Microscopy

Question 1

What is the equation for maginification?

Answer 1

i = am

Question 2

How do you convert mm to micrometers?

Answer 2

x1000

Question 3

What is magnification?

Answer 3

the increase in the apparent size of an object

Question 4

What is resolution?

Answer 4

the ability to distinguish between two points as separate

Question 5

Why are light microscopes limited?

Answer 5

the wavelength of light is too long

Question 6

What does TEM stand for?

Answer 6

transmission electron microscope

Question 7

What does SEM stand for?

Answer 7

scanning electron microscope

Question 8

How are the lenses focused in TEM and SEM microscopes?

Answer 8

with electromagnetic lenses

Question 9

What is the maximum magnification of a light microscope?

Answer 9

x1500

Question 10

What is the maximum magnification of a TEM/SEM microscope?

Answer 10

up to x500,000

Question 11

What is the resolving power of a light microscope?

Answer 11

200nm

Question 12

What is the resolving power of TEM microscopes?

Answer 12

1nm

Question 13

What is the resolving power of a SEM microscope?

Answer 13

20nm

Question 14

Name the status of the sample in all 3 microscopes

Answer 14

living or dead in light/ dead in TEM and SEM

Question 15

Which one of the microscopes does the specimen have to be extremely thin for?

Answer 15

TEM

Question 16

Which microscopes are more expensive?

Answer 16

TEM and SEM

Question 17

Which one is a 3d image?

Answer 17

SEM

Question 18

What colour is the image of the microscopes?

Answer 18

coloured - light/ black and white - TEM and SEM

Question 19

Why does TEM and SEM have to be in a vacuum?

Answer 19

As they use electrons to illuminate the specimen, the electrons would hit air particles

Question 20

Why does the sample have the be extremely thin?

Answer 20

To allow the electrons to penetrate the sample

Question 21

What are the limitations of electron microscopes?

Answer 21

vacuum has to be used to prevent scattering of electrons so the sample has to be dead/ complex preparation and staining of specimens are required/ image only in black and white/ the image may contain artefacts such as distortion of key features if specimen was dehydrated

Question 22

What is an eyepiece graticule?

Answer 22

a small piece of glass with a measurement scale that fits inside a microscope

Question 23

Say what is special about the scale of an eyepiece graticule

Answer 23

the scale remains constant no matter the magnification

Question 24

What is used to calibrate the eyepiece graticule?

Answer 24

a special microscope slide called a stage micrometer

Question 25

What is calibration?

Answer 25

involves fixing known points and constructing a scale between them

Question 26

What is cell fractionation/ differential centrifugation?

Answer 26

the process of cells being broken up to allow the different organelles that they contain to be separated

Question 27

Explain how to use an eyepiece graticule to calculate the size of a structure

Answer 27

place the micrometer on the stage and line up the scales on the graticule and micrometer, count how many graticule divisions are in 100micrometers of the micrometer - length of 1 eyepiece division = 100micrometers/no of divisions, use values to calc the size

Question 28

What is the first step to differential centrifugation?

Answer 28

chop up fresh tissue in an ice cold, isotonic buffer solution

Question 29

Name and explain the conditions of the solution used in step 1 of d.c

Answer 29

ice cold - slows down enzyme activity / isotonic - water potential stays equal to tissue so that organelles won't shrink or burst as a result of osmotic water loss/gain / buffered - so the pH doesn't fluctuate which could alter the structure of organelles or affect the functioning of enzymes

Question 30

What is the second step to differential centrifugation?

Answer 30

put the chopped up tissue into a blender/homogeniser which breaks open the cells

Question 31

What is the third step to differential centrifugation?

Answer 31

filter the mixture to remove the debris/homogenate

Question 32

What is the fourth step to differential centrifugation?

Answer 32

pour the mixture into a centrifuge with tubes and spin very quickly, the denser parts get separated to the bottom of the tube where they form a pellet called sediment

Question 33

What is within the sediment?

Answer 33

the nuclei

Question 34

What is the fifth step to differential centrifugation?

Answer 34

the liquid layer on the top (called the supernatant) is poured into a fresh tube leaving the sediment behind

Question 35

What is the sixth step to differential centrifugation?

Answer 35

the supernatant is spun again for longer and at a faster speed to produce another sediment containing mitochondria and chloroplasts

Question 36

What are the lightest organelles separated by differential centrifugation?

Answer 36

ribosomes