1. ELECTRON MICROSCOPY Introduction Principles of operation of TEM
Sample preparation for TEM
Advantages and disadvantages of TEM
Principles of operation of SEM
Sample preparation for SEM
Advantages and disadvantages of SEM
School of Biological Sciences
Investigating micrographs

2. INTRODUCTION
With the invention of the light microscope it was discovered
that plant and animal tissues were made up of aggregates of
individual cells. However, light microscopes are limited to
approximately x1000 magnification and have poor resolution.
Therefore not all the internal structures of a cell can be seen
with a light microscope.
In 1924 a French physicist by the name of de Broglie stated that a beam of electrons should behave in a similar way to a beam of light i.e with wave properties the wavelength should be shorter. Therefore an electron beam should give better resolution.

3. RESOLUTION
When there is sufficient light, two points 0.2mm apart or more
can be distinguished with the naked eye as being separate
points. When this distance is less than 0.2mm, only one point is
seen. This distance is called the resolving power (or resolution)
of the eye.
In other words resolution is the closeness two objects can be in
proximity and still be perceived as two separate objects.
Can’t see two separate objects
Can see two separate objects

4. The invention of the electron gun
led to the development of the
electron microscope.
The metal tungsten filament is
heated to about 2500oC which
causes it to release electrons.
Due to the large voltage
difference between the
filament and the anode plate
the electrons are forced to
flow in the direction of the
arrow.
The cathode shield increases
the electron flow further and
concentrates the electrons into
a narrow beam.
Back to principles of operation
Filament
Cathode
High voltage generator
Electron beam
Anode

5. Two main types of electron microscopes;
TEM Transmission
SEM Scanning
TEM produces a high resolution image of the internal structures of cells. TEM uses the electrons that have passed through the specimen to form an image.
SEM produces a three dimensional image of the specimen surface. A beam of electrons scans the whole specimen which then emits low energy, secondary electrons. This technique can be used to study whole cells.

6. When an electron beam strikes a specimen a number of events occur
When an electron beam strikes a specimen a number of events occur. Electrons are scattered depending on the nature of the material.
If the electrons hit a dense array they are scattered out of the main beam and fewer electrons will reach the viewing screen. There is no fluorescence and that area appears dark.
If the electrons pass a scarcity of atoms they travel straight through, hitting the viewing screen and causing fluorescence. That area will appear light. The image comes from the arrangement of light and dark patches on the screen.

7. PRINCIPLES OF OPERATION OF TEM
Electron gun
Specimen holder
Projection chamber

8. The main components of a TEM are:
THE ELECTRON GUN – produces
an electron beam.
THE COLUMN – uses electromagnetic
lenses to control the beam and
produce a magnified image
IMAGE VIEWING AND RECORDING
The image is produced on a
fluorescent screen below which a
shutter and camera are located. e e
Condenser lens e e
Specimen e
Objective lens e e e
Projector lens e e
e e e
e e e e
e e e e e e
Vacuum
Fluorescent screen

9. Electrons only behave like light when they are manipulated in vacuum
Electrons only behave like light when they are manipulated in vacuum. Therefore the whole column is evacuated since atoms such as O2 and CO2 scatter the electrons.
e e e
e e
e e

10. SAMPLE PREPARATION FOR TEM
The aims of sample preparation are as follows:
To preserve the material in its natural state
To ensure that the material withstands changes which might occur on exposure to atmosphere, vacuum and electron beam.

11. Fixation for TEM It is then placed into fixing solution
The tissue is cut into tiny pieces
It is then placed into fixing solution

12. Dehydration and embedding of TEM
Tissue is placed in final embedding mixture and the resin is polymerised in the oven
It is then placed in a dilute solution of resin embedding media
Tissue is dehydrated in alcohol
Specimen vials

13. Section cutting of TEM
Sections are cut on an ultramicrotome with a glass or diamond knife. The sections are floated off the edge of the knife onto the surface of a water trough.
The colour of the sections vary with thickness. When the sections are gold they are picked off the surface with a copper grid.
The section on the copper grid is now ready for staining and viewing in the electron microscope.
3.05mm

14. Advantages Disadvantages
TEM
Very good resolution
Thin sections are effectively two dimensional slices of tissue and do not convey the three dimensional arrangement of cellular components
magnification
Artefacts may be created
Can see sub-cellular components and measure them

15. PRINCIPLES OF OPERATION OF SEM
Electron gun
Detector
Detector
Image viewing
Specimen chamber
Control panel

16. SEM uses electrons that are emitted from the specimen surface.
The specimen is scanned with
a very fine beam of electrons.
These are scattered as they hit high and low points in the specimen. The scattered electrons are measured by a detector and used to control a second beam which forms an image on a TV screen
Electron source
T.V. Monitor e
Electron beam e e e e e
eee
Specimen
Detector
Vacuum

17. Sample preparation for SEM
The preservation used will usually determine which
drying process to use.
There are two basic methods of drying the specimen:
Freeze-drying – used after freezing
Critical point drying – used after chemical fixation and dehydration

18. Freeze drying for SEM
Sample placed in nitrogen slush to maintain it’s structure
Then placed in liquid nitrogen to allow easy handling
Sample placed in copper holder and……….
…..placed in freeze drier
Sample is mounted on a stub

19. Critical point drying for SEM
Sample is chemically fixed
Then dehydrated with alcohol
Sample is placed in critical point drier. Here the sample is flushed several times with liquid CO2. The pressure and
temperature is then raised which converts the liquid CO2 to gas. The gas is then vented off slowly.
The sample is removed and mounted on a stub

20. Coating the specimen for SEM
Most biological specimens are poor conductors and poor
emitters of secondary electrons therefore the surface of the
sample needs to be coated with a thin layer of a conducting material.
There are two ways to do this:
Sputter coating
Evaporation of carbon

21. Sputter coating for SEM
When power passes to the anode, the noble metal evaporates (called the plasma effect) and the metal falls onto and coats the specimen.
ANODE
METAL
A sputter coater
CATHODE

22. Evaporation of carbon for SEM
Two carbon rods are placed end to end. One of the rods is
sharpened to a point. These are placed in a vacuum and the
specimen is placed below them. When electricity passes
through the carbon rods, the carbon tip evaporates and the
carbon falls onto and coats the specimen.
CARBON RODS
CARBON RODS

23. Advantages Disadvantages
SEM
Provides great depth of focus
Micrographs show a 3D image of specimen
Smaller and simpler in comparison to TEM
Only surface features seen
Resolution attainable is not very high (approx 10nmn)

24. Investigating Micrographs
Transmission Electron Micrographs
Scanning Electron Micrographs
Can you spot the differences between the two types of electron microscopy?

25. Transmission Electron Micrographs
Virus particles
Plasma membrane
Endoplasmic reticulum
Golgi membranes
Mitochondrion
Section of mammalian cell

26. Scanning Electron Micrographs
Sample of geranium petal showing the cone shaped projections and the internal structure.

27. Measuring Micrographs
How to work out the size of an organelle?
Measured size
Magnification
Magnification of micrograph is X100000
Measured size = 80mm
Convert to m = 80000
 m
100000
= 0.8m or 800nm

28. for using this programme. We hope that it has been useful!
This programme was developed as part of a work placement project by Sumerah Khan and Sheerin Dariani
THANKS TO:
Chris Gilpin, Ian Miller
Les Lockey, Samantha Newby

29. References
B. Schotanus (1980) Electron microscopy, what is it ? Marketing electron optics. Philips Export B.V. Eindhoven.
Dr Yvonne Miller (1998) Preparation of specimens for TEM and SEM.
Mike Mahon, Chris Gilipin, Ian Miller (2000) Microscopy and analysis University of Manchester - School of Biological Sciences.
Sam Newby (2000) Freeze drying and critical point drying EMPGU.
5) Specimen preparation (1991) (21/1/00) world/SEM/ specimenprep.html pages 1-2.
6) Dr. Ron Butler (1980) Transmission electron microscopy, What an SEM is ?, Aims of specimen preparation and Electron microscopy unit. EMPGU

30. 1. What is the resolving power of the naked eye?
Questions 1 & 2
1. What is the resolving power of the naked eye?
A 200 m
B 0.02m
C 0.002m
D 2mm
2. The filament of a transmission electron microscope is made up of which element?
A copper
B tungsten
C carbon
D gold

31. True or false? true or false ?
Questions 3 & 4
3. Electrons flow away from the filament because of the large voltage difference between the filament and the anode plate
True or false?
4. In electron microscopy, the lenses used to magnify the image are made of electromagnets
true or false ?

32. 6. A vacuum is needed in the electron microscope to
Questions 5 & 6
5. Which of the following is the first step in the processing of biological material for transmission electron microscopy?
A Dehydration
B Sectioning
C Fixation
D Embedding
6. A vacuum is needed in the electron microscope to
A Pull the electrons onto the specimen
B Eliminate molecules of nitrogen, oxygen or carbon dioxide
C Pull the specimen into the column
D Prevent secondary radiation affecting the microscope control panel

33. Questions 7 & 8
7. The difference between TEM and SEM is that in TEM, secondary low energy electrons are used to produce an image.
True or false
8. Which of the following structures could not be seen with a light microscope but could be seen with a transmission electron microscope?
A Nucleus
B Cell wall
C Ribosome
D Golgi apparatus

34. 9. Which of the following is not an advantage of TEM?
Questions 9 & 10
9. Which of the following is not an advantage of TEM?
A High resolution
B High magnification
C Three dimensional detail
D Detail of sub cellular components
10. Which of the following statements about SEM is not true?
A The specimen is usually coated with gold
B Resolution is excellent
C Samples for SEM can be chemically fixed or freeze dried
D Thin sections of tissue are not necessary

35. wrong please choose again
Try again
wrong please choose again 

36. Answer question 1
Answer: A this is the same as the value quoted in the text i.e. 0.2mm 

37. Answer question 2
Answer: B a tungsten filament is used 

38. Answer correct
Correct well done 

39. Answer question 7
Answer: this is false as secondary electrons are used to form an image in SEM 

40. Answer question 5
Answer: the material has to be fixed to prevent distortion and decomposition 

41.  Answer: B molecules in the air would cause scattering of electrons
Answer question 6
Answer: B molecules in the air would cause scattering of electrons 

42. Answer question 8
Answer: C it is possible to see the other structures with a light microscope 

43. Answer question 9
Answer: C TEM gives a two dimensional image 

44.  Answer: B SEM has relatively poor resolution compared with TEM
Answer question 10
Answer: B SEM has relatively poor resolution compared with TEM 

45. Scanning Electron Micrographs
Sample of geranium petal showing the cone shaped projections and the internal structure.

46. Transmission Electron Micrographs
Virus particles
Plasma membrane
Endoplasmic reticulum
Golgi membranes
Mitochondrion
Section of mammalian cell

47. Q 11What is the size of the virus
Questions 11 & 12
Q 11What is the size of the virus
Q12 What is the size of the mitochondrion
Magnification X100000
Magnification X13000
Measured size = 9mm
Measured size = 11mm
A 90mm
A 846mm
B 90nm
B 8.46nm
C 90m
C 8.46m
D 900nm
D 846nm