1. Microscopes Teacher PowerPoint AS Biology – Module 2
2.1.1 Cell structure
Microscopes
Teacher PowerPoint

2. Lesson 1 You should be able to:
describe how to prepare slides for use in microscopy and explain the reasons for staining specimens for use in microscopy;
draw accurate and clear diagrams from a light microscope;
evaluate the advantages and disadvantages of using a light microscope.

3. Eye

4. Seeing cells under a light microscope

5. The light microscope Ocular lens x 10 Resolution = 200nm or 0.2um
A compound light microscope has two lenses:
The objective lens – this is placed near the specimen;
An eyepiece (ocular) lens – this is the lens through which the specimen is viewed.
The objective lens produces a magnified image, which is magnified again by the eyepiece lens.
Ocular lens x 10
Objective lens x4 x10 x40 x150
A B C D
Magnification A = 10 x 4 = 40times
B = 10 x 10 = 100times
C = 10 x 40 = 400times
D = 10 x 150 = 1500times
Resolution = 200nm or 0.2um
Max mag = 1500X

6. Slide preparation for light microscopes

7. Slide preparation for light microscopes
Dry mount
Solid specimen either cut thin or whole with a cover slip on top.
Wet mount
Specimens suspended in water or oil (with refractive index same as glass). Cover slip on top from angle to prevent bubbles.
Squash slides
Wet mount but using a lens tissue apply pressure to the cover slip to squash the specimen.
Smear slides
Edge of a slide is used to smear a specimen then a cover slip is added.
Questions
1. Suggest reasons for the following:
Specimens must be thin.
When using a wet mount, the refractive index of the medium should be roughly the same as glass.
A cover slip must be placed onto a wet mount at an angle.
2. Why might two microscope slides be used instead of a cover slip and microscope slide when preparing a squash slide?
So light can shine through it and details can be seen
To prevent refraction between liquid and glass and prevent distortion of image
To prevent air bubbles being trapped.
Squashing could break a cover slip. This is less likely with two microscope slides.

8. Microscope activity
Prepare a microscope slide with the onion tissue provided.
Ensure you have stained the tissue and have used a cover slip.
View this under your microscope.
Accurately draw what you can see using the rules below:
use a sharp pencil and plain paper.
use at least 50% of available space
use correct proportions
continuous lines
no shading
use ruled label lines.
labels outside diagram
label lines should not cross over others
include a title
state the magnification of the image

9. Staining sample
STAINING = with coloured/fluorescent chemicals that bind to cell organelles and makes them visible; STAINING = Allows us to identify different types of cells and different organelles; STAINING = provides contrast;
LEARN!!!
Crystal violet and methylene blue are positive dyes so attracted to negative charged material in the cytoplasm.
This means cell components become stained.
Nigrosin and congo red are negative dyes so are repelled by negative cytoplasm. They do not enter cells so are background stains.

10. Differential staining sample
Gram stain technique Used to see gram-positive (bacteria with thick peptidoglycan cell walls) and gram-negative bacteria (with very thin peptidoglycan cell walls); Gram negative need to have a counter stain;
These are killed by penicillin as it works by breaking down the cell wall
These are NOT killed by penicillin as cell wall is not essential
alcohol

11. Differential staining sample
Acid-fast technique Identified mycobacterium like (TB); Carbol fuchsin dye (red) is added to cells using a lipid solvent; Acid/alcohol wash will remove stain from any bacteria but not mycobacterium;
mycobacteria
Non-mycobacteria

12. LIGHT MICROSCOPE
DISADVANTAGES LOW RESOLUTION (200nm) WAVELENGTH OF LIGHT TOO LONG CAN NOT SEE ULTRA STRUCTURE (SMALL ORGANELLES)
ADVANTAGES PORTABLE TAKES UP LITTLE SPACE SPECIMENS EASY TO PREPARE CAN SEE CELLS AND OFTEN NUCLEUS

13. Exam question
Why is staining used when preparing specimens to be examined under a microscope? [2]
so the cells and their contents are visible;
to increase contrast;
so organelles can be seen, for example the nucleus;
so different types of cells can be seen.

14. Summary questions Textbook page 13. Complete Questions 1-2.
Mark and annotate your answers (see page 692 for the link to the answers).

15. Can you?
Learning outcome   
What can I do to improve?*
Describe how to prepare slides for use in microscopy and explain the reasons for staining specimens for use in microscopy.
Draw accurate and clear diagrams from a light microscope.
Evaluate the advantages and disadvantages of using a light microscope.
What can I do to improve ideas – check through notes, read the textbook, complete questions in my question pack/textbook, ask a friend or ask the teacher.

16. Lesson 2 You should be able to:
state the difference between magnification and resolution;
distinguish between images produced by a range of microscopes; light microscope, transmission electron microscope, scanning electron microscope and laser scanning confocal microscope;
state the differences in resolution and magnification that can be achieved by a light microscope, a transmission electron microscope and a scanning electron microscope.

17. Magnification Degree to which the size of an image is larger than the object itself.
Resolution The ability to see two objects that are close together as separate objects and see in detail.

18. LIGHT MICROSCOPE V’S ELECTRON
Electrons are used to increase resolution as they have a much shorter wavelength than light so have a higher resolution
About 0.2nm

19. Electron microscopes
- Beam of electrons is focused by ELECTROMAGNETS through or onto the surface of a specimen
- Transmission electron microscope (TEM): ULTRA STRUCTURE OF CELLS AND ORGANELLES – SMALLER ORGANELLES AND THEIR DETAILS CAN BE SEEN;
- Scanning electron microscope (SEM): 3D SHAPE OF THE SURFACE OF CELLS and ORGANELLES and their SURFACE FEATURES;
SEM
SEM
TEM

20. Size matters
TEM vs. SEM
Transmission electron microscopes (TEM) are used mainly to see the ULTRASTRUCTURE OF ORGANELLES and CELLS;
SMALLER organelles become visible;
SHAPES and DETAILS of organelles can be seen;
Mag = 500,000x
RES = 0.2nm
2D images
Scanning electron microscopes (SEM) are used to study the 3D SHAPE OF THE SURFACE OF ORGANELLES and CELLS
Can see SURFACE FEATURES of cells;
Mag = 100,000x
RES = 0.2nm
3D images
LEARN!!!

21. LEARN!!! Light microscope TEM SEM Max resolution 200nm 0.2nm
Max magnification
x1500 X X
LEARN!!!

22. Laser scanning confocal microscopy
High intensity laser illuminates the specimen;
This causes fluorescence from specimen dye;
Emitted light from specimen is filtered through a pinhole aperture;
Only light from the most focused place (focal plane) is detected;
More runs at different focal planes allows 3D images;
Reflect laser wavelength only

23. Staining sample
STAINING = with coloured/fluorescent chemicals that bind to cell organelles and makes them visible; STAINING = Allows us to identify different types of cells and different organelles; STAINING = provides contrast;
LEARN!!!

24. Electron microscopes sample
Fixation –using chemicals or freezing to solidify sample;
Staining - with Heavy Metal (lead) salts that reflect or stop electrons;
Dehydration – removing water molecules
Set in resin to preserve;
No colour images so this one has been altered after with a PC (false colour image)

25. DISADVANTAGES Cannot look at living cells Must be in a vacuum No colour images Specimens must be thin Artefacts may end up in the image Have to be treated with metal (lead) salts
ADVANTAGES Higher resolution Higher magnification Shorter wavelength Can see ultra structure
Artefact – structures produced due to the preparation process.

26. Light and electron card sort

27. Samples not often distorted Sample often distorted
Light
Electron
Cheap
expensive
Portable and small
Large and fixed
Sample prep simple
Sample prep complex
Samples not often distorted
Sample often distorted
Vacuum not needed
Vacuum needed
Coloured images
No colour (added digitally)
Up to x1500
TEM = x
SEM = x
Res 200nm
TEM = 0.2nm
SEM = 0.2nm
Specimens can be living or dead
Dead only

28. TEM or SEM? Image produced is 2D. Electrons bounce off the sample.
Electron beam passes through a very thin prepared sample.
Magnification possible is about x
Electron beam is directed onto the sample, but the electrons don’t pass through.
The resolution possible is 0.5nm.
Image produced is 3D.
Magnification possible is about x
The resolution possible is 3nm.

30. How is an image produced by a laser scanning confocal microscope different to that produced by an electron microscope?
The image produced by a laser scanning confocal microscope:
has a lower resolution than the electron microscope;
can have a fluorescent tag; 
can show movement as it can be used on living cells;
can see different layers, at different depths of the sample.

31. Summary questions Textbook page 25. Complete Questions 1-4.
Mark and annotate your answers (see page 692 for the link to the answers).
Extension work
Read pages 23 and 24 for information about atomic force microscopy and super resolved fluorescence microscopy.

32. Can you?
Learning outcome   
What can I do to improve?*
State the difference between magnification and resolution.
Distinguish between images produced by a range of microscopes; light microscope, transmission electron microscope, scanning electron microscope and laser scanning confocal microscope.
State the differences in resolution and magnification that can be achieved by a light microscope, a transmission electron microscope and a scanning electron microscope.
What can I do to improve ideas – check through notes, read the textbook, complete questions in my question pack/textbook, ask a friend or ask the teacher.

33. Lesson 3 You should be able to: convert between µm, nm and mm;
calculate the magnification of an image using the magnification formula;
manipulate the formula for magnification to calculate image size/actual size.

34. What are the following units?
micrometre
nanometre
millimetre µm nm mm
Put them in order from smallest to biggest.

35. smallest nm µM mm
nanometre
micrometre
millimetre
BIGGEST

36. Rob’s easy conversion System
1mm
1000um (micrometer)
nm (nanometers)
1um
0.001mm
1nm mm
X 1000
X 1000,000
÷ 1000
÷ 1000,000

37. mm um(micro) nm(nano) DOWN X 1000 UP ÷ 1000 DOWN X 1000000

38. Example conversions
1. A hair is 1mm thick, what is this in micrometers(um) and nanometres'(nm)
mm – um (down x 1000) mm = 1000um
mm – nm (down x ) 1mm = nm
2. A cell is 0.05mm thick, what is this in micrometers(um) and nanometres(nm)
mm – um (down x 1000) mm = 50um
mm – nm (down x ) mm = 50000nm
3. A cell membrane is 0.05nm thick, what is this in micrometers(um) and millimetres(nm)
nm – um (up ÷ 1000) nm = um
nm – mm (up ÷ ) nm = mm

39. How do we calculate magnification?
The actual size of an object is 5mm, the image size when seen drawn a microscope is 50mm. What is the magnification?
Magnification = image size
actual size
Can you rearrange this equation?

40. magnification
An organelle that is 5um in diameter looks 1mm under a microscope. How many time has it been magnified?
Mag = size of image ÷ size of object
Mag = 1mm ÷ 5um
(must convert 1mm to um, x 1000)
Mag = 1000um ÷ 5um
Mag = 200 times
Size of image
magnification
Actual Size X ÷

41. 2. A mitochondria is about 60nm in diameter
2. A mitochondria is about 60nm in diameter. When viewed under an electron microscope that magnifies it x , what would the diameter of the image be in mm?
Size of image = mag x size of object
Size of image = 400,000 x 60nm
Size of image = 24,000,000nm
(convert nm to mm, ÷ 1,000,000)
Size of image = 24mm
Size of image
magnification
Actual Size X ÷

42. ÷ Let’s try an example: Image size Magnification = Actual size
Actual size 70 μm
Magnification =
Image size
Actual size
Whitefish cell during metaphase of mitosis
Measure the image size in mm and then convert to the same units as given for the actual size.
Image size = 66 mm = μm
Size of image
magnification
Actual Size X ÷
66 000
Magnification =
= 943 x 70

43. ÷ … and another example ... Image size Magnification = Specimen size
Actual size 120 μm
Glomerulus and kidney tubules
Measure the image size in mm and then convert to the same units as given for the actual size.
Image size = 51 mm = μm
Size of image
magnification
Actual Size X ÷
51 000
Magnification =
= 425 x
120

44. And another….. ÷ Actual size = image mag Actual size = 50mm mag
Size of image
magnification
Actual Size X ÷
Actual size = 50,000ųm
1200
Actual size = um

45. … and looked at another way …
Magnification =
Image size
Actual size
Transmission electron micrograph of a mitochondrion from mouse lung tissue (magnification x). Public Domain image. Louisa Howard.
Our formula can be rearranged as:
Actual size =
Image size
Magnification 5
10cm
So, if you know the magnification factor and can measure the image size …
Image size = 10 cm = 100 mm = μm = nm
Actual size =
= 500 nm

46. Microscopes recap
What is the formula for calculating the magnification of an image?
How do you calculate magnification when using a microscope?
What is meant by a microscope’s resolution?
A mitochondria is 2µm long. In a book, a picture of the organelle is 3cm long. Calculate the magnification of the image.

47. Can you?
Learning outcome   
What can I do to improve?*
Convert between µm, nm and mm.
Calculate the magnification of an image using the magnification formula.
Manipulate the formula for magnification to calculate image size/actual size.
What can I do to improve ideas – check through notes, read the textbook, complete questions in my question pack/textbook, ask a friend or ask the teacher.

48. Lesson 4 You should be able to:
state the difference between an eyepiece graticule and stage micrometer;
use a stage micrometer to calibrate an eyepiece graticule;
use the calibrated eyepiece graticule to measure the length of cells under a microscope.

49. Key terms
Eyepiece graticule = placed into the eyepiece lens. This will not change size (like the image does) when you change the magnification. Stage micrometer = goes on the stage of the microscope and is used to calibrate the eyepiece graticule. It is a microscope slide with a scale etched into it. It is a known size (e.g. 1mm)

50. An amoeba seen under a light microscope; (a) with a x10 objective and x10 eyepiece; MAG = x100 We use an eyepiece graticule to measure the actual size;

51. An amoeba seen under light microscope;
(b) With objective x 40
And eye piece x 10;
MAG = x400
Eyepiece graticule stays the same;
Image increases;
IF WE KNOW THE VALUE OF each EPGU we can work out the Object size;

52. X 40 mag The stage micrometer is 1mm long divided into 100 divisions;
Each division is 0.01mm or 10μm;
The eye piece graticule is divided into 100 divisions (epu = eye piece unit);
In (a) 40 eye piece units (epu) = 1mm (1000 μm);
Therefore 1 epu = 1000/40 = 25 μm;
The nucleus is 20epu;
20 x 25 μm = 500 μm;
X 40 mag

53. X 100 mag The stage micrometer is 1mm long divided into 100 divisions;
Each division is 0.01mm or 10μm;
The eye piece graticule is divided into 100 divisions (epu = eye piece unit);
In (b) 100 eye piece units (epu) = 1mm (1000 μm);
Therefore 1 epu = 1000/100= 10 μm;
The nucleus is 50epu;
50 x 10 μm = 500 μm;
X 100 mag

54. Mag of eyepiece lens
Mag of objective lens
Total mag
Value of 1EPU ųm
X 10 X4
x40 25
X10
x100 10
X40
x400
2.5

55. Summary questions Textbook page 17. Complete questions 1-3.
Mark and annotate your answers (see page 692 for the link to the answers).

56. Can you?
Learning outcome   
What can I do to improve?*
State the difference between an eyepiece graticule and stage micrometer.
Use a stage micrometer to calibrate an eyepiece graticule.
Use the calibrated eyepiece graticule to measure the length of cells under a microscope.
What can I do to improve ideas – check through notes, read the textbook, complete questions in my question pack/textbook, ask a friend or ask the teacher.