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BIOL 266 > Unit 2: How Cells are Studied > Flashcards

Unit 2: How Cells are Studied Flashcards

1
Q

Optical pathway of compound optical microscope:
1. Iris diaphragm

A

Restricts amount of light entering lens

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2
Q

Optical pathway of compound optical microscope:
2. Condenser lens

A

Focuses the light onto the specimen – no magnification

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3
Q

Optical pathway of compound optical microscope:
3. Objective lens

A

Picks up the light transmitted by the specimen and focus it on the focal plane of the objective lens, creating a magnified image

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4
Q

Optical pathway of compound optical microscope:
4.

A

Reflecting prism

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5
Q

Optical pathway of compound optical microscope:
5. Ocular lens

A

(eyepiece)
The image on the objective focal plane is further magnified by the ocular lens, or eyepiece, and projects it onto the human eye

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6
Q

Total magnification = ____*_____

A

Objective lens * Ocular lens

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7
Q

Most important property of microscope is NOT it’s ______, but it is it’s _____. Why?

A

Magnification,
Resolving power

Because resolution can differentiate between structures at a certain magnification. The clearer the image (more resolution).

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8
Q

Resolution (D) is the

A

ability to see two nearby points as distinct images. The smaller the value of D, the better the resolution.

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9
Q

How is resolution determined?

A

Resolution is determined by the objective lens and its ability to gather the “cone of light” coming from the specimen. The light comes into the objective lens as a cone due to diffraction by the specimen:

D = (0.61* lambda)/(n*sin(alpha))
* lambda is the wavelength of incident light in nm
* n is the refractive index of the medium between the specimen and the objective
* alpha represents half the angle of the cone of light
objective lens specimen

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10
Q

Why does electron microscope have more resolution than a light microscope?

A

Because of electrons wavelength being super tiny, much smaller than light wavelengths.

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11
Q

The lower the wavelength, the better the

A

Resolution

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12
Q

The resolution of such an electron microscope is times greater than that of the light microscope.

A

~100,000

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13
Q

fundamental limitation on all microscopes:

A

A given type of radiation cannot be used to probe details smaller than its own wavelength (l).

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14
Q

The best alpha is ___degrees, why?

A

70 degrees, because it can take 140 degrees of scattered light

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15
Q

1 of 2 ways to partially circumvent the fundamental limitation on all microscopes:

A

By increasing alpha, which will decrease D. Best alpha is thus 70deg

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16
Q

2 of 2 ways to partially circumvent the fundamental limitation on all microscopes:

A

increase the refractive index of the medium between the specimen and the objective lens (n).
(e.g. n = 1.0 for air, n = 1.5 for oil). Thus, using oil increases resolution by 50%.

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17
Q

No matter how many times the image is magnified, the light microscope can never resolve objects that are less than

A

~ 0.2 μm in size (not good enough to see proteins interacting in a cell)

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18
Q

3 types of light microscopy:

A
  1. Brightfield: no contrast. can put a stain but are usually toxic and can only see dead cells. Good for counting cells and see overall observations
  2. Phase-contrast: requires phase plate (light and dark) can play with emergent light
  3. Differential interference contrast (DIC) or Nomarski interference: polarized light used, gives it more dimensions (one side more illuminated than the other). Good for thicker samples.
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19
Q

TEM vs. light microscope: (5 items

A
  1. TEM larder
  2. TEM uses electrons (not light)
  3. Beam of electrons projected downwards (light upwards)
  4. TEM uses electromagnetic coils to focus beam of electrons and to magnify image (light uses glass lenses)
  5. TEM in vacuum (light operates in air).
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20
Q

Optical path of TEM

A
  1. Cathode (heated) to disrupt electrons
  2. Electrons emitted to anode (electric potential of zero, drop in V causes electrons to accelerate towards it
  3. Electromagnetic condenser lens focuses electrons onto specimen plane.
  4. Specimen (thin and dead)
  5. Electromagnetic objective lens picks up electrons passed thru specimen, magnifies the image in focal plane of it.
  6. Electromagnetic projector lens (like ocular lens) picks up electrons from focal plane of obj lens. It then focuses and magnifies them onto specimen detector.
21
Q

Why must TEM specimens be stained?

A

Epoxy slices with elecrons passing through with low atomic number, so details need to be seen with staining of high atomic numbers. It will define parts of organelles, etc.

22
Q

Resolution (D) for TEM =

A

0.2nm; 1000x more resolution than proteins. Allows to see interactions of proteins.

23
Q

Fluorescent microscopy

A

Uses fluorescents of excitation (lower wavelength) and emission of higher wavelength. If a compound is illuminated at it’s excitation wavelength and viewed through a filter (for just excitation wavelength to pass), it is seen to glow against a dark background.

24
Q

Energy of a photon of light is inversely proportional to

A

its wavelength.

25
Q

Three steps of fluorescence

A
  1. Excitation
  2. Energy dissipation
  3. Fluorescence emission
26
Q

Why can’t we inject the fluorescent chemical into cell?

A

Because it would bind to all proteins. So attach it to antibody specific to that protein.

27
Q

Optical path in fluorescence microscope

A
  1. Light source goes through first barrier filter
  2. and 3. Fluorescent dye (fluorescein) in specimen excited to emit light (fluoresce) at a specific (longer) wavelength
  3. Beam splitting mirror reflects light below 510 nm but transmits light above 510nm
  4. Second barrier filter cuts out unwanted fluorescent signals, allowing through excitation wavelengths
28
Q

Confocal microscopy improves

A

Image of conventional fluorescence microscope…

29
Q

Confocal light microscopy optical path

A
  1. Light from a laser passes through pinhole,
  2. dichroic mirror and objective lens focuses the light at specific depth in the specimen.
  3. Emitted light from specimen focused at second confocal pinhole, reaches detector
  4. Emitted light from elsewhere of specimen largely excluded form this second confocal pinhole.
30
Q

Using antibodies to bind to specific proteins (how is it done)?

A

Purify protein X, secondary detects any (other animal) antibody so you don’t have to tag your own every (other animal) antibody. Primary antibody recognizes antigen, wherever it is in cell. Secondary antibody binds to any other animal’s primary antibody.

31
Q

Secondary antibody allows to

A

amplify the signal.

32
Q

Fixation of sample (locks) two main types

A
  1. Cross-linking
  2. Pecipitation (dehydrates sample)
33
Q

Different animals antibodies raised in useful in immunofluoroscopy because:

A

Can tag different parts/components with different colours (make sure different wavelengths).

34
Q

Immunogold electron microscopy

A

Put gold on the antibody because the atomic number is super high. You need to stain it with a high atomic number which will effectively deflect those electrons and show where that antibody is

35
Q

Live cell imaging via

A

green fluorescent protein linkage

36
Q

Subnuclear fractionation steps

A
  1. Disrupt plasma membrane in isotonic solution, on ice to preserve proteins, organelles, etc… Then blend, sonicate or homogenize (grinding manually) but important to leave organelles intact.
  2. Results in suspension of homogenate, extract, lysate.
  3. Differential centrifugation forming pellet (sedimentation) which extracts at rate that depends on size and density.
    -Low speed start, takes bigger things out to fractions. Then speed it up
    -Only nuclei sediment at 10min for 1000g’s
    -Results in post-nuclear supernatant
  4. Next is mitochondria, peroxisomes, lysosomes, golgi (intermediate sizes and densities): sediment at 20min 20000g’s.
37
Q

Differential centrifugation does NOT

A

yield totally pure organelles,

38
Q

Equilibrum density-gradient centrifugation

A

Pellet resuspended in buffer and differentiated organelles strictly via their density by subjecting pellet to a gradient of solute (sucrose) density (top tube lower density and lower in tube higher in density. Densities of the components of pellet separate depending on density at it’s equilibrium position

Then tube is punctured, and fractions are collected.

39
Q

Western blotting analysis steps

A
  1. Separate proteins on a polyacrylamide gel
  2. Transfer the proteins onto a thin membrane
  3. Probe the membrane with a specific antibody
  4. Detect the antibody

Requires an antibody, separated based on molecular size.

40
Q

Gel electrophoresis (2 types)

A
  1. Native gel electrophoresis (PAGE) is without denaturant
  2. SDS PAGE
41
Q

PAGE

A

keeps protein complexes intact, migrating in electric field dependent upon: size, shape, charge.

42
Q

SDS binds

A

Uniformly to proteins

43
Q

SDS PAGE migration in electric field

A

Strictly dependent upon size. Larger proteins migrate through the polyacrylamide gel more slowly while smaller proteins migrate more quickly.

44
Q

A reducing agent (b-mercaptoethanol or dithiothreitol (DTT)) is used in _____ included to break _______

A

SDS PAGE,
Disulfide bonds

45
Q

3 types column chromotography

A
  1. Ion exchange excludes based on charge using salt gradient and beads: non charged pass first, charged will interact and elute slower and begin to increase salt gradient.
  2. Size exclusion (gel filtration) excludes just on molecular size larger ones don’t interact in beads
  3. Affinity chromotography: Specific sequence within the protein due to presence of ligand that binds specific to protein of interest in the column.
46
Q

Three ways purified protein interactions can be studied:

A
  1. Immunoprecipitation
  2. Yeast two-hybrid
  3. FRET (Fluorescence resonance energy transfer)
47
Q

FRET

A

Asking whether Z and Y interact (proteins). So we drag YFP and CFP together and then suggest interaction if the protein is close enough together and energy is only absorbed if it is close enough together. If they are close enough, CFP close enough to YFP will transfer energy and the excitation wavelength will be yellow, implying an interaction.

48
Q

How do you assess the function of the protein?

Gene silencing!

A

facilitated by examining the effects of removing that protein from the cell. One such method is called RNA interference (RNAi).

BIOL 266 (9 decks)

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