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molecular basis of disease > microscopy > Flashcards

microscopy Flashcards

1
Q

What is microscopy?

A
  • using microscope to view objects/specimens that are not visible to the naked eye.
  • Hooke, 1665
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2
Q

What are parts of the microscope?

A
  1. detector (PMT, CCD)
  2. objective (+/- immersion medium)
  3. specimen (cover glass)
  4. Light conditioning system
    - kohler illumination
    - phase ring
    - wollaston prism and plarizers
    - filter cubes (for fluorescence)
  5. light source (halogen, XBO)
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3
Q

How are specimen in light microscope kept alive?

A

“the box” controls the conditions to keep specimen alive:
maintenance of CO2 atmosphere , air tight table top.
-O2
-temp

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4
Q

What are problems with experimental time scales?

A
  1. Difference of seconds cause :
    - artefacts in multichannel/4D imaging
  2. stability, viability, possibility of multi-position timelapse.
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5
Q

Define triangle of frustration

A
  1. temporal resolution
    - low exposure time,
    - low pixel number
    - binning
  2. spatial resolution
    - high pixel number
    - no binning
  3. sensitivity
    - high exposure time
    - binning

All detections have their benefits and limitations, what is best depends on the application requirement.

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6
Q

Define binning

A

a technique to boost camera frame rate and dynamics, whilst reducing noise by sacrificing resolution .

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7
Q

What are markings on objectives?

A
  1. magnification : 100x
  2. application : DICH
  3. coverslip thickness
  4. working distance
  5. Numerical aperture/ immersion medium (oil)
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8
Q

What is objective resolution?

A
  • Aperture of the objective determines resolution.

- The higher the numerical aperture the better the resolution power of the objective.

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9
Q

What is the difference between magnification and resolution?

A

resolution does NOT = magnification

  1. Magnification = ability to enlarge the sample
  2. Resolution = ability to clearly distinguish between two points.
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10
Q

What do we use light microscopy for?

A
  1. histology - using antibodies
  2. phase contrast -cell morphology
  3. time -lapse (heart cell differentiation, cell migration)
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11
Q

What is the limitation of light microscopy histology?

A

-gives general pattern but not enough detail.

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12
Q

What is the limitation of phase contrast -cell morphology?

A

-we can only see a fixed timeline and we can’t define how these cells have gone from intact collagen to denatured collagen.

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13
Q

What are 2 types of electron microscopy?

A
  1. Transmission EM (2D)
    2.Scanning EM (3D)
    => hit specimen with beams of electron
    => areas that are more dense (have microtubules), harder for electrons to pass through so appear as darker spots.
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14
Q

Outline the fluorescence absorption and emission cycle.

A
  1. when molecule absorbs light at particular wavelength it will be excited and reach higher level of energy
  2. the high level of energy wont be maintained for too long so releases energy.
  3. At lower energy light is emitted.
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15
Q

What is stroke shift?

A
  • due to energy loss the emitted light is shifted to longer wavelength relative to the excitation light.
  • difference in energy between ye lowest energy of absorbence and highest energy of emission = stroke shift.
  • emitted light wavelength is always longer wavelength than excitation wavelength.
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16
Q

What is photobleaching?

A
  • bleaching of fluorochromes

- due to high intensity illumination the fluorophores might lose permanently their ability to emit light.

17
Q

How can you overcome photobleaching?

A
  • create an environment where we can work with reduced excitation light intensities
  • use shorter exposure
  • use anti-bleaching in our mount.
18
Q

What are Green fluorescent proteins (GFPs) and what are they used for?

A
  • proteins naturally found in light-producing cells of cnidarins
  • GFPs can be fused with other proteins and introduced in cells via transfection. This allows live study of fluorescent tags in living cells
19
Q

What are 2 ways fluorescence can be used as tags?

A
  1. attached to antibodies

2. fused with protein

20
Q

What is the problem with using GFP attached to antibodies?

A
  • you can only visualise those specimens which are fixed so NOT alive
  • alternative way to to visualise live specimen is by using plasmid constructions
21
Q

compare widefield and confocal microscopy

A

+ve: higher z- resolution and reduced out -of-focus- blur make confocal pictures crisper and clearer
-ve : only a small volume can be visualised by confocal microscopes at once. Bigger volumes need time consuming sampling and image reassembling.

22
Q

What are examples of intracellular live imaging?

A
  1. microtubule dynamics (GFP-tubulin)

2. vesicle transport through microtubules (GFP-kinesin)

23
Q

What is the limitation of time lapse light microscopy?

A

Can be hard to differentiate between cardiomyocytes like cells from adipocytes

molecular basis of disease (29 decks)

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