Microscopy

Question 1

Why is cell fractionation needed

Answer 1

• Needed to study the structure and function of various organelles that make up a cell
• we need a large number of isolated organelles which we can gate from cell fractionation

Question 2

Define cell fractionation

Answer 2

The process in which cells are brocken up and the different organelle they contain are separated out

Question 3

Describe the process of homogenisation

Answer 3

• tissue place in cold Isotonic, buffered solution
• cells Brocken up by a homogeniser/blender
• breaks up cells in tissue, releases organelles from cells
• homogenate is filled to remove complete cells and large debris

Question 4

Why is isotonic, buffered solution cold

Answer 4

To reduce enzyme activity’s that could break down organelles
Eg lysozymes

Question 5

Why is isotonic, buffered solution buffered

Answer 5

• so pH doesn’t fluctuate

- change in pH could alter the structure of organelles affecting the functioning enzymes

Question 6

Why is isotonic, buffered solution have the same water potential as the tissue

Answer 6

• Prevent organelles from bursting or shrinking as a result of osmotic gain or loss of water

Question 7

Principle of ultracentrifugation

Answer 7

Heavy organelles will fall to the bottom first in the sediment leaving the clear supernatve which will be removed and re spun for loner and faster

Question 8

What would u find in sediment 1

Answer 8

Heaviest organelles eg nuclei

Question 9

What happens to supernatent 1

Answer 9

Taken and re-spun at medium speed

Question 10

What would u find in sediment 2

Answer 10

Next heaviest organelles eg mitochondria

Question 11

What happens to supernatent 2

Answer 11

Taken and spun at high speed

Question 12

What would u find in sediment 3

Answer 12

Contains next heaviest organelles eg lysosomes

Question 13

How do u get greater resolution

Answer 13

Shorter wavelengths

Question 14

What does a greater resolution do to the image

Answer 14

Make it the image clearer

Question 15

Define resolution

Answer 15

Minimum distance apart 2 objects can be in order for them to appear separate

Question 16

What happens after resolution reaches it limit

Answer 16

Image gets larger and blurred

Question 17

Define magnification

Answer 17

Ho many times bigger is the image than the actual object

Question 18

Method of using a light microscope

Answer 18

• add a drop of water to a glass slide
• obtain a thin section of specimen and place on slide
• stain with iodine of potassium iodide
• lower cover slip using a mounted needle

Question 19

Why when using a light microscope must the specimen be stained

Answer 19

Thin section are transparent staining them means you can see them

Question 20

Why are specimen thin on a light microscope

Answer 20

So light can pass through the specimen and so you can only focus of one layer of cell

Question 21

Weaknesses o a light microscope

Answer 21

• Poor resolution

- very little intercellular detail can be seen

Question 22

Poor resolution

Answer 22

Long wavelength of light

Question 23

What are the 2 types of electron microscope

Answer 23

• TEM

- SEM

Question 24

Differences between light and electron microscopes

Answer 24

• uses a beam of electrons rather than light
• electron microscope has higher resolution power whereas light microscope has poor resolution
• electron has shorter wavelength whereas light has long wavelengths

Question 25

Why a electron beam can be focused more precisely than a light beam

Answer 25

electrons are negatively charged some o can be o used through a electromagnets whereas light has no charge

Question 26

What TEM microscope specimen stained with

Answer 26

Stains containing heavy metals

Question 27

2 advantages of using a transmission electron microscope

Answer 27

• high resolution images are produced

- small objects can be displayed

Question 28

2 disadvantage of using a transmission electron microscope

Answer 28

• can only be used of thin specimens

- microscope only works on non living specimens

Question 29

Give 2 disadvantage of a scanning electron microscope

Answer 29

• lower resolution images given

- only can be used on living specimens

Question 30

Why is an image consisting of bright and dark areas produced from a TEM

Answer 30

The specimen is very thin and will allow some electrons to penetrate but not others this results in the detection of light and dark areas

Question 31

Why can’t we use electron microscope to view live specimens

Answer 31

Because the entire process takes place in a vacuum. This is because electrons are absorbed or deflected by molecules of air, and this would prevent them from reaching the specimen

Question 32

How does a SEM work

Answer 32

Scanning electron microscopes fire electrons across the surface of the specimen to be reflected back and forth

Question 33

Wha type of image does a SEM produce

Answer 33

• 3D image

- image primary black and white but colour can be added by a computer

Question 34

Similarities between SEM and TEM

Answer 34

• Better than light microscopes
• both may contain artefacts
• beam of electrons used
• vacuum
• complex staining process

Question 35

Differences between SEM and TEM

Answer 35

• specimens don’t need to be as thin in SEM
• SEM produces 3D images whereas TE produces 2D images
• SEM = X 100 000 TEM = X 500 000
• SEM = 20nm TEM = 0.1nm

Question 36

How do u calibrate a graticule

Answer 36

• Use a stage micrometer
• line up scales on graticule and micrometer
• then calculate length of divisions on eyepiece graticule

Question 37

Why are graticule needed

Answer 37

• needed to measure size of objects under objective lens

- you need to calibrate the graticule as each objective will magnify to a different degree