????? ???????????????? Electron Microscopy technique

1
????? ???????????????? Electron
Microscopy technique

• ????? / ????? ??????
• ??????? ???????- ????? ?????? ????????? ??????

2
??? ?? ????? ??? ??? ??? ????? ??? ??????? ???
??? ?????? ???? ??? ?????? ??????????

• ?????? ?????? ?????? ????? ??? ????? ???? High
  vacuum ??? ??? ?? ??????? ?????? ??? ??? ???? ??
  ????? ??? ?? ???? ???? ???? ?????????? .
• ????? ???? ?????? ??? ?????? ??????? ??? ??????
  ???????????? ???? ???? ??????? .

3
1924
???? ??? ??? ??????? ???????? ??????????? ???
???? ????? ???? ???????? ??????? ??????? ???????.
???? ??? ?????? ???????
????? ????? ????? ???? ???? ?? ???? ?? ???? ???
????? ?? ????? ???? ????? ???? ?? ???? ????????
?? ????? ???? ?????? ?????? ??? ????? ??????
?????? ????? ?? ( - ) ???? ?????? ??? ??????
????? ????? ?????? ????? ?????? ??? ????? ??????
???? ?????? ????? ( / - ) ?? ?????? ????? ?????
????? ????? ???? ??? ????? ??????? ??? ???? ??
??? ???????? ?????? ??? ????? ??? ???? ?????
??????? ??????? ?????? ?? ???????? ?????? ???
???? ???????? ??? ????? ??????? ?? ????? ???? ??
???? ????
???? ?? ??? ?????? (???? ?? ??????) ????
???????????? ???? ??????? ???????
4
1955

• - ??? ???? ??? ?????? ????? ????? ???? ?????
  ??????? ?? ????? ??? ?????? ?? ????? ????????
  ??????? ???? ?????? ????? ?? ????? ?????? ???????
  
• ??????? ?????? ???????? ????????? ??????????
  ?????? ?????? ????? ?????? ?????? ?????? ????????
  ?????? ???????????? ?????? ?? ??? ?????? ????????
  ??? ???? ???? ?? ??????? ?????? ??? ???????
  ??????? ??????????? ???????? E.M. ????? ???? ??
  ???????? ????????????? ??????? ??????? ? ???????
  ???? ?? ?? ??? ???
• ???? ???? ???????? ?? ????? ??? ????????? ??????
  ?????? ?????? ????? ???????? ??? ????? ????????
  ??? ?????? ?????????? ??????
• ???? ?? ??? ????????? ????? ??????? ??????

5
1980
??? ???? ?????? ?????????? ??????
1986
???? ???? ???????? ???? (SEM) ?????? ??? (35JSM)
6
????? ??????? ? ???????
L.M ?????
E.M ?????????
7
L.M?????
E.M?????????
1-Transmission Electron Microscopy
Compound L.M
Simple L.M
(TEM)
1- Inverted L.M
Single lenses
2- Scanning Elect Microseopy
(SEM)
2- Dark field L.M
3- Phase Contrast Microscop
3- Enery Dispersiv X-ray Spectroscop
4- U.V.M.
5- Immuno flourecent I.F.L.M.
4- Field Emission Scanning Electron
Microscopy
(F E.SEM)
6- Descetting L.M attaching
5- Electron Probe Micro- Analyzer.
7- Camera L.M
(EPMA) (SEMEDS)
8- Cooliscop L.M
8
?????? ??????

• ??? ????? ??? ????? ????? ?????? ?????????? ???
  ?????? ????
• ???? ?????? ?????? ?? ??????? ?????????? ???
  ????? ???????
• ???????? ????????.
• ??????? ???? ??????? ?????? ?? ?? ??? ???????
  ?????????
• ????? ??????.

9
??????????? ??? ??? ??????? ???????? ??? ????????

• ??? ??????? ??? ?????? ?????? ?????? ???
• 1500x ?????? ?????? ???? ?????? , 25x ??????
  ?????? ?? ?????? ????????
• ??? Magnification power (M.P.)
• ?????? ???? ??? ??????? ???????
• ???? ???? ???? ??? ??????? ??? ?????? ??? ??? ???
  ?? ???? ?????? ????? ????
• ?????? ????????? ???? ?? ???????? ??????? ?????
  ??? ???????? ? ?????
• ?????? ??? ??????? ??? ??? ??????? ? ???????
• ???? ???? ?? ???? ???? ??????? ?? ??? ???????
  Resolution power (R.P.)

10
??? ?? ??? ??????? ??? ?????? ?????????? ??????
???? ?? ??? ????? ??? 600,000 ??? ?????? ?? ????
??????? ??? ?? ????? ??? ??? ?? ??? ?? ????? ???
??? ????? ???? ??????? ??????? ???? ???? ?? ????
???? ??????? ??? ????? ?? ??? ??????? ?? ??????
?????? ?????? ??????? ??? ??? ??? ?? ?? ????
?? ??? ??????? ?? ???? ??????? ???????- ? ???
??????? ?? ??? ??????? (Resolution power) ??
(Resolving power) ?? ???? ????? ??? ???? ?????
???????? ???? ?? ?????? ????? ??? ??????? ??????
?? ?????? ?? ??? ?? ?????
11
??? ??? ??? ?????? ??????????....?

• ??? ??? ?????? ?????? ?????????? ????? ????? ??
  ??? ?????? ??? ?????
• ??????? ??????????? ??? ??????? ?? ??? ???????
  ???????? ???????? ????? ???
• ??????? ?????? ???? ????? ??? (R.P.) ??? ?????
  ?????? ?????????? ????
• ????? ?????? ???? ( ?????? ) ?????? ??? ????
  ????? ???? ????? ??? ??
• ????? ??? ????? ?? ??? ??????? ???? ???????
  ??????????

12
?????? ?? ???????? ?????????

• 1m 1000mm
• 1mm 1000um
  0,1mm 100um
• 1nm 10 um 1um 1000nm
  0,1um 100 nm
• 1nm 1000A

13
???????????? ???????? ??? ???????? ?? ?????
???????

• ??? ??????? ????? ??????? ????? ??? ??? ??????? ?
  ???????

???? ??? ??????? ?? ??????? ??????? ????? ???
??????? ???????(????? ?????? ??????)
- ????? ??? ??????? ?? ??????? ??????????? ?????
??? ????? ????????? ( ??? ?????? ) ??? ??????
?? ?????? ????? ???? ?? ???? ??????? ????? ???
?????? ?????????
14
??? ??????? ?? ?????? ??? ?????? (?? ?????? ??
??????????) ????? ?????? (????? ??????
wave length)( ? ???? )
?????? ??????? ????? ?????? ????? ????? ? ????
????? ( 500 ) ??????? ???????
(0,5 um) ????? ?????? ?? ??????
???? ??? ??????? ??? ?????? (0,2 um) ???? ???
??????? ?????? ??? ????????? ?? ??? ???????
U.V.M. ????? ????? ?????? W.L.0,3um ?
???? R.P.0,1um ????? ?? ???????? ??? ???? ??
????? ?????? ???? ???? ???? ??? ??????? ???????
????
?
R.P
??????? ????????? ???? ????? ?????? ????
???? ?? ??????? ? ??????? ??????
?????? ?????????? ????? ?????? ?????? ??????????
? ???? ???? ?????????? ??? ???? ????
?????? ?????????? ?????? ??????
15
?????? ????
?? ????? ?????? ?????? ?????????? ????? ???
??????? ???????? ???? ????? ????? ??? ??????
????? ??????? ????? ?
??? ????? ??? ?? ???????? ?????????? ?????????
?????? ???? ???????? ?? ???? ????
????? ????? ?????? ?????? ?????????? ??????
?????????? ????????? ??????? ??? ? ?????
?????? ( ???? ) V ???????? ???????
??????
16
???? ??????? 60,000 ???? (60K.v.) ?????? ????????
??? ?????...........?
????
???? ??? ??? ????? ????? (0,5nm) ?? (0,05um)
???? ?????? ??? ?????? ?? ???? ??????? ?? ??????
?????? ??? ????? ?????? ??? ????????
17
?? ??????? ??? ???????? ?????? ?????? ???? ??
???? ??????? ??? ?????? ?????? ?? ?????? ?????
?????? ??? ????? ?? ??? ????? ?????? ?????. ???
???? ?? ?????? ?????? ?? ???? ??? ?? ????? ?????
??? ??????? ??? ??? ??? ?? (250nm) ??????
???? ?? ???? ??? ??? ?????? ???? ????????
???????? ??? ????? ?? (250nm) ?? ?? ??????
??????? ?????? ?????? ??? ?????? ??? ?????
??????? ???? ??? ??????? ??? ???????? ?? 250
???????
18
???? ??? ?? ?????? ?????? ???? ??????? ?? ??????
?????? ?? ????? ?????? ?????
????
???? ????? ?????? ???? ??????? ?????? ?????? ???
?? ???? ?? ???? ????? ?????? ?????? ?????? ?????
??? ????
19
??? ??????? ???? ????? ?????? ??????????
????????? ????? ?????? ?????? ?????????? ???
?????? ??????? ????? ??????? ????? ?? ?????
?????? ??? ?? ??????????? ??? ????? ?????? ?????
????? ??? ??? ???????? ????????? ?? ?????
??????????? ???? ??????? ??????????? ????? ????
?? ?????? ???? ?????? ??? 60.000 ???? ??
(60K.V) ??? (180K.V) ????? ??? ??????? ??????
???? ???? ????? ???? (1000K.V)
20
????? ??? ??????? ?? ??? (600,000X) ???? ???? ???
(1,600,000X) ?????? ????????? ???? ???????
??????????? ??? ?? ??? 0,2-0,1nm ?? ????? ???
??? ????? ??????? ????? ??????? ???????. ??????
?????? ?????????? ?? ??? ??????? ???? ????? ????
?????? ?????? ??? ?? ???????? ?? ???? ????? ??
?? ????? ???????? ??? ?????? ???? ??? ???
??????. ??? ???????? ???????? ??????? ????????
???? ????? ?? ?? ??? ???????????? ??? ??? ????
????? ???? ?????? ???? ??????. ????? ????? ???
????? ??? ??? ????? ??? ??? ????? ????
??????????? ??? ????? ??????? ??????? ??? ????
????? ??? ?????? ??????????.
21
?????? ?????? ???? ??? ?????? ??? ??????
?????????? ??????? ??????
E.M L.M





22
???? ???? ?????? ??????????

• ????? ????? ?????? ??? ??? ???? ????? ???????
  ??????????? ???? ?????? ????? ???? ??????
  ???????????.
• ??? ?? ?????? ?????? ??? ????? ?????? ????
  ??????? ????????? ????? . ?? ?? ??????? ???? ???
  ????? ??? ?? ????????? Photons ???????? ?? ???
  ????? ?????? Different wavelengths ?? ??????
  ?????? ??????

23
???? ???? ?????? ??????????
24
Basic structure

• Electron microscope divided into the following
• Basic components -
• 1- Illumination system.
• 2- Imaging system.
• 3- Specimen stage.
• 4- Vacuum pump system.
• 5- Image recording system.

25
1- Illumination system.

• 1- Cathode housing.

Electron gun
Cathode (filament)
Anode
2- Electromag Alignment system
26
???? ??????? ?? ???? ?????? ?????????? ??????? ??
????? ??????

• ????
• ????? ?? ??? ?????? Cathode???? ???? ???? ??????
  (??????) ?????? ???-
• 1- ????? ??????????? Electron gun???? ????? ???
  ?????? Filament
• ??? ?????? Cathode ??? ??? ??? ??? V .
• ?????? / ?????? ???? ???? ?? ??? ????? ?????
  ?????? ????? ??????????? ??? ??????
• ( ???? ???? ?????? ??? ????? ????? ???? ?????
  )
• ???? ??? ????? ???????? ??? ????? ???? ?????
  
• ??? ??? ???? ?? ( 3000 ???? ????? )
• ????? ????? ????? ??????? ?? ??? ??????
  ????? ??? ??? ?????????.
• ?????? ?? ?????? ????????? ????? ????????
  ?????????
• ?? ??? 40 K.V ???? 12000 40000) K.V????(
• ????? ????? ????? ??? ???? ?????? (???
  ?????? ) ??? ????? ????.

27
???? ??????? ?? ???? ?????? ?????????? ??????? ??
????? ??????

• 2- ??????(wehneltyl cylinder) Anode ??? ????
  ?????? ??????? ????? ??? ??? ????
• ?? ?????? ???? (1mm Dia.)?? ???? ??? ?????
  ??? ?????? ??????????
• ??? ???? ???? ??? ??? ???? ?? ?????? ????
  ??????? ?????? ????? ??????????? ???
• ?????? ??????? ????? ??? ?????? ?? ??????
  ???????

28
??????- Electromagnetic Alignment system ??
?????? ?????????????? ?????? ?? ????? ????????
????? ??? ???? ?????? ???? ???? ???? ????????
???? ?????? ?????????? ?????? ?????? ?????? ??
???????? ?? ???? ????? The electron beam can be
focused by passing trough a magnetic
field
29
2- Imaging system.
???? ????? ??????-
Lenses
??????? -
????? ?????? ?????????? ?????? ?? ???? ?????
????????? ???? ??? ????? ?????? ???????
???????? ??
1- ?????? ??????? Objective lens ( O.L. )

2- ?????? ??????? Projector lens ( P.L. )

3- ?????? ???????? Intermediate lens ( I.L.
)
4-?????? ??????? Condenser lens (C.L. )

30
???? ?? ???? ?? ???? ???? ??????? ?????? ??- 1-
??? ??????? ??? ????? ?? ??? ????? ????
?????? ???? ??????? ???? ??? ???? ???????
??? ???? ??? ???? ????? ? ?????? ??????????
?????? ?? ???? ?????? ????????? ???? ?????? ?????
?????? ????? ???? ???? ?????? ?????? ??
????? ??? ????? ??????????? ??? ?? ?????? ????
?????? ??????? ???? 2- ?????? ??????? ???? ????
??????? ??????? ?????? ?? ???? ??????
?????? ????? ?????? ?????? ??????????? ??? ???
?????? ????????? ???? ??? ???? ?????
????? ?? ???? ????? ?????? ?????? ??? ??????
??????? ??? ????? ?????? ??????? ???????????
??? ??? ????????? (3-5mm)
31
????? ????? ??????? ????????

• 1- ???? ??? ?????? ??????? ?? ?????? ????????????
  
• ?????? ??????? ??????? projector lens
• ??? ???? ????? ?????? ??????? ?? ??????
  ??????.
• 2- ????? ?????? ???????? Intermediate lens
• 3- ?? ?????? ??????? Projector lens
• ??? ?????? ???????? ?????? ???????? ????
  ?????? ??????? ??????? ??????
• ????? ???? ????????/ ???????
• 4- ??????? ?????? ??????? Objective lens
• ??? ???? ???? ?? ?????? ?? ???? ??? ???
  ?????? Cathode
• ??? ???? ??? ?????? ???? ????? ???????????
  ??? ?????? ?? ?????? (??????)
• ???? ?????? ?????????? ?? ???? ????? ?????
  ????.

32

• ???? ?????? ??????? ?? ???????? ????? ??? ??????
  ??????? ??????? ????????
• ????? ?????? ?? (Aperture)
• ????? ??? ??? ?????? ???? ????? ??????? ???
  ???????-
• Apertune 125um, Apertune 250um Apertune
  3100um

• ?????? ???? ?????? ??????? (??????) ??????
  ?????? ??????????? ??????? ????? ??? ??
• ???? ?????? ???????? ????? ???? ??????? ?? ??????
  ????????
• ???? ???? ???? ??? ??????? ???? ??????? (contrast
  diaphragm)

33

• ??????? ???? ?????? ?????? (??????) ?????? ??????
  ??????????? ????????
• ???? ?????? ??????? ????? ??????? ??? ??????
  ??????? ?????? ??????
• ????? ???? ???? ?????? ??????? (selective
  diaphragm)
• ??? ??? ?? ????? ?????? ???????? ?????? ???
  ?????? ???????? I.L.

• ??????? ??????? ????? ?????? ???? ???? ?????
  ?????? ????
• ????? ???? ??????? ??? ??????? ?????????

34
????? ???? ??????? Magnification instrument
???? ???????Magnification instrument ????? ??
????? ????? ??? ?????? ??? ????? ?????? ???????
??????? ???? ??????? ??? ??????? ???? ??????
?????? ?????? ???????
35
?????? ?????????? ??????
36
?????? ?????????? ??????

• ?????? ???? ????? ??????? ?????? ??????
• ???? ?????? ??? ??????? ?? ?????? ??????
• ????? ??? ?????? ??????? ????? ??????????? ???
  ?????????? ???? ?????? ?????? ?????
• ????? ??? ?????? ?? ??????? ??? ?? ?????? ??????
  .
• ???? ???? ??????? ??????? ???? ???? ??? ?????
  ???? ???? ?? ???????????
• ??? ????? ??????? ????? ????? ????? 5nm
• ??????? ?? ??????? ??????? ?? ????? ????? ?? ???
  ?????? ???? ??????
• ???? ?????? ?????? ???? ????? ?? ???? ??????
  ???????? ?? ????? ?????? ?????? ????? ??? ?????
  ?????? ?? ?????? .

37
???? ??????? Resolving
????? ?????? ?????? ?????? ?? ???? ??????? ??????
???? ?? ??????? . ??????? ??????? ( Condenser
lens ) ???? ??? ????? ????? ??? ???? ???????
????? ????? ???? ??????? ???? ?? ????? ???????
??? ???????? ??????? ???????? ( Objective
eye Piece lens ) ??? ?? ???? . ???? ???????????
???? ????? ?????? ??? ??? ?????? ???? ?????
??????? , ??????? ????? ??????? ????? ???? ?????
x25 ?x1500 ????? ?????? ??? ??? ?????? ??
???????? ??????? ???? ????? ??????? ?? ???? ????
????? ??????? , ????? ????? ???? ????? ??? ?????
???????? ???? ?? ?????? ??????? ?? ?????? ????? .
38
???? ??????? Resolving

• ????? ??????? ??? ??????? ????? ??????? ???? ????
  ????? ?? ??????? . ???? ???? ???? ????? ???????
  ???? ?? ??? ???? ????? ???????? ?????? ??????? ,
  ????? ?????? ??????? ???? ?????? ??????? .
• ??????? ???? ????? ?????? ?? ??????? ??? ?? ???
  ???? ???? .

39
?????? ?????????? ??????
?? ??? ????? ?? ??????? ??? ?????? ??? ???? ????
?? ??????????? ????? ?????? ?? ?? ???????????
????? ?????? ?????? ????? ??? ??????
???? ?????? ???? ?? ??? ??????????? ???? ?????
????????? ?????? ????? ????????? ?????? ??????
???? ??? ?????? ?????? ?????? ??? ???? ????
?????? 1965? ???? ??? ????? ???? ?? ??? ???? ??
??? ??????? ??????? ??????????? ????????? .
?????? ?????? ?????? ???? ???? ??????
40
?????? ???? ??????? ?? ?????? ?????? Resolving
Power
??? ???? ???? ??????????? ???? ???? ??????
???? ?????. ???? ???? ?????? ???????
????? ?????? ???????? ???????? ???? ?????? ????
???? ???????????
???? ?????? ??? ???? ??????? ?? ?????? ??? ???
10nm
41
Tissue Processing
????? ????? ?????? ???????? ???????? ????? ??????
?????? ??????????
42
Tissue Processing

• ???????
  Fixation
• ???? ???????
  Washing
• ??? ?????
  Dehydration
• ??????? ??????? ?????? Critical Point
  drying
• ?????? ?? ??? ???????
• Processing Specimens after drying
• ??????? ??? ??????? ??????? Specimen stubs

43
??????? Fixation

• ???? ??? ???????
• ?????? ????? Primary
  Fixation
• ?????? ??????????? 2-3 Bufferd glutraaldhyd
• ??? ???? ????? ?????? ??? 2-3 ????? ?? ????
  ????? ?? ???????
• ??? ??? ?? ????? ???? glycogen protein ??
  ?????? ????????

44
??????? Fixation

• ?????? ??????? Secondary
  Fixation
• ???????? ??? ????????? 1 Bufferd Osmic acid
• Osmium tetroxid OsO4
  
• ??? ???? ????? ??????
• ??? ??? ??
• 1- ????? ?????? ??????? Lipid ?? ?????? ????????
  
• 2- ????? ????? ?????? ?????? ?????????? ??????
  ???????? ????????
• 3- ???? ?? ????? ??????? ????????? ??? ??????
  ????? ????? ??????????? ???? ???? ??? ?????
  ??????? ???? ????? ??????? ?????????

45
????? ??????? ??????

• ?????? ??? ?????? ?? ??????? ??????? .
• ?????? ??? ?????? ?? ?????? ?????? ???? ?????????
  ?????????? .
• ????? ?????? ?? ?? ??????? ??????? ????? ?????
  ?????? ?????? ??? ??? .
• ????? ?????? ?? ???? ???????? .
• ???? ????? ??? ???????? ??????? .
• ???? ?????? ??? ?????? ?????? ?? ????? .

46
????? ???? ???? ??? ???????

• ????? ?????? ?????? ??? ??? ??????
• ??? ?????? ( ???? ????? )
• ???? ??? ?????? ?? ??????
• ??? ??????? ??? ?? ?????? ?? ??? ??????
• ???? ???????
• ???? ?????? ( ???? ???? ?????? ?? ?? ????
  ??? ???? ???? )
• ??? ??????? ??????
• ???? ???????
• ?????????

47
???? ??????? Washing

• ???? ?????? ??? ???????? ?????? ??? ??? ???? ???
  ?????? ?????? O.A , ??? ????? ?? ????? ?????
  ????? ???? ??? ??????
• ????? ?? ?????? ?????? ?????? ?? ?????? ????
  ????? ????? ?? ??? ??????? ?????? ?? ??????
  ?????? ??????? .
• ???? ????? ??? ?????? ???? ????? ???????

48
??????? dehydration

• ????? ??? ????? ?? ??????
• ?? ????? ?????? ?? ?????? ????? H2O ?? ??????
  ???????? ????????? ????????? ???? ?????? ???
  ????? ???????? ??? ????????? ??????????
• ?? ?????? ???? ???????? ???? ?????? ???????? ??
  ????? ??????
• ???? ??? ?? ??? ???? ?????? ?????? ??????? ??????
  ??? ??????? ??????? ????????
• ???? ?????? ?????? ???? ??? ??? ???? ?????? ?????
  ??? ????? ???? ???? ???????
• ????? ??? ??? ??? ????? ???? ???? ????? ????????

49
???? ?????? ??? ?????? ????? ?????? ????????
10 MIN 2X 30 ETHANOL
10 MIN 2X 50 ETHANOL
10 MIN 2X 70 ETHANOL
10 MIN 2X 90 ETHANOL
10 MIN 2X 95 ETHANOL
10 MIN 3X 100 ETHANOL
50
????? ???? ?????? ??? ?????

• ?????? ?????? ????? ?????? ???????? ???????? ??
  30 ?????? ????????? ?? ?? ??? ????? ????????
  ???? ?? 5 ?? 10 ???? 100 ??? ?? ??????
  ??????????? ??? ???????? ???????? ?? ????? ??????
  ????? ??? ?????? ?? ???? ??? ????????? ???????
  ??????
• ???????- ???????? ????????
• ????????? ??? ??? ???????? ???? ??? ?????? ??
  ????? ??????
• ????? ??????? (????? ??? ??? ??????)
• ???????? ??? ???
• ??????? (?????-????-?????) ???? ???.

51
???? ??? ?????????? ?????? ???? ??? ????? ???????
??????? ??????????
????? ?????? ???? ?????? ?? ???????? ?????????
??????
??? ?????? ?? ?? ???? ????? ??? ???? ?? ???????
????? ??? ???????
??????? ???? ?? ??? ??????? ?????? ??? ???????
52
?????? ?? ??????? ?????? ???? ??????? ??????
????? ??????? ?????????? ??????
53
(No Transcript)
54
(No Transcript)
55
(No Transcript)
56
(No Transcript)
57
??????