Electron microscopy guide


TEM alignment

STEM alignment

Wave interference


Diffractive imaginging



If you have read and fully understood the previous sections of this manual, you probably know everything you need to know about alignment. However, alignment is often regarded as a deep and complex thing; and is always made to seem extremely tortuous in the manufacturer's manuals.

Very roughly speaking, to align a column, start with the illumination (the gun and condenser system, condenser aperture), choose the specimen height (eucentric height), get the beam shifts and tilts pivoting correctly, get the beam tilted accurately down the optic axis of objective lens (rotation centre, objective aperture, if required), and finally refine the focus of the diffraction lens, if you need it. Correct astigmatism for each lens appropriately at each point. And then start again from the beginning: if in doubt, re-iterate.

Remember: The aim is to get a stable condition as lenses are changed in strength: that is, non-moving illumination as a function of spot size, stable rotation centre in image mode, and an on-axis diffraction pattern in SAD mode. If whatever you are looking at moves when a lens is changed, something is not properly on axis.

Some microscopes have alignment procedures that can be followed on the computer screen. You would be well-advised to understand any alignment procedure you follow.


If you lose the illumination, then:

  • check the HT and emission current
  • take out all apertures
  • go down in magnification
  • think about the specimen: if it is solid except for a small hole, then it is probably the problem: in extremis, take it out. Note that on some microscopes, the minimum spot size is limited by the computer when the specimen is removed. This is to reduce the danger of X-rays being emitted from the polepiece. If you really have drastically lost the beam (say after a filament replacement), then it is best to replace the empty specimen holder so that low spot size is accessible.
  • Go down in spot size (low C1) and spread the beam with C2, and look for any hint of intensity on the phosphor screen.
  • Explore gun shift and tilt (this is a last resort)

If you can't find an aperture:

  • On each aperture mechanism, know which fine adjustment knob moves the aperture in and out, and which moves it laterally.
  • Go down in magnification or camera length
  • watch the screen as you put in the aperture blade: if any light flies across the screen, you know the aperture is laterally on-line; to find it, move the adjustment mechanism in and out.
  • If you see nothing as you move the blade in and out continue to move it in and out as you change the lateral adjustment, until you see a flash of light.

Other hints:

Astigmatism, aperture alignment and the effects of 'wobbling' any particular lens are all interrelated. Since astigmatism is the most difficult alignment, always leave it till last. Wobbling a lens with an out-of-line aperture always causes an image shift, even if the lens itself is on line. Again, whenever in doubt, re-iterate.

If you ever change the specimen height, or radically alter the setting of the objective lens, restart the whole alignment from the beginning.

When you switch modes, many lenses can change setting. Because of hysteresis, the alignment will almost certainly not be right when you return to a previously-aligned mode. It's tedious, but if you are a perfectionist, always re- align when you change mode.

Which apertures to use

When you understand the real effects of the two main apertures - the condenser and the objective - then you really understand all of electron microscopy. In this brief introduction, we don't have space to go into all the details, because all of this is specimen related, and depends on what you want to find out about.

Just know that the most important aperture is the objective aperture. A small objective aperture tends to increase contrast in the image but decrease resolution. Very high resolution imaging is often best done without an objective aperture: but if this is your interest, you should understand the nature of the envelope function which attenuates the contribution of high-angle rays. What goes through the objective is crucial. If you are imaging a crystalline material, you should always know which diffraction spots are excited in the back-focal plane, and which ones are being allowed through the objective aperture.

The condenser aperture pretty obviously affects the maximum achievable flux or intensity on the phosphor screen: but a large condenser also reduces contrast in high-resolution imaging (although this depends on spot size and the convergence angle, which sometimes has a separate control). Often the best electron images come out fine on a photographic film or on the CCD camera, but are virtually invisible on the phosphor to the naked eye. That is because, although intensity is increased by increasing the condenser aperture, contrast is reduced. Just try looking at some gold islands on a carbon film with high-intensity illumination (focussed almost to point on the phosphor screen): even the heavy gold particles virtually disappear. Try the same experiment with different sizes of objective aperture.

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Copyright J M Rodenburg