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211: Cell Biology > 1. Imaging in cell biology > Flashcards

1. Imaging in cell biology Flashcards

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1
Q

define resolution

A

the smallest structure you can see

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2
Q

what is the resolution of a light microscope

A

200 nm

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3
Q

what is the resolution of a TEM

A

0.1 nm

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4
Q

what is the resolution of a SEM

A

1 nm

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5
Q

what is a limitation of brightfield microscopy

A

samples must be preserved, sectioned and embedded. This process alters cell structure, so only gives a snapshot of dead cells

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6
Q

what is the purpose of ocular and objective lens’

A

magnify images
ocular lens = x10
objective lens and ocular couple to produce high mag.

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7
Q

what is the function of a microtome

A

sections specimens which have been preserved and embedded

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8
Q

what is a cryo-microtome

A

a microtome inside a freezer, can freeze tissues without using chemicals

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9
Q

why is a cryo-microtome good in immunolabelling

A

does not use chemicals to preserve tissues - only freezing

this does not alter epitope structure and so antibody binding is unaffected

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10
Q

why use a chemical stain (2 points)

A
  • to see ultrastrucrure detail

- most cellular material is not capable of absorbing light

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11
Q

how do chemical stains work

A

bind to a class of molecules within a cell and absorb light, generating a visual image

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12
Q

give two examples of stains

A

haematoxylin and eosin

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13
Q

why do you fix tissues

A

prevents the autolysis

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14
Q

what is a common tissue fixative

A

formaldehyde

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15
Q

how thick are specimens generally sectioned

A

5 microns thick

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16
Q

how does fluorescent microscopy work

A

ultraviolet light excited fluorochrome in the dye, making it fluoresce

17
Q

how does SYBR green work

A

binds to the groove in double stranded DNA, when a laser is shone on it, it emits lights = allows measure conc of ds DNA

18
Q

advantages of fluorescent microscopy: 2

A
  • cells may be fixed or living

- many fluorescent dyes can be used at once to label different cellular components

19
Q

what are the limitations of fluorescent microscopy

A

the whole of the cell will fluoresce - creating a blurry image

20
Q

what is immunolabelling used for

A

to identify and localise a particular antigen in a tissue

21
Q

how do primary and secondary antibodies work

A

the primary antibody binds directly to the antigen

the secondary antibody is marked with fluorescent dye and binds to the primary antibody making them visible

22
Q

how can we immunolabel the cytoskeleton

A

actin filaments can be stained with rhodamine-labelled phalloidin, this tag fluoresces red

microtubules can be stained with a tag that fluoresces green

23
Q

what is the advantage of confocal microscopy

A

facilitates optical sectioning - we get fluorescence from only a section of the cell

produces a sharp image

24
Q

what is a limitation of confocal microscopy

A

very expensive

25
Q

what can you study with advanced light microscopy

A

transparent living cells

26
Q

TEM:

  • how does it operate?
  • the state of the specimen
  • what are specimens stained with
  • how are the electrons released and focused
A
  1. under vacuum
  2. dead > fixed, embedded, sectioned, stained
  3. electron-dense materials (e..g. lead and uranium)
  4. released from tungsten filament, focused using magnetic lenses.
27
Q

how thick are TEM sections

how do we cut them

A

ultrathin = 50 nm

using a ultramicrotome

28
Q

what does metal shadowing allow us to visualise with TEM

A

viruses, molecules, cell components

-> coats them with platinum

29
Q

what are TEM grids coated with

A

formvar - allows small samples to be mounted onto the grid

30
Q

what is the blade used on a ultramicrotome

A

glass or diamond

31
Q

describe cryo-electron microscopy

A

uses frozen biomolecules (usually proteins), electron beam pass through and structure inferred

32
Q

describe x-ray crystallography

A

x-rays scatter as they pass through a crystallised protein, waves interfere creating a diffraction pattern, this shows the atom position

33
Q

limitation of x-ray crystallography

A

can’t focus x-rays, must use crystallised sample to produce a diffraction pattern

34
Q

SEM:

  1. how does it operate
  2. resolution
  3. image
  4. state of specimen
A
  1. under vaccum
  2. 1 nm
  3. 3D
  4. dead (under vacuum): fixed, dehydrated, gold coated
35
Q

perk of SEM:

A

only method of visualising nanoparticles

36
Q

limitation of SEM:

A

resolution limited due to thickness of metal coating

37
Q

what does a dichroic mirror allow

A

selectively observe emitted fluorescence

38
Q

how does formaldehyde work

A

cross-links amino groups on adjacent molecules

211: Cell Biology (20 decks)

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