Techniques in Cell Biology

Question 1

What are the two types of microscopes?

Answer 1

• Light microscopy

- Electron microscopy

Question 2

What is a light microscope?

Answer 2

Uses physical light to visualise sample

Question 3

What is an electron microscope?

Answer 3

Uses electrons due to electrons being more sensitive to air molecules which the reactive with immediately when hit

Question 4

What is optical resolution limit?

Answer 4

Minimum distance that allows recognition of object details

Question 5

What does optical resolution depend on?

Answer 5

Wavelength of the light/beam used

Question 6

Is you have a smaller wave length is the resolution better or worse?

Answer 6

Better

Question 7

Is you have a larger wave length is the resolution better or worse?

Answer 7

Worse

Question 8

What microscope uses a visible light?

Answer 8

Light microscopy

Question 9

What microscope uses an electron beam?

Answer 9

Electron microscopy

Question 10

What is the wavelength of the visible light in light microscopy?

Answer 10

390-700 nm

Question 11

What is the wavelength of the electron beam in electron microscopy?

Answer 11

0.0025nm

Question 12

What is used to focus light in light microscopy?

Answer 12

Glass lenses

Question 13

What is used to focus the beam in electron microscopy?

Answer 13

Electromagnetic lenses

Question 14

What is the resolution limit of the light microscopy?

Answer 14

200nm

Question 15

What is the resolution limit of electron microscopy?

Answer 15

0.05nm

Question 16

What is the advantage of light microscopy?

Answer 16

Cell is alive

Question 17

What is the disadvantage of light microscopy?

Answer 17

Low resolution

Question 18

What is the advantage of electron microscopy?

Answer 18

High resolution

Question 19

What is the disadvantage of electron microscopy?

Answer 19

Cell is dead

Question 20

Why is the cell dead in electron microscopy?

Answer 20

Due to vacuum conditions

Question 21

Why is an electron microscopy used?

Answer 21

Used for structure and organisation of cell

Question 22

Why is a light microscopy used?

Answer 22

Used to understand how structure move around

Question 23

What are the different types of electron microscopy?

Answer 23

• Scanning electron microscopy

- Transmission electron microscopy

Question 24

What is scanning electron microscopy?

Answer 24

Scanning over the surface with electron beam

Question 25

What is transmission electron microscopy?

Answer 25

Goes through the specimen with electron beam

Question 26

Which type of electron microscopy gives 2D images?

Answer 26

Transmission electron microscopy

Question 27

Which type of electron microscopy gives 3D images?

Answer 27

Scanning electron microscopy

Question 28

What do advanced electron microscopy make use of?

Answer 28

-Make use of resolution limit of microscopy

Question 29

What do advanced electron microscopy allow us to do?

Answer 29

- Single molecular analysis - Analysis of membrane topology using freeze fracture electron microscopy - Reconstruction of ultra-structures

Question 30

What do animated movies enhance our understanding of?

Answer 30

Ultrastructure of the cell

Question 31

What is ultrastructure of a cell?

Answer 31

3D reconstruction of serial electron microscopy images

Question 32

What understanding does life cell imaging provide?

Answer 32

New dimension of the cell

Question 33

How can you have life cell imaging?

Answer 33

- Video-enhances light microscopy | - Fluorescent light microscopy

Question 34

What is structural analysis?

Answer 34

X-ray structure analysis electron cryo-microscopy giving detached structural image

Question 35

What is protein-protein interaction study?

Answer 35

Shows dynamic interaction

Question 36

What is microarray technology expression profiling?

Answer 36

Gives global approach

Question 37

In what ways can we study and understand a molecule?

Answer 37

- Structural analysis - Protein-protein interaction study - Microarray technology expression profiling

Question 38

What is fluorescence?

Answer 38

The emission of light by a substance that has absorbed light

Question 39

Out of emission and excitation which has a higher wavelength?

Answer 39

Emission

Question 40

What occurs to energy in relation to light when emission at a higher wavelength than excitation?

Answer 40

Energy gets lost before light is emitted

Question 41

What is excitation?

Answer 41

Light used to excite the electron in probe

Question 42

What does fluorescent microscopy do?

Answer 42

excites specimen and collect emission light

Question 43

What do different fluorescent proteins have?

Answer 43

Different excitation and emission spectra

Question 44

What are excitation and detection dependent on?

Answer 44

Used filters

Question 45

What do fluorescent microscopy allow us to do?

Answer 45

- Visualise a single molecule | - Multiple proteins can be analysed at once

Question 46

Describe the process of fluorescent microscopy?

Answer 46

- Excitation filter allows broad spectrum light down the objective - Excites the dye - Dichroic mirror = mirrors excitation light down the objective - Probe in spectrum gets excited (electrons fall back to ground state) - Sends out a higher wavelength back through objective and through mirror - Goes through emissions filter to be detected by detector

Question 47

Who got the noble prize in 2008 in chemistry?

Answer 47

Drs. Roger Tsien, Osamu Shimomura and Martin Chalfie

Question 48

Why did Drs. Roger Tsien, Osamu Shimomura and Martin Chalfie get the Nobel prize?

Answer 48

Contributed to developing and expanding the use of fluorescent proteins in biological application

Question 49

Where is the green fluorescent protein from?

Answer 49

The jellyfish = Aequorea victoria

Question 50

What can GFP proteins be used for?

Answer 50

Reporter to analyse proteins in the living cell

Question 51

What did Chalfie discover?

Answer 51

GFP can be used to tag protein

Question 52

How does GFP act as a reporter to analyse proteins in living cells?

Answer 52

- Excites blue | - Emissions green

Question 53

What does GFP allow us to do?

Answer 53

observe subcelluar and cellular dynamics in living cells

Question 54

What did Roger Tsien develop?

Answer 54

Palette of fluorescent proteins

Question 55

What does having a palette of fluorescent proteins allow us to do?

Answer 55

View multiple proteins at the same time

Question 56

What are the common photobleaching and photo activation techniques?

Answer 56

- Fluorescent Recovery After Photobleaching (FRAP) - Fluorescent Loss in Photobleaching (FLIP) - Photoactivation

Question 57

What does FRAP reveal?

Answer 57

Differences in membrane fluidity and protein mobility

Question 58

What can be seen on a FRAP graph?

Answer 58

Intensity of fluorescence after bleaching increase but many not increase to full intensity again

Question 59

How does FRAP work?

Answer 59

- High energy laser destroys ability of GFP to be excited and send out light - Area is dark - If structures move around then unbleached molecules will diffuse - If structures are not mobile then unbleached areas will not diffuse

Question 60

What does FLIP reveal?

Answer 60

- Photobleach selected area that contains GFP | - GFP of neighbouring areas will go down if communication occurring

Question 61

What is photo activation?

Answer 61

Fluorescent proteins become visible after laser radiation

Question 62

What wavelength is fluorescent protein invisible and need activation at?

Answer 62

400nm

Question 63

What wavelength can fluorescent protein become detectable?

Answer 63

488nm

Question 64

How does photo activation work?

Answer 64

- 400 nm laser light induces a chemical reaction - About 100-fold increase in fluorescence after photo-activation - Photoactivatable red fluorescent protein becomes available

Question 65

What does photactivation allow us to visualise?

Answer 65

Sub-population of signals provide detailed insight into motility behaviour