1. Imaging in cell biology

Question 1

define resolution

Answer 1

the smallest structure you can see

Question 2

what is the resolution of a light microscope

Answer 2

200 nm

Question 3

what is the resolution of a TEM

Answer 3

0.1 nm

Question 4

what is the resolution of a SEM

Answer 4

1 nm

Question 5

what is a limitation of brightfield microscopy

Answer 5

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

Question 6

what is the purpose of ocular and objective lens’

Answer 6

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

Question 7

what is the function of a microtome

Answer 7

sections specimens which have been preserved and embedded

Question 8

what is a cryo-microtome

Answer 8

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

Question 9

why is a cryo-microtome good in immunolabelling

Answer 9

does not use chemicals to preserve tissues - only freezing

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

Question 10

why use a chemical stain (2 points)

Answer 10

• to see ultrastrucrure detail

- most cellular material is not capable of absorbing light

Question 11

how do chemical stains work

Answer 11

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

Question 12

give two examples of stains

Answer 12

haematoxylin and eosin

Question 13

why do you fix tissues

Answer 13

prevents the autolysis

Question 14

what is a common tissue fixative

Answer 14

formaldehyde

Question 15

how thick are specimens generally sectioned

Answer 15

5 microns thick

Question 16

how does fluorescent microscopy work

Answer 16

ultraviolet light excited fluorochrome in the dye, making it fluoresce

Question 17

how does SYBR green work

Answer 17

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

Question 18

advantages of fluorescent microscopy: 2

Answer 18

• cells may be fixed or living

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

Question 19

what are the limitations of fluorescent microscopy

Answer 19

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

Question 20

what is immunolabelling used for

Answer 20

to identify and localise a particular antigen in a tissue

Question 21

how do primary and secondary antibodies work

Answer 21

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

Question 22

how can we immunolabel the cytoskeleton

Answer 22

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

microtubules can be stained with a tag that fluoresces green

Question 23

what is the advantage of confocal microscopy

Answer 23

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

produces a sharp image

Question 24

what is a limitation of confocal microscopy

Answer 24

very expensive

Question 25

what can you study with advanced light microscopy

Answer 25

transparent living cells

Question 26

TEM: - how does it operate? - the state of the specimen - what are specimens stained with - how are the electrons released and focused

Answer 26

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.

Question 27

how thick are TEM sections | how do we cut them

Answer 27

ultrathin = 50 nm | using a ultramicrotome

Question 28

what does metal shadowing allow us to visualise with TEM

Answer 28

viruses, molecules, cell components | -> coats them with platinum

Question 29

what are TEM grids coated with

Answer 29

formvar - allows small samples to be mounted onto the grid

Question 30

what is the blade used on a ultramicrotome

Answer 30

glass or diamond

Question 31

describe cryo-electron microscopy

Answer 31

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

Question 32

describe x-ray crystallography

Answer 32

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

Question 33

limitation of x-ray crystallography

Answer 33

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

Question 34

SEM: 1. how does it operate 2. resolution 3. image 4. state of specimen

Answer 34

1. under vaccum 2. 1 nm 3. 3D 4. dead (under vacuum): fixed, dehydrated, gold coated

Question 35

perk of SEM:

Answer 35

only method of visualising nanoparticles

Question 36

limitation of SEM:

Answer 36

resolution limited due to thickness of metal coating

Question 37

what does a dichroic mirror allow

Answer 37

selectively observe emitted fluorescence

Question 38

how does formaldehyde work

Answer 38

cross-links amino groups on adjacent molecules