Microscopes

Question 1

what are microscopes?

Answer 1

instruments that produce an magnified image of an object

Question 2

what is magnification?

Answer 2

the degree to which an image increases in size from its original image and size

Question 3

what is resolution?

Answer 3

the ability to distinguish between two points

Question 4

formula for magnification

Answer 4

actual size

Question 5

formula for actual size

Answer 5

magnification

Question 6

to convert millimetre to nanometre …

Answer 6

x1000

Question 7

to convert nanometre to centimetre …

Answer 7

/10,000

Question 8

what is cell fractionation?

Answer 8

process by which cells are broken up and organelles are separated out

Question 9

what type of solution is the tissue placed in before fractionation?

Answer 9

cold, buffered solution with same water potential

Question 10

why is the solution cold?

Answer 10

to reduce enzyme activity that might break up cells

Question 11

why is the solution buffered?

Answer 11

to prevent changes in pH so that structure/function of enzymes is not altered

Question 12

why is the solution isotonic?

Answer 12

to prevent organelles shrinking/bursting by osmosis

Question 13

how are the cells broken up?

Answer 13

in a homogeniser to release organelles

Question 14

why is the resultant fluid filtered?

Answer 14

to remove debris and complete cells

Question 15

what happens during ultracentrifugation?

Answer 15

organelles in filtered homogenate are separated out in a centrifuge

Question 16

how does a centrifuge work?

Answer 16

• spins homogenate at low speed for 10 mins - heaviest organelles, nuclei, forced to bottom forming a sediment
• supernatant removed leaving sediment of nuclei
• supernatant spun again at faster speed - removes mitochondria
• process continues increasing speed each time

Question 17

how to prepare a slide

Answer 17

• add drop of water onto slide (temporary mount) using pipette
• use tweezers to place specimen on top of water drop
• add drop of stain
• add coverslip by standing upright next to drop and carefully lowering onto drop without creating air bubbles

Question 18

what are the disadvantages of preparation of specimen for microscopy?

Answer 18

due to complex process:

• artefacts can form (e.g. air bubbles, fingerprints)
• resolution affected

Question 19

name 5 types of stain

Answer 19

• methylene blue
• eosin
• iodine
• sudan red
• acetic orcein

Question 20

what is methylene blue used for?

Answer 20

makes nuclei more visible

Question 21

what is eosin used for?

Answer 21

stains red blood cells and cytoplasmic materials pink

Question 22

what is iodine used for?

Answer 22

stains cellulose yellow

Question 23

what is sudan red used for?

Answer 23

stains lipids

Question 24

what is acetic orcein used for?

Answer 24

stains chromosomes red

Question 25

name two advantages of using electron microscopes

Answer 25

- electrons have shorter wavelength than light so higher resolving power - electrons have negative charge so can be focusing using electromagnets.

Question 26

why do electron microscopes have vacuum inside?

Answer 26

prevent electrons colliding with air molecules

Question 27

name two types of microscopes

Answer 27

- transmission electron microscope | - scanning electron microscope

Question 28

describe how a transmission electron microscope works

Answer 28

- electron gun fires beam of electrons which are focused by condenser magnet onto specimen - specimen absorbs some electrons which makes it dark and lets some electrons pass through making it light - 2D image projected onto fluorescent screen producing a photomicrograph

Question 29

describe how a scanning electron microscope works

Answer 29

- electron gun fires beam of electrons which is directed onto surface of specimen - beam is passed back and forth across portion of specimen in pattern - electrons are scattered and pattern of electrons depends on contour of specimen's surface - 3D image formed by computer analysis of pattern of scattered electrons

Question 30

resolution of TEM

Answer 30

0.1 nm

Question 31

resolution of SEM

Answer 31

20nm

Question 32

maximum magnification of TEM

Answer 32

x 500,000

Question 33

maximum magnification of SEM

Answer 33

x 100,000

Question 34

limitations of TEM

Answer 34

- artefacts - vacuum - thin specimen - 2D - staining - black and white - expensive

Question 35

limitations of SEM

Answer 35

- artefacts - vacuum - staining - black and white - expensive - lower resolution than TEM

Question 36

advantages of light microscopes

Answer 36

- specimen can be alive | - inexpensive

Question 37

limitations of light microscopes

Answer 37

- poor magnification/resolution