[3.1-3] microscopes + methods of studying cells

Question 1

what is magnification?

Answer 1

the number of times which an image of an object can be enlarged greater than the object’s original size

Question 2

what is resolution?

Answer 2

the ability to discern between 2 separate objects and to see them as separate from each other (clarity)

Question 3

why would you use an electron microscope? (AQA question) [3]

Answer 3

1. an electron microscope uses a beam of electrons but a light microscope uses a beam of light
2. a beam of electrons has a shorter wavelength than the beam of light
3. therefore, the electron microscope has a greater resolving power than the light microscope

Question 4

why might the sample be damaged if it is <0.1nm?

Answer 4

• you would have to use high frequency and high energy,
• the sample would be damaged because on the EM spectrum, high frequency waves are dangerous and radioactive

Question 5

principles of a TEM

Answer 5

• the e- beam is fired through a very thin sample from an e- ‘gun’ (e- source)
• electromagnets focus the e- beam
• denser parts absorb more electrons so appear darker
• e- have shorter wavelengths so give a higher resolution than light microscopes

Question 6

why must the sample be very thin in TEM?

Answer 6

• otherwise e- cannot penetrate
• this means only 2D slices are viewable, not 3D images

Question 7

how are images produced using the electron beam?

Answer 7

• e- are absorbed = darker areas on image
• e- pass through = lighter areas on image

> only has monochrome black and white images and 2D

Question 8

why is the process for making EM samples long and complex?

Answer 8

• EM samples need a long processing and staining process, which is complex
• this can also produce ‘artefacts’; things that weren’t in the sample originally but were produced as a result of chemicals

Question 9

why can you not view living issue in an EM?

Answer 9

because the entire column is in a vacuum

Question 10

similarities and differences between SEM and TEM

Answer 10

• can see 3D structures (unlike TEM)
• still can’t see living tissue
• still need processing and staining for samples

Question 11

how does the SEM (scanning electron microscope) work?

Answer 11

• electron beam scans back and forth
• they are reflected and scattered off the surface
• they are collected by detectors
• it is converted into an image

Question 12

units of length

Answer 12

• 1m = 1000mm (1 × 10⁻³ m)
• 1mm = 1000µm (1 × 10⁻⁶ m)
• 1000µm = 1000nm (1 × 10⁻⁹ m)

Question 13

describe the process of cell fractionation and differential (ultra) centrifrugation (6)

Answer 13

1. break up the cell/tissue sample by chopping + blending it (‘homogenisation’)
2. add ice cold isotonic buffer solution to the mixture
3. filter the solution to remove debris and whole cells
4. put the resulting liquid into a centrifruge and spin at increasing speeds
5. remove pellet formed and re-spin supernatent at progressively higher speeds
6. repeat the process; each run will yield a pellet of progressively smaller/lighter/less dense organelles (likely first pellet is nucleus, likely last pellet are ribosomes)

Question 14

why does an ice cold, isotonic buffer solution need to be added to the mixture during cell fractionation?

Answer 14

• ice cold - reduces enzyme activity that might break organelles down eg. lysozyme
• isotonic - prevents water moving in/out by osmosis so no lysis
• buffer - maintains pH to prevent proteins (eg. enzymes) denaturing temporarily

Question 15

what is an eyepiece graticule?

Answer 15

• a glass disc that is in the eyepiece of a microscope that has a scale etched on it
• this scale is typically 10mm long and divided into 100 sub-divisions

Question 16

what is a stage micrometer?

Answer 16

• a special slide that has a scale etched onto it
• the scale is usually 2mm long and its smallest sub-division is 0.01mm (10µm)

Question 17

how do you calibrate an eyepiece graticule? (eg.)

Answer 17

1. line up the eyepiece graticule and the stage micrometer
2. 10 units on the micrometer scale are equivalent to 40 units on the graticule scale
3. 1 unit on the micrometer scale equals 4 units on the graticule scale
4. as each unit on the micrometer scale equals 10µm, each unit on the graticule equals 10/4 = 2.5µm

Question 18

how do you calculate the scale for different objective lenses using the magnification?

Answer 18

if an objective lens magnifying x40 gives a calibration of 25µm per graticule unit, then an objective lens magnifying x400 (10 times greater) will means a graticule unit is equivalent to 25µm/10 = 2.5µm