METHODS OF STUDYING CELLS -Microscopes

Question 1

Describe how optical (light) microscopes work?

Answer 1

1. lenses focus a ray of light and magnify the view of a thin slice of specimen
   2.different structures absorb different amounts of wavelengths of light
2. reflected light is transmitted to the observer via the objective lens and eyepiece.

Question 2

suggest the advantages and limitations of using an optical microscope?

Answer 2

+ colour images
+ can show living structures
+affordable apparatus
- 2D image
-lower resolution than electron microscope = cant see ultra structure

Question 3

describe how a transmission electron microscope works?

Answer 3

1. pass a high energy beam of electrons through a thin slice of specimen.
2. more dense structures appear more darker since they absorb more electrons
   3.focus image on fluorescent screen or photographic plate using magnetic lenses.

Question 4

suggest the advantages and limitations on TEM?

Answer 4

+ electrons have shorter wavelength than light = high resolution, so ultra structure is visible
+ high magnification (x 500000)
- 2D image
- extensive preparation may introduce artefacts
-requires a vacuum = so it cannot show living structures
- no colour image

Question 5

describe how a scanning electron microscope works?

Answer 5

1. focus a beam of electrons onto a specimens surface, using electromagnetic lenses
2. reflected electrons hit a collecting device and are amplified to produce an image on a photographic plate.

Question 6

suggest the advantages and limitations of SEM

Answer 6

+ 3D image
+ electrons have a shorter wavelength than light = higher resolution
- requires a vacuum = cannot show living structures
- no colour image
- only shows outer surface

Question 7

define magnification

Answer 7

factor by which the image is larger than the actual specimen

Question 8

define resolution

Answer 8

the shortest distance between two points on a specimen

Question 9

state an equation to calculate the actual size of a structure from microscopy?

Answer 9

actual size = image size/magnification

Question 10

outline what happens during cell fragmentation and ultracentrifugation?

Answer 10

1. mince and homogenize (blend) tissues to break open cells and release the organelles
2. filter homogenate to remove debris
   3.perform a differential centrifugation:
   a) spin homogenate in centrifuge
   b) the most dense organelles in the mixture form a pellet
   c) filter off the supernatant and spin again at a higher speed

Question 11

state the order of sedimentation of organelles during differential centrifugation.

Answer 11

nucleus
mitochondria
lysosomes
RER
plasma membrane
SER
ribosomes

Question 12

explain why fractionated cells are kept in a cold, buffered, isotonic solution?

Answer 12

cold: slow action of hydrolase enzymes
buffered: maintain constant pH
isotonic: prevent osmotic lysis/ shrinking of organelles