techniques in cell biology

Question 1

electron microscopy

Answer 1

Electron beam (0.0025nm). Provides very high resolution- 0.05nm
-material is always did and fixed or dried--> organelles would explode due to vacuum

Question 2

Scanning electron microscopy

Answer 2

3d and only shows the surface

Question 3

transmission electron microscopy

Answer 3

cross-section

Question 4

light microscope

Answer 4

cells are alive- uses visible light (390-700nm). glass lenses focus light. Resolution limit of 200nm.

Question 5

GFP

Answer 5

green fluorescent protein

Question 6

GF was discovered by

Answer 6

osamu shomura

Question 7

calcium activated aequorin (blue) what activated it to become GFP

Answer 7

aequorin is activated by blue light to go green

Question 8

what is GFP used for

Answer 8

as a reporter to analyse proteins in the living cell. Martin chalfie responsible for fusing GFP gene to the genes, so it is expressed in other organisms

Question 9

what else can GFP be used to do

Answer 9

observe sub cellular structures and cellular dynamics in living cells –> activated by blue light
–> used to show that sub cellular in eukaryotic cells are dynamic

Question 10

roger Tsien responsible for

Answer 10

development of a palette of fluorescent proteins–> the palette can be then inserted into different genes to see how different proteins interact or where their role takes place in the cell
-means multiple proteins can be analysed at the same time

Question 11

photoactivation

Answer 11

fluorescent proteins that become visible after laser radiation

• fluorescent protein is invisible and needs activation at 400nm before it can be detected at 488nm
• 400nm laser light induces a chemical reaction about 100x increase in fluorescence after photo activation

Question 12

photobleaching

Answer 12

when the fluorescent part of the protein in a membrane is removed

Question 13

FRAP

Answer 13

fluorescent recovery after photobleaching

Question 14

FLIP

Answer 14

fluorescent loss in photobleaching

Question 15

FRAP

Answer 15

MOTILITY reveals differences in membrane fluidity and protein mobility

Where fluorescent proteins or dyes get locally photo-bleached and the diffusion of unbleached proteins get monitored, in a fluid environment

Question 16

FLIP

Answer 16

COMMUNICATION- diffusion from an unbleached part of the cell results in a decrease of fluorescence in the unbleached part, which gives insight into communication between organelles. e.g. used to show if proteins in the golgi enter the ER

Question 17

what can advanced election microscopy achieve

Answer 17

single molecule analysis e.g. analysis of myosin motors

Question 18

how can we analyse membrane topology

Answer 18

using freeze fracture electron microscopy e.g. the average of any images allows reconstruction of ultra structures

Question 19

electron microscopy can provide

Answer 19

ultrastructural information eg. the membrane of a mitochondria. 3D reconstruction of serial electron microscopy images can provide even more detail and can provide a new dimension of understanding of the cell.

Question 20

why is electron microscopy not so good

Answer 20

cells are dead so images may not look how they do n textbooks

–> information combined is what gives us the full image

Question 21

why can we see membranes

Answer 21

they are stained with heavy metals and the electron beam cannot get through the membranes

Question 22

fluorescence

Answer 22

the emission of light by a substance that has absorbed light-> emission at a higher wavelength than excitation (energy gets lost before light is emitted)

Question 23

how do fluorescent microscopes work

Answer 23

they exit the specimens and collect the emission light

e.g. GFP in jellyfish. When blue light is supplied the blue FP will go up in energy and as it loses energy it will become green

Question 24

main difference between FLIP and FRAP

Answer 24

The major difference between these two microscopy techniques is that FRAP involves the study of a cell’s ability to recover after a single photobleaching event whereas FLIP involves the study of how the loss of fluorescence spreads throughout the cell after multiple photobleaching events. This difference in purpose also leads to a difference in what parts of the cell are observed. In FRAP, the area that is actually photobleached is the area of interest. Conversely, in FLIP, the region of interest is just outside the region that is being photobleached. Another important difference is that in FRAP, there is a single photobleaching event and a recovery period to observe how well fluorophores move back to the bleached site. However, in FLIP, multiple photobleaching events occur to prevent the return of unbleached fluorophores to the bleaching region. Like FLIP, FRAP is used in the study of continuity of membranous organelles. FLIP and FRAP are often used together to determine the mobility of GFP-tagged proteins.[3] FLIP can also be used to measure the molecular transfer between regions of a cell regardless of the rate of movement. This allows for a more comprehensive analysis of protein trafficking within a cell. This differs from FRAP which is primarily useful for determining mobility of proteins in regions local to the photobleaching only.[5]

Question 25

light microscopy wavelengths

Answer 25

390-700nm

Question 26

electron microscopy wavelength

Answer 26

0.0025nm

Question 27

light microscopy resolution

Answer 27

200nm

Question 28

electron microscope resolution

Answer 28

0.05nm