1. Cell Biology Practical TEM&SEM
Khamis AL-Riyami

2. Electron microscope
The electron microscope is a type of microscope that uses a beam of electrons to create an image of the specimen.
It is capable of much higher magnifications and has a greater resolving power than a light microscope, allowing it to see much smaller objects in finer detail.
They are large, expensive pieces of equipment, generally standing alone in a small, specially designed room and requiring trained personnel to operate them.

3. TRANSMISSION ELECTRON MICROSCOPE

4. Out Line Introduction Applications Sample Preparation
Specifications of JEM-1400

5. Light Microscope
Transmission Electron Microscope
(TEM)
Price
Cheap
Expensive
Size and portability
Small and Portable
Large and requires Special room
Type of Specimens
Living or Dead
Dead
Samples Preparation
Simple and Easy
Lengthy and Complex
Vacuum
Not required
Required
Image color
Natural color of sample maintained
Black and White
Source of illumination
Light
Electron
Magnification
Up to 2000X
Up to 1,300,000X
Resolution
200nm
0.2nm

6. Introduction
Electron Microscopy is defined as a specialized field of sciences that employs the electron microscope as a tool.
The transmission electron microscope (TEM) is a scientific instrument that uses electrons instead of light to study objects at very fine resolutions. They were developed in the 1930s when scientists realized that electrons can be used instead of light to "magnify" objects or specimens under study.

7. 1. Electron Gun, producing a stream of
monochromatic electrons.
2. This stream is focused to a small, thin,
coherent beam by the use of condenser lenses.
3. This coherent beam will hit the sample and
parts of it will transmitted.
4. This transmitted portion is focused by
the objective lens into an image.
5. The image is passed down the column
through the intermediate and projector
lenses, being enlarged all the way.
6. The image strikes the phosphor image screen
and light is generated, allowing the user to see
the image.

8. Applications
A TEM can be used in any branch of science and technology where it is desired to study the internal structure of specimens down to the atomic level.
Biology and life sciences:
Clinical diagnosis
2. Biological samples examinations (animals & plants)
Nanoparticals & Material sciences

9. SAMPLES PREPARATION Extraction of tissue Fixation Dehydration
Infiltration
Embedding
Polymerization
Sectioning
Staining
Observation (Screening)

10. Extraction of Tissue
Fresh Tissue
Fragment thickness 1mm3

11. Fixation
Primary Fixation with 2.5% glutaraldehyde in Sodium Cacodylate Buffer
Secondary Fixation with 1% Osmium Tetroxide

12. Dehydration Dehydration with Alcohol (Acetone or Ethanol) 25% 75% 95%
Absolute Alcohol

13. Infiltration Infiltration with Resin 1Alcohol : 1Resin Pure Resin

14. Embedding
Embedding with Epoxy Resin

15. Polymerization
Polymerization in oven under 60oC over night

16. SECTIONING
Semithin 0.5um
Ultrathin 60-90nm

17. Staining
With heavy metals
Urinayl Acetate
Lead Citrate

18. Observation (Screening)
Using JEOL
JEM-1400
Electron Microscope

19. JEM-1400
Is a high performance, high contrast, 120kV TEM with excellent imaging and analytical capabilities in one compact.
Acceleration voltage of 40 to 120kV.

20. JEM-1400 Specifications Resolution 0.2nm Accelerating Voltage 40-120KV
Magnification x200-x1,200,000
Along with this instrument implanted the Tomography System

21. Tomography System Three-dimensional imaging techniques
Electron tomography is similar in approach to larger scale medical imaging technologies such as CT scans and MRI.
Tomography acquires a series of projected images from different perspectives as the sample is rotated incrementally about an axis perpendicular to the viewing direction.
A computer then combines these images into a three-dimensional model of the sample.
In tomographic analysis the entire acquisition of the tomographic series can be automated.

22. Scanning Electron Microscope

23. Outline Introduction Application of SEM Sample preparations
SEM JSM-6510LA specifications

24. Introduction
The scanning electron microscope (SEM) uses a focused beam of high-energy electrons interact with the surface of solid specimens.
From electron-sample interactions reveal information about the sample including texture, chemical composition, and crystalline structure
Qualitative and quantitative chemical analysis information is also obtained using an energy dispersive x-ray spectrometer (EDS) with the SEM

25. Principle of SEM
A beam of high energy electrons generated by an electron gun
Beam processed by magnetic lenses, focused at the specimen surface and systematically scanned across the surface of a specimen

26. Used to analyze characteristic X-ray by measuring energies of X-ray
Principle of EDS
Used to analyze characteristic X-ray by measuring energies of X-ray
The detector is cooled by LN2 in order to reduce electric noise
The advantage of EDS is that the X-ray from a wide range of elements from B to U are analyzed simultaneously.
Can result wit semi-quantitative and qualitative analysis of the sample
also result with X-ray mapping of the sample.

27. Applications
The scanning electron microscope (SEM) gives the user with an advantage over the light microscope (LM) in three areas:
Resolution at high magnification. The best resolution possible in
LM is about 200 nm
SEM has a resolution of better than 10 nm (typically 5 nm) .
Depth of field . greater depth of field up to 100 times that of light microscopy
Microanalysis. analysis of sample composition including information about chemical composition, crystallographic, magnetic and electrical characteristics.

28. Practical uses for the SEM
SEM is used in most of the scientific fields and industrial field, in Forensic science, biology, chemistry, engineering, Geological science, archaeology and even in art.

29. Sample Preparations
The specimen must meet the following requirements befor it is loaded to the stage:
SEM has vacuum 10-4 Pa so the samples need to be dried
The sample should be not more than 150mm in diameter
The surface to observe is exposed.
The specimen is firmly fixed to the specimen mount .
The specimen has conductivity.

30. Critical point Draying
Biological samples
Fixation
Dehydration
Critical point Draying
Coating
Mounting
observation

31. Fixation
glutaraldehyde
Osmium tetroxide

32. Critical Point Draying
Dehydration
Critical Point Draying
Coating
Mounting

33. Metals, ceramic and glass
If the sample dry and conductive
It dose not need preparation
If the sample not conductive
Need to coat with Platinum or gold thickness nm

34. SEM specifications JEOL JSM-6510LA
Observation of specimens up to 150mm in diameter.
high resolution of 3.0nm at 30kV
Magnification 5x to 300,000
JEOL (energy dispersive X-ray spectrometer (EDS) provides elemental analysis.
Low Vacuum (4.0nm)(30Kv))
analyze non-conductive specimens without conductive coating.
image display of the secondary electron image and a backscattered composition image allow the user to contrast and compare specific details

35. Thank you