1. The TEM system and components:
Vacuum Subsystem
Electron Gun Subsystem
Electron Lens Subsystem
Sample Stage
More Electron Lenses
Viewing Screen w/scintillator
Camera Chamber

2. TEM Illumination control
Filament saturation
Filament centering
Spot size (Condenser Lens Current)
Condenser aperture

3. Focusing the TEM Control objective lens current
Adjust astigmatism correction coils too
Use large screen at low mags
Use small screen at high mags
Beware of lingering on an area too long
Iterate focus and stigmators
Can take through-focus-series

4. Focusing on a hole in a thin carbon film
(Fresnel Fringes)
(a) underfocused objective lens (bright fringe);
(b) at focus (no fringe);
(c) overfocused objective lens (dark fringe).
Note also the change in appearance of the carbon fine grain.
Magnification ~750,000X.
(From Agar,p.137).

5. Astigmatism Correction
Sort of like the SEM in that astigmatism shows up as a directional defocus
Must correct condenser lens, objective lens and projector lens separately
Use both objective stigmator selectors
Use the first one
Use the second one
Refocus
Repeat

6. Contrast Considerations
Resolving power is good (why?), but…
To see image features contrast is needed
How to increase contrast…?
(SEM contrast is derived from local topography and/or differential interactions of beam with sample)

7. Contrast Considerations
Mass-thickness Contrast (absorption)
Density x thickness
More dense or thicker areas look darker due to absorption of beam electrons.
Thickness fringes due to destructive interference as beam traverses the sample
Use stains to highlight specific areas
Uranium, manganese, osmium
Coat and/or shadow areas to generate contrast

8. Contrast Considerations
Other “deficiency” contrast mechanism
Electron scattering
Random
Amorphous materials change electron trajectories
Regular
Crystalline materials change trajectories uniformly

9. Contrast Considerations
Most of the beam is NFS
Goes right through the sample unperturbed
Other elastic interactions change the direction of NFS electrons which can be selected for or eliminated from the image forming beam. More on this later…

10. Brightness Considerations
Type of source (W, LaB6, FE)
Higher mags means a strong Int-Proj lens, which means lower intensity
Hard to see on fluorescent screen
Ways to mitigate
use lower mags
converge beam with condenser lens
align beam as needed

11. Beam Energy Considerations
Higher voltages produces
shorter wavelength
better resolution
greater depth penetration of sample

12. Beam Energy Considerations
Lower voltages produces
greater contrast due to larger scattering angles for slow(er) moving electrons
less depth penetration
larger proportion of electrons involved in inelastic collision events

13. Electron Beam-Sample Interaction

14. Magnification Considerations
Higher mag-> features look larger (duh)
But intensity drops off and ability to properly focus and stigmate do too
Use LOWEST practical magnification

15. Most photographic emulsions used in electron microscopy
can resolve image details of ~20µm, thus the resolution
of object details will depend on the image magnification
as shown in the table (resolution = 20µm/magnification):

16. Exposure Considerations
Low intensity situations lead to longer exposure times
Vibration will make edges blurry
High intensity situations lead to short exposure times (and concomitant error)

17. Sample Stability Considerations
High intensity or long exposure situations may cause sample to degrade (bonds break, polymer chain-scission, etc.)
Remember the contamination square in the SEM??? Same thing happens in the TEM- you will grow a nice carbon bump on samples as you look at them.

18. TEM Sample Prep for Materials

19. TEM Sample Prep for Biologicals
Almost always a microtome is involved

20. Imaging Modes in the TEM
Bright Field Mode
Dark Field Mode
Diffraction Mode

21. Bright Field Imaging
If the main portion of the near-forward scattered beam is used to form the image
transmitted beam
000 beam
zero-order beam

22. Dark Field Imaging
If the transmitted beam is excluded from the image formation process
off-axis imaging
tilted beam imaging

23. TEM Imaging:
Ray Paths

24. Electron Diffraction Elastic Scattering Events Bragg diffraction
nl=2d sinq

25. Electron Diffraction
Four conditions in Back Focal Plane (BFP) of the objective lens:
No sample No reflections (only transmitted beam)
Amorphous Transmitted beam + random scattering
Polycrystal Transmitted beam + rings
Single crystal Transmitted beam + spots

26. Electron Diffraction Angle of incidence ~1/20 to even come close to
satisfying the Bragg condition.
Therefore only the lattice planes close to parallel to the
beam are involved in diffraction.

27. Electron Diffraction Rd=lL Think of TEM as a diffraction camera
R is measured
d is the unknown
l is the electron wavelength
L is the camera length
(lL is the camera constant)
Transmitted Beam L
Diffracted Beam
Reciprocal relationship between
lattice spacing and distance from
the transmitted spot. R

28. Electron Diffraction Au (111) ring [2.35 Å d-spacing]
With 200KV and L=65cm the (111)
ring should be at about 7.5mm from
the transmitted beam
Rd=lL
R=0.027A*650mm/2.35A

29. Ray Paths in the TEM

30. TEM Imaging Modes: Diffraction vs BF
Electron Diffraction
TEM Imaging Modes: Diffraction vs BF

31. Metal particles
Polymer mix
Electron Diffraction
TEM Images