
  2. Alexa 568
  3. Cy5
  4. Cy3
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21
Q

Which 4 fluorescent proteins are often used in genetic engineering?

A
  1. GFP
  2. CFP
  3. YFP
  4. RFP
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22
Q

What is “autofluorescence”? What is a downside of this?

A

A phenomenon where cells can be excited to fluoresce at a specific wavelength. Can interfere with added fluorescence.

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23
Q

What are the 3 main elements of a fluorescent microscope? What light does each part interact with?

A
  1. 1st barrier filter: passes blue
  2. Beam-splitting mirror: reflects blue, passes green
  3. 2nd barrier filter: passes specific green
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24
Q

How does confocal fluorescence microscopy work? How can it be used?

A

Detects only the in-focus light which passes through a pinhole. Can be used to make sections which can be combined to form a 3D image.

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25
Q

What 3 strategies can we use to detect specific molecules in a microscopy sample?

A
  1. Dyes/stains
  2. Antibodies
  3. Genetic engineering: fluorescent proteins
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26
Q

When would you want to use a dye instead of a different identifier (antibodies, GFP, etc.)?

A

When you want to detect all of a specific class of molecule (ex: DNA).

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27
Q

What is most often used as a primary antibody?

A

Rabbit antibody.

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28
Q

What is most often used as a secondary antibody?

A

Sheep antibody.

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29
Q

How do fluorescent-tagged antibodies work?

A

1º antibody detects antigen “A” on cell surface. 2º antibody with fluorescent marker binds to 1º antibody.

30
Q

Do all organisms produce antibodies?

A

No. Only animals, and more specifically vertebrates, make antibodies.

31
Q

How can antibodies be used to identify DNA at centromeres?

A

By targeting the histone proteins that are centromere-specific.

32
Q

How can GFP be used as a reporter sequence?

A

By adding it into a gene, we will observe GFP fluorescence every time that gene is expressed.

33
Q

What strategy can we use to determine intracellular localization of a synthesized protein?

A

Insert a GFP sequence into the gene so that synthesized protein has fused GFP and fluoresces wherever it ends up.

34
Q

What is photoactivated GFP? Why might this be used?

A

GFP which must be activated by light in a certain area. Can be used to visualize protein diffusion.

35
Q

What is photobleaching? Why might this be used?

A

Permanent deacivation of fluorescence. Can be used to visualize protein mobility/diffusion in the cell.

36
Q

Briefly describe fluorescence resonance energy transfer (FRET).

A

Protein interaction can be identified depending on the colour of light which is emitted by the fluorescent proteins.

37
Q

Why might we use total internal reflection (TIRF) microscopy?

A

Gives a crisp image which shows only the surface molecules. Other molecules are invisible.

38
Q

How come electron microscopes have better resolution than light microscopes?

A

Electrons have a shorter wavelength than light.

39
Q

What are some limitations/disadvantages to electron microscopy?

A

Specimen must be dead, can only observe in a vacuum, specimens must undergo complex preparation.

40
Q

How do we prepare specimens for electron microscopy? (4 steps)

A
  1. Fixation (glutaraldehyde or osmium tetroxide)
  2. Dehydration
  3. Permeation with resin
  4. Slice into thin sections
41
Q

How can we label samples for electron microscopy?

A

Not with fluorescence but instead with gold. Density of gold reflects electrons differently to allow contrast.

42
Q

What are some specific characteristics of scanning electron microscopy?

A
  • Cheaper than tEM
  • Looks at surface anatomy only
  • Must coat w/ gold layer otherwise no contrast
  • specimens frozen and dehydrated
  • vacuum chamber required
43
Q

What is the scanning transmission electron holographic microscope (STEHM)?

A

Built at UVic, can create a holographic image of a subject. Best resolution of any microscope. Combines EM with computer tomography.

44
Q

How can we isolate a specific cell from a tissue? What 2 steps are involved?

A
  1. Disrupt extracellular matrix w/ enzymes or EDTA

2. Sort cell types w/ FACS based on charge

45
Q

What is an FACS?

A

Fluorescence activate cell sorter.

46
Q

What is an alternative way to separate specific cells from tissue without using FACS?

A

Laser capture microdissection.

47
Q

How can we isolate organelles from cells?

A

By centrifugation at progressively increasing speeds.

48
Q

What organelles will precipitate when centrifuged at low speed?

A

Whole cells, nuclei, cytoskeletons.

49
Q

What organelles will precipitate when centrifuged at medium speed?

A

Mitochondria, lysosomes, peroxisomes(/chloroplasts).

50
Q

What organelles will precipitate when centrifuged at high speed?

A

Microsomes, small vesicles.

51
Q

What organelles will precipitate when centrifuged at very high speed?

A

Ribosomes, viruses, large macromolecules.

52
Q

How can proteins be isolated from cells?

A

Column chromatography.

53
Q

What are the 3 types of column chromatography, ordered by size of molecule isolated (largest to smallest)?

A
  1. Ion-exchange chromatography
  2. Gel-filtration chromatography
  3. Affinity chromatography
54
Q

Describe ion-exchange chromatography.

A

Positively charged beads in column bind negatively charged molecules.

55
Q

Describe gel-filtration chromatography.

A

Porous beads in column capture small molecules and allow large ones to pass.

56
Q

Describe affinity chromatography.

A

Beads in column have attached substrate and so capture enzymes.

57
Q

What was the “neuronal doctrine” argument?

A

The early 20th cent. disagreement between the idea that 1 cell could grow into a tissue vs the idea that many cells fused to make a tissue.

58
Q

What differentiates a 1º culture from a 2º culture?

A

1º: cells derived from the original donor tissue

2º: re-cultured cells in new colony.

59
Q

In cell biology, how do we define a cell culture described as being “in-vitro”?

A

Parts of cells or fractions in a test tube.

60
Q

In cell biology, how do we define a cell culture described as being “in-vivo”?

A

Regards whole cells, even those which are grown in a dish (different from medical defn.).

61
Q

What human cell line is commonly used for cell cultures? Where does this cell line come from?

A

HeLa cells. Grown from tumour cells taken from Henrietta Lacks (against her knowledge).

62
Q

What is meant by “replicative cell senescence”?

A

Cells can only replicate a limited number of times before they are programmed to die.

63
Q

What is the advantage of replicative cell senescence?

A

Helps to reduce the risk of cancer and unwanted mutations.

64
Q

What replication machinery (related to senescence) do many prokaryotes and other organisms have that human cells lack?

A

Telomerases which can elongate telomeres to preserve replicative ability.

65
Q

What is a hybridoma cell? Why use them?

A

A fusion of cells from 2 different organisms often used for antibody production.

66
Q

What is one major application of hybrid cell use?

A

Gene mapping.

67
Q

What does CRISPR stand for?

A

Clustered regularly interspaced short palindromic repeats.

68
Q

How can CRISPR-Cas9 be used?

A

For gene-editing.

69
Q

In CRISPR, what is the purpose of the Cas9?

A

To cause a double strand break, opening up the genome for transgenic gene integration.

70
Q

How can Cas9 be modified in CRISPR?

A

With an activation or repression domain so that it can influence gene expression instead of causing a double strand break.

UVic BIOL 360 - Fall 2019 (10 decks)

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