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

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

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

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).

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

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)

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

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

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…

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

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

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

Electron Beam-Sample Interaction

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

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):

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)

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.

TEM Sample Prep for Materials

TEM Sample Prep for Biologicals Almost always a microtome is involved

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

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

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

TEM Imaging: Ray Paths

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

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

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.

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

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

Ray Paths in the TEM

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

Metal particles Polymer mix Electron Diffraction TEM Images