Visualising cells

Question 1

What are the advantages of light microscopy?

Answer 1

light is normally non-destructive to sample

Question 2

What sort of objects can a light microscope be used for?

Answer 2

cells and limited features of sub cellular components

Question 3

what is the smallest size a light microscope can resolve and why?

Answer 3

250nm
due to diffraction limit

Question 4

how does a light microscope work?

Answer 4

light focused on specimen by lenses in condenser
objective and eyepiece lenses focus an image of specimen in eye

Question 5

what are the modes in light microscopy?

Answer 5

bright field
phase contrast
Nomarski differential interference-contrast
dark field

Question 6

why is bright field on a light microscope useful?

Answer 6

can be done while cell is still alive

Question 7

what can dyes be used for in light microscopy?

Answer 7

help locate features in a cell
enhance images

Question 8

how can we enhance contrast?

Answer 8

using dyes or different phases of light

Question 9

what is an example of a dye?

Answer 9

H&E

Question 10

How do fluorescent dyes work?

Answer 10

small molecules or proteins are added to an antibody that bind specifically to a cellular component
light of particular wavelength is shone on sample which emits light at another wavelength for collection

Question 11

what is an antibody?

Answer 11

targeting molecule

Question 12

What is immunostaining?

Answer 12

using antibodies as dyes

Question 13

why can antibodies be used in immunostaining?

Answer 13

they can specifically bind to targets

Question 14

What is confocal microscopy?

Answer 14

very similar to fluorescence microscopy except a laser is used to illuminate a small pinhole whose image is focused at a single point in the 3D specimen

Question 15

what are the advantages of confocal microscopy and why do we have this advantage?

Answer 15

very sharp images obtained as emitted light from elsewhere is not focused so doesn’t contribute

Question 16

what is confocal microscopy used for?

Answer 16

reconstruct objects in cells

Question 17

how does confocal microscopy work?

Answer 17

sample scanned in z direction to take individual images
a stack of separate confocal images is assembled

Question 18

what does GFP stand for?

Answer 18

green fluorescent protein

Question 19

what is GFP?

Answer 19

naturally occurring protein from jellyfish

Question 20

what is GFP used for?

Answer 20

create “fusion proteins”

Question 21

how are fusion proteins formed?

Answer 21

genetic engineering used to attach GFP to other genes

Question 22

what is electron microscopy used for?

Answer 22

resolving smaller structures within cells

Question 23

what must happen in electron microscopy?

Answer 23

sample must be prepared

Question 24

how is a sample prepared in electron microscopy?

Answer 24

thin section of “tissue” is treated with carbon (or other electron dense material)
sample is dehydrated

Question 25

what can preparation of a sample lead to in electron microscopy?

Answer 25

loss in volume and some structure

Question 26

what are the disadvantages of electron microscopy?

Answer 26

large and expensive sample preparation is complex

Question 27

what are the advantages of electron microscopy?

Answer 27

can resolve structures down to ~1nm

Question 28

what methods can be used to scan surface features?

Answer 28

SEM TEM

Question 29

how does SEM work?

Answer 29

electrons are scattered or emitted from surface

Question 30

how does TEM work?

Answer 30

electrons are passed through sample

Question 31

how can contrast be enhanced in SEM/TEM?

Answer 31

using specific agents or gold nanoparticle labelled antibodies

Question 32

how does cryo EM work?

Answer 32

a droplet of pure protein in water is placed on a small EM grid that is plunged into a vat of liquid ethane at 180'C this freezes proteins in a thin film of ice and rapid freezing ensures surrounding water molecules have no time to form ice crystals

Question 33

what can cryo EM be used for?

Answer 33

resolving protein structures and larger macromolecular structures reconstruct macromolecular structures

Question 34

what is suprresolution microscopy used for?

Answer 34

transforming traditional light microscopy to get below the diffraction limit

Question 35

how does superresolution microscopy work?

Answer 35

fluorophores are switched on and off sequentially in different regions of the specimen as a function of time

Question 36

what is an example of a superresolution microscopy technique?

Answer 36

STORM