1. Microscopes and Cells
IB HL Biology

2. 2.1.4 Comparison of relative sizes of molecules, cell membrane thickness, viruses, bacteria, organelles and cells, using the appropriate SI unit.
Refer Figure 1.18, Clegg p20
Size relationships
of biological and chemical
levels of organization are
compared
-Notice the diversity and
how the power of 10 is used
-Although sizes are expressed
in length and diameter, cells
and organisms are 3-D

3. Size of various cells and structures:
Molecules: 1 nm
Membranes (on organelles): 10 nm
Viruses: 100 nm
Bacteria: 1 um
Organelles: up to 10 um
Most cells: up to 100 um
Measurements above in 2D, remember all structures have 3D shape.

4. The Metric System Know how to convert from one unit to another.
Basic
Unit
Kilo-
1000
Units
Hecto-
100
units
Dek- 10
Deci-
0.1
Centi-
0.01
Milli-
0.001
Multiply
Divide

5. What units are used to measure cells?
1 mm = 1000 micrometers (um)
1 mm = 1,000,000 nanometers (nm)
Or…
A micrometer is 1 x 10-3 mm (0. 001)
A nanometer is 1 x 10-6 mm ( mm)

7. Magnification and Scale bars
Specimen size how large the specimen actually is
Image size how large the specimen appears in a drawing or photograph
Magnification how much larger the image is than the actual size
Formula used for these calculations:
Magnification = size of image
size of specimen
Microscopy Calculations Youtube video

8. Calculating Linear Magnification
What is the actual size of this specimen in um?
60mm/5 = 12mm
12mm x 1000 um =
12,000 um
Magnification x5
60 mm
Measuring picture

9. Refer to image on p16 and problem 9 on p17
2.1.5 Calculate the linear magnification of drawings and the actual size of specimens in images of known magnification
Magnification could be stated (for example, ×250) or indicated by means of a scale bar
Magnification = Image size = 58mm = 2.5mm x 1000 = x Size of specimen mm(size of specimen meas with scale bar)
Scale bar
1um
Refer to image on p16 and problem 9 on p17

10. Calculating image size using Scale Bar
Magnification = Image size = 129mm = 460um
Size of specimen 28mmX100um (scale bar)
Refer to problem 5, p12
Scale bar
0.1mm

11. If we want to see a cell… We have to magnify it
Magnification: making something that is small appear larger
A cell from the inside of your cheek

12. Antony vanLeeuwenhoek
Father of microbiology
Eyeglass maker who invented first microscope in 1600’s

13. Eyepiece Body tube Arm Stage Base Stage clips Diaphragm Light source
Revolving nosepiece
Arm
Objective lens
Stage
Stage clips
Coarse adjustment knob
Fine adjustment
Diaphragm
Light source
Base

14. Light Microscopy Advantages
Can view living specimens
Inexpensive and easy to use

15. Light Microscopy Disadvantages
Resolution is limited.
Resolution: the ability to form separate images of objects that are close together
Resolving power: the minimum distance two points can be separated and still be individually distinguished as two separate points.
The smaller the resolving power, the better the resolution.

16. Light Microscopy Disadvantages
Can only magnify a limited number of times (ours go up to 1000x; best light microscopes magnify up to 4000x)
Limited by focal length of lens

17. Electron Microscopes
To magnify an image a large number of times, you must use an electron microscope.
Specimen has a beam of electrons passed through it

18. Electron Microscopes There are different types of electron microscopes
In a transmission electron microscope (TEM), an electron beam passes through a very thin section of material
An image is formed because the electrons pass through some parts of the section but not others
In a scanning electron microscope (SEM), a narrow beam of electrons is scanned in a series of lines across the surface of the specimen
The electrons that are reflected or emitted from the surface are collected by a detector and converted into an electrical signal, which is used to build a 3-D image, line by line, on a TV screen

19. Electron Microscope Advantages
Images can be magnified thousands of times (up to 250,000x)
A lot of detail can be seen

20. Electron Microscopy Advantages
Can magnify 1000’s of times
Details are easily visible
HIV, magnified 24,000x

21. Electron Microscopy Disadvantages
Expensive ($$$$$)
Must use heavy metal dyes, which kill organisms

22. Transmission vs. Scanning EM
Transmission EM’s view cross-sections
SEM’s view surfaces only

23. Comparison of Light and Electron Microscopes
Light Microscopes
Electron Microscopes
Material can be prepared easily for examination. Often, a sample can simply by placed on a slide with a few drops of water and a cover slip. An image can be obtained within seconds
Preparation of material for examination always involves a long series of procedures. These take several days to complete and often involve the use of toxic chemicals
Living material can be examined, so specimens do not always have to be killed. There is less danger of artificial structures appearing and causing confusion if the specimen is still alive
Living material cannot survive in the vacuum inside electron microscopes. Tissues therefore have to be killed as the first stage in the preparation of them for examination
Movement can be observed if living material is examined, including the flow of blood, streaming of cytoplasm inside cells and the locomotion of microscopic organisms
No movement can be observed as the material is always dead. Movement can only be deduced indirectly by complex experiments, often involving radioactive tracers
Colors can be seen – both natural colors and artificial colors caused by staining
Only monochrome images are produced, with black, white, and shades of grey
The field of view (the area which can be observed at once) is relatively large ~2mm across at low power with typical microscopes
Only a small field if view can be examined at once – in a TEM the max uninterrupted view is about 100mm across
The resolution of light microscopes is relatively poor – about .25mm so the max useful magnification is only about x600. Many structures within cells cannot be seen clearly
The short wavelength of electrons gives very good resolution – about .25nm. This allows magnification of up to x500,000. Very small objects therefore become visible including many of the details of cell structure

24. To calculate total magnification:
Multiply the magnification on the objective by the magnification found on the eyepiece
You will need this for every specimen you draw under the scope!

25. Field of View (FOV)
Field of View:  Sometimes abbreviated "FOV", it is the diameter of the circle of light that you see when looking into a microscope.
As the power gets greater, the field of view gets smaller. 
You can measure this by placing a clear metric ruler on the stage and counting the millimeters from one side to the other.  Typically you will see about 4.5mm at 40X, 1.8mm at 100X, 0.45mm at 400X and 0.18mm at 1000X. 

26. Calculating FOV Measuring the microscope field of view on lowest power
Place a clear plastic ruler with mm markings on top of the stage of your microscope. 
Looking through the lowest power objective, focus your image. 
Count how many divisions of the ruler fit across the diameter of the field of view. 
Multiply the number of divisions by 1000 to obtain the field of view in micrometers (µm).
Record this in µm (1mm = 1000 µm ).
Magnified at 40X, the lines of the ruler are clearly visible.

27. FOV Mathematical Calculation
Total Magnification Low Power = FOV at Other Power
Total Magnification at Other Power FOV at Low Power

28. Practice calculating FOV
Example:
If a 5x FOV is 3 mm, what is the 40x FOV of that microscope?
Total Magnification Low Power = FOV at Other Power
Total Magnification at Other Power FOV at Low Power
5 = FOV at Other Power
40 3mm
(3)(5) = (FOV of higher power)(40)
=0.375 mm FOV of higher power

29. Website for microscope calculations
Calculating with microscopes