1. Cells and Microscopy What is magnification and resolution?
What is cell fractionation?
How does ultracentrifugation work?
How do electron microscopes work?
What are the differences between a transmission electron microscope and a scanning microscope?
What are the limitations of the transmission and the scanning electron microscope?

2. In 1665 – Robert Hooke (Brit) – developed a simple compound microscope using several lenses. He used it to examine thin slices of cork, taken from under the bark of an oak tree.
He saw that the sections were made of a number of repeating, small, fairly rectangular spaces. These had the appearance of the shape of the rooms where the monks lived.
These rooms were called cells. So he called the tiny chambers in the cork “cells”.

3. 1838 – Schleiden (German) – a botanist, said all plants are made of cells.
1839 – Schwann (German) – a zoologist, said animals made of cells.
From this emerged The Cell Theory =

4. “The basic unit structure and function of all living organisms is the cell.”

5. Added to this in 1855 by Virchows’ theory: = all cells arise from pre-existing cells by cell division. So all living things are made of cells. If something is not made of cells, then it is not living. E.g. a virus.

6. Organisms that are made of one cell = Unicellular.
E.g. 1: -
E.g. 2: -
Organisms made of more than one cell = Multicellular.

7. Largest single organism = giant redwood tree – up to 100m height.

8. Largest animal – blue whale – up to 30m long.
Adults can be 2 metres high.

9. 2. Microscopy
a. Use of Measurement in cell studies.

10. Domestic cat – 0.3m height
Goldfish – 0.1m long
Housefly – 0.01m long

11. The internal systems of units (SI units) are used.
To measure objects in the microscopic world use very small units of measurements
The internal systems of units (SI units) are used.
The basic unit of length = metre (m)
Other units used in multiples of a thousand times larger or smaller.
E.g. kilo means 1000 times.
Therefore 1km = 1000 m
1 micrometre is a thousandth of a millimetre.
1 nanometre is a thousandth of a micrometre.

12. µ = Greek letter mu Equivalent in metres. Unit Symbol 103 kilometre Km
One thousandth = = 1/1000 = 10-3
millimetre mm
One millionth = = 1/ = 10-6
micrometre µm
One thousand millionth = =
1/ = 10-9
nanometre nm
µ = Greek letter mu

13. Relative size.
The smallest structure seen with the human eye = 50 – 100 µm in diameter.
Human body has approx 60 million million cells. Each cell is 10-30µm in diameter.
Mitochondria is 1µm = ________ nm
Bacteria (average diameter) approx 0.5µm = _____ nm
Smallest organelle is the ribosome = 20nm diameter approx. the cell membrane is approx 10nm wide.
Viruses are about nm in diameter.

14. b. Microscopes.
Cell biology = study of cells – using different types of microscopes.
Microscopes = magnify the image of an object.
2 kinds: light microscopes and electron microscopes
Both uses form of radiation to create image.

15. i) Light/Optical microscopes – uses light as a source of radiation
i) Light/Optical microscopes – uses light as a source of radiation. Light microscopes use light rays, which have a long wavelength.
They can only distinguish between 2 objects if they are 0.2µm or _____nm, or further apart.
The specimen needs to be thin, (1 cell thick), transparent (see through) and flat. It can be kept flat by putting a cover slip, a thin piece of glass), over it.

16. Limitations of Light Microscopes.
The light rays are passed through the specimen. Different parts of the specimen absorb different wavelengths of light. So different shades and intensities of light pass through the 2 lenses to the eye.
Maximum resolution = 0.2µm or 200nm.
Maximum magnification = X1250 to X1500

17. Image seen with a light microscope

18. A light microscope

19. How a light/optical microscope produces an image

20. TEM

21. Transmission Electron Microscope
uses electrons as a source of radiation. This is a shorter wavelength, so they can distinguish between 2 objects as close together as 0.1nm.
Light travels in waves and light has a longer wavelength than a beam of electrons.

22. electromagnetic spectrum.
Whole range of different wavelengths = electromagnetic spectrum.
Visible light is only part of this spectrum.
Human eyes range = 400nm (violet light) - 700nm (red light).
In brain the differences are converted into colour.
Some animals see different wavelengths.
E.g. bees see UV light - many flowers have UV markings. We can’t see them. Bees can.
Cobras see infrared radiation. So they see us in the dark!

23. Electrons are a suitable form of radiation..
Electrons have very short wavelength: the greater the energy, the shorter the wavelength.
Electrons have negative charge means can be focused well using electromagnets.
Electrons are absorbed by molecules in the air so a near vacuum has to be created in a special chamber of the electron microscope.
As water boils at room temp in a vacuum, the specimens to be placed into a vacuum need to be dehydrated - so must be dead.

24. Transmission and scanning electron microscopes
2 types of electron microscopes. They both use magnets (instead of lenses), to focus the beam of electrons onto the specimen. They have a greater resolving power than a light microscope, because the electron beam has a shorter wavelength than light.
Image produced is known as an electron micrograph.
This can be photographed = photomicrograph.

25. Transmission Electron Microscope (TEM)
Consists of an electron gun that produces a beam of electrons that is focused on the specimen by a condenser electromagnet.
The electrons are passed through a thin specimen (or transmitted), before being viewed. The thinner bits allow the electron to pass through more easily.
The parts of the specimen that absorb the electrons appear darker, whilst other parts that allow the electrons to pass through appear bright, so giving some contrast. They give a 2 dimensional image.

26. Limitations: Cannot observe living organisms.
Image is only in black and white.
Specimen must be extremely thin, so need specialists who are trained to section and use the equipment.
The creation of artefacts = something observed that is not naturally present, created as a result of the process.

27. artefacts Can be caused by:
Dehydrating the cell. Removing the water can distort the cell or organelles.
Cutting the thin sections can damage them.
The specimens have to be stained with heavy metals, which are selective and don’t always reflect the true nature of the specimen.
At high magnifications it is only possible to view a very small field of view. This may mean the fields being viewed isn’t characteristic of the whole sample.
The sample can be damaged by the electron beam.

28. Image taken using a TEM Maximum resolution = 0. 1nm
Image taken using a TEM Maximum resolution = 0.1nm. Maximum magnification = X

29. Scanning Electron Microscope. (SEM)
Here electron beam is directed onto the surface of the specimen from above, rather than penetrating it from below.
The beam is passed back and forth (SCANNED)across a portion of the specimen in a regular pattern. The electrons are scattered by the specimen and the pattern of this scattering depends on the contours of the specimen surface.
They do not pass through, like it does with the TEM, so the specimen does not have to be as thin as it does for the TEM.
They give a 3D image, by computer analysis of the pattern of scattered electrons.

30. Image taken using a SEM Colour enhanced as image is black and white!
Maximum resolution
= 20nm.
Maximum magnification = X

31. A dust mite

32. SEM
Disadvantage of the SEM – has a lower resolving power than the TEM, at around 20nm and a lower magnification.
Advantages of the SEM over the TEM
Surface structures can be seen.
Great depth of field obtained, so bigger picture.

33. Electron Microscopy.
Only see black, white and grey with both TEM & SEM, so use a “false colour” image produced by the use of a computer.
Both electrons microscopes are very expensive, compared with a light microscope.
They are large and non-portable.
Preparing the samples and using the electron microscope requires a high level of skill and training, compared with a light microscope, which even an AS student can use! (Joke!!)

34. SEM – how the image is produced

35. Questions
1. Explain the advantages and disadvantages (limitations) of using an electron microscope rather than a light microscope to study cells.

36. 2. Complete the table: Type of microscope
Maximum resolution that can be achieved
Best effective magnification that can be achieved
Light microscope
Transmission electron microscope
Scanning electron microscope

37. 3. List the similarities and differences between light and electron microscopes.
4. Explain why both light and electron microscopes are used widely in biology.

38. Answers.
1. Advantages – greater resolution/ more detail - Greater magnification. Disadvantages – electron microscopes can’t be used to study living tissue, specimens must be dead. - Natural colours can’t be seen. - They are not portable - They are expensive. - Training is required before use.

39. Maximum resolution that can be achieved
Type of microscope
Maximum resolution that can be achieved
Best effective magnification that can be achieved
Light microscope
200nm
X 1400
Transmission electron microscope
0.1nm X
Scanning electron microscope
20nm X

40. 3. Similarities Both magnify images.
Both use a radiation source passed through the specimen.

41. Differences Light microscope Electron microscope
Beam of light (longer wavelength)
Beam of electrons (shorter wavelength)
Small
Large and non portable
Relatively inexpensive
Expensive
Not a lot of training required to use
Training required
See colour images
Black and white images
Specimen can be alive and unharmed
Specimen must be dead
Lower resolving power
Greater resolution
Lower magnification
Greater magnification

42. 4. To gain knowledge of living things structure and function at a microscopic level.

43. Freeze-Fracture TEM can only see a cross section.
SEM can only see surfaces, not internally within structures.
To do this the cell needs to be cracked open. Done by freeze fracturing.
Here specimen is immersed in liquid nitrogen at -1960C and pushed against a sharp blade in a precise way.
The frozen tissue splits along lines of weakness, often in the middle of a membrane.
The fractured surfaces are etched with a heavy metal, so they can be seen. Enabled us to know that proteins are embedded in the cell membrane.
Images produced through freeze fracturing, enabling the organelles to be seen.

45. c. Magnification To observe a specimen: Place slide on stage.
Light focused onto specimen using the condenser lens.
Light passes through the specimen and is captured and refracted by an objective lens.
Light rays up to eyepiece lens. Gives final image.
Our microscopes have 3 different objective lens; X4, X10 & X40. The eyepiece lens has a magnification of x10.
Some have a X100; this has to be used as an oil immersion lens.
To calculate the magnification of a microscope = lens objective X eyepiece lens.

46. Overall magnification
So our microscopes can magnify at what magnifications? Complete the table below.
Mag of objective lens
Mag of eyepiece lens
Overall magnification

47. The greater the magnification,
the smaller the field of view.

48. SPECIMEN lots of different kinds, including: Small living organisms
Thin sections of larger plants and animals.
Smear preparations of blood or cheek cells.
Can stain specimen to see them better. Stains are chemicals which bind to either the chemicals in the specimen or in the specimen itself.

49. Calculating Magnification
Magnification = the number of times larger an image is compared with the real size of the object.
Material put under the microscope = object.
Appearance of this specimen when viewed under the microscope = image.
Magnification = Size of image Size of object

50. OR
Size of object = Size of image
Magnification
Size of image = Mag X Size of object
Remember always that all the sizes must be in the same units. Normally best to convert into µm first.
1mm = 1000µm
1µm = 1000nm

51. Questions 5. Distinguish between magnification and resolution.
6. An organelle that is 5µm in diameter appears under a microscope to have a diameter of 1mm. How many times has the organelle been magnified?
7. A cell organelle called a ribosome is typically 25nm in diameter. If viewed under an electron microscope that magnifies it X400. What would the diameter of the ribosome appear to be in millimetres?
8. At a magnification of X a structure appears to be 6mm long. What is its actual length?
9. Why do sections of tissues need to be cut into thin sections for examination under a microscope?
10. Suggest why light microscopes are so useful in biology.
11 A person makes a drawing of an incisor tooth. The width of the actual tooth is 5mm. the width of the tooth in the drawing is 12mm. Calculate the magnification of the drawing.

52. 12. The actual maximum diameter of this cell is 50μm
12. The actual maximum diameter of this cell is 50μm. Calculate the magnification of the diagram.

53. Transverse section through leaf blade.
13. This is a photomicrograph, (a photograph taken using a light microscope), of a transverse section through a leaf. Use the scale bar to calculate the magnification of the photomicrograph.

54. If we know the magnification, we can turn the equation round so that we can calculate the real size of something from its magnified image.  
Real size of object = Size of image
Magnification
Use your value for the magnification of the photomicrograph above to calculate the thickness of the leaf.

55. 14. If a nucleus measures 100mm on a diagram, with a magnification of X10 000, what is the actual size of the nucleus?

56. 15. Complete the following table:
Metres (m)
Millimetres (mm)
Micrometres (μm) 5
0.3 23 75

57. Answers
5. Magnification is how many times bigger the image is compared to the original object. Resolution is the ability to distinguish between 2 objects very close together. The higher the resolution - the greater the detail that can be seen.

58. Answers
6. Organelle that is 5µm in diameter. Appears under a microscope to have a diameter of 1mm. 1mm = 1000µm 1000 ÷ 5 = X Ribosome = 25nm in diameter (size of object) Mag = X400 Diameter (or size of image?) Mag = size of image ÷ size of object Rearrange = size of image = Mag X size of object Size of image = 400 X 25 =10000nm or 10µm = 0.01mm

59. 8. Mag = X12 000
A structure appears to be 6mm long = size of the image.
What is its actual length or size of object?
Mag = size of image ÷ size of object OR Size of object = size of image ÷ magnification = 6mm = 6000µm
Size of object = 6000 ÷ = 0.5µm or 500nm
(1µm = 1000nm)

60. 9. Light cannot penetrate thick sections of tissue and so no detail could be seen.
10. Easy to use. Can observe living cells and single celled organisms. Can also observe arrangements of tissues in organisms using it.
Small and portable and enable us to see images in their natural colours.
11. Actual diameter = 5 mm
Diameter on diagram = 12mm
Magnification = size of image ÷ size of specimen = 12 ÷ 5 = X 2.4
12. Actual diameter = 50μm – size of object.
Measure: Diameter on diagram = 2.8cm – size of image.
Convert: 2.8cm = 28mm = µm = ÷ 50 = X560

61. 13a. Length of scale bar = 1.5cm = 15mm We are told that scale bar = 100µm or 0.1mm So magnification = 15/0.1= X150 b. Thickness of leaf on micrograph = 4.6cm = 46mm = µm Magnification = X150 So real thickness = /150 = 307μm

62. 14. Nucleus = 100mm = μm Magnification = X10 000, The actual size of the nucleus = / = 10μm

63. 15. Metres (m) Millimetres (mm) Micrometres (μm) 0.000 005 0.005 5 0.3
300
0.023 23
23 000
0.075 75

64. RESOLUTION
= the minimum distance apart that 2 objects can be in order for them to appear as 2 separate points. If 2 points can’t be resolved they will be seen as 1 point.
Depends on the wavelength or form of radiation used in the microscope.
Resolution for the human eye = 100μm.

65. General rule = Resolution = the limit of resolution is about 0
General rule = Resolution = the limit of resolution is about 0.50 times the wavelength.”
So wavelength of light = 400nm -700nm
Minimum wavelength of light = 400nm.
So minimum resolution = half minimum wavelength of light = 200nm.anything smaller than 200nm cannot be seen using a light microscope.

66. The mitochondrion with a diameter of 1000 nm interferes with light waves and so can be seem. The ribosomes with 22 nm diameter do not and so can’t be seen.

67. So the max resolution of light microscope = 0
So the max resolution of light microscope = 0.2µm or 200 nm = if 2 objects are closer than 0.2µm or 200 nm apart then they can’t be distinguished as separate and will be seen as 1 object.
Max resolution of electron microscope = 0.1 nm. (TEM)
Greater resolution = greater clarity.

68. An increase in magnification increases the size of the image but not necessarily accompanied by increase in resolution. Can make image larger but not clearer. Magnification up to limit of resolution can reveal further detail but further magnification just increases blurring. To see very small objects you need a microscope with a very high resolution.

70. Questions
16. Explain why ribosomes are not visible using a light microscope. (3 marks)
17. Why is the electron microscope able to resolve objects better than the light microscope?
18. Why do specimens have to be kept in a near vacuum in order to be viewed effectively using an electron microscope?

71. 19. Which of the biological structures in the following list: - Plant cell (100µm) - DNA molecule (2nm) - Actin molecule (3.5nm) - A bacterium (1µm) - Virus (100nm) Can in theory be resolved by the following? a. A light microscope b. a transmission electron microscope c. a scanning electron microscope 20. In practise the theoretical resolving power of an electron microscope cannot always be achieved. Why not?

72. Answers
16. Resolution of a microscope is limited by the radiation used to view the specimen.
Resolution = the limit of resolution is about 0.5 times the minimum wavelength (of visible light).”
Shortest wavelength of light = 400nm.
Therefore resolution of a light microscope is 0.5 X 400 = 200nm.
The diameter of a ribosome is much smaller than this at nm.
17. The EM uses a beam of electrons that has a much smaller wavelength than light, therefore it has a better resolution.
18. Electrons are absorbed by the molecules in air and if present, this would prevent the electrons reaching the specimen.
19. a. Plant cell and bacteria.
b. all of them.
c. plant cell, bacterium and virus.
20. The preparation of the specimens may not be good enough.

73. 3. Cell fractionation.
= breaking up a cell to isolate all the organelles that it contains.
This allows us to study the cell organelles structure and relate to function.
Based on theory that the mass of different organelles vary and depend on their size.

74. What is an organelle?
Answer – a structure found in the cytoplasm of the cell, with a specific function.

75. Cell Fractionation.
To fraction the tissue, it is first placed in a cold isotonic buffered solution.
Isotonic solution = a solution with the same water potential (Ψ) as the tissue.
(Water potential – Ψ = a measure of the ability of a solution to give out water.)
This is done to prevent the organelles, (not the cell), from bursting or shrinking as a result of gaining or losing water. The cells themselves are burst open in the blender anyway.

76. Treatment of tissue
Cold – slows down enzyme activity, which may break down the organelles before they are extracted and so reduces the rate of metabolic reactions in the cell.
Buffered – to maintain a constant pH. The neutral pH prevents damage to the structure of proteins, including enzymes.
Buffer = a solution or substance which resists changes in pH by taking in or releasing hydrogen ions.

77. There are 2 stages to Cell fractionation:
Homogenation
Ultracentrifugation

78. i) Homogenation
A homogeniser or blender breaks up the cells, releasing the organelles.
Resulting fluid = homogenate. This is filtered to remove any cells or large pieces of broken cells.

79. A homogeniser (industrial size)

80. ii) Ultracentrifugation Machine = ultracentrifuge.

81. Spins tubes of homogenate at very high speed to create a centrifugal force, in order to separate the fragments from the homogenate.
Process:
Tube of filtrate spun at low speed.
Heaviest organelles – the nuclei settle at the bottom as a sediment/pellet.
Fluid at the top = supernatant is removed leaving sediment.
Supernatant transferred to another tube and spun at a faster speed.
Next heaviest organelles – the mitochondria settle put next as sediment.
Continue process so at each increase speed the next heaviest organelle is sediment and separated out.

82. Organelles to be separated out
Speed of centrifugation / Gravitational force
Duration of centrifugation/mins
Nuclei
1000 10
Mitochondria
3 500
Lysosomes
16 500 20
Ribosomes 60

84. Cell Fractionation
So heavier organelles are isolated at slower speeds. The supernatant is spun at high speeds to separate out the lighter organelles.

85. Question
21. Using table 2, suggest which organelle or organelles would most likely be found in each of the following: Sediment 1 Sediment 3 Supernatant 1 Supernatant 3

86. Answer 21. a. Nuclei b. lysosomes
c. mitochondria, lysosomes and ribosomes.
d. ribosomes.