Focus/stigmation
Micrograph quality is affected by aperture size, focus, stigmation, and use of image collection software.
The image of rat’s liver on the left was taken without an objective aperture inserted. The image on the right had an objective aperture inserted. Note the increased contrast in the right image.
Achieving correct focus of an image is important.
The image on the left shows a hole in a resin support film that is close to focus (suitable for collection). On the right the image is out of focus: note the added white ring (also known as a Fresnel fringe) on the inside edge of the hole that provides extra contrast which is appealing to the eye but incorrect.
To achieve good focus by eye, at high magnifications, it is helpful to focus on the ‘grain’ of the sample support film. This supplies information about both focus and stigmation. The presence of stigmation results in a streaking or stretching of the normally roundish grains (see arrows on the left and right in the image) which makes achieving focus more difficult.
Correcting stigmation involves rounding the cross-section of the beam with the X and Y stigmators. Often both X and Y stigmation need adjustment, however, each results in a stretching of the grains along a different axis.
In the image the top and bottom images are out of focus.
When the beam cross-over point is slightly above the sample or slightly below the sample, the beam is not at focus (see top and bottom frames in the image for out-of-focus images). The contrast rings around the grains will be negligible at focus but as we move through focus from over to under, the contrast ring colour will invert (white to black).
An image can be brought into focus either optically, as discussed above (by using binoculars attached to the TEM to observe the viewing screen) or by using the Fast Fourier Transform (FFT) application available in imaging software packages for TEMs equipped with digital image collection equipment.
Detail on using FFT
The image shows an FFT display from a biological sample where the image in the central panel has correct stigmation. To the left and right the image needs correcting.
Alternatively, an image at focus will have the least contrast, as was seen in the image of the hole above. The selected area diffraction aperture is used to enhance contrast when working in the low magnification mode, in the same way that the objective aperture is used at higher magnification. By taking the selected area diffraction aperture out, the point of lowest contrast can be seen more clearly as the focus is changed. This can be used as a guide to when focus is reached.
The presence of an artifact known as a Fresnel fringe can be used as a guide to help focus an image. The image below shows this in practice, where the inner fringe denotes an under focused image, and a thicker outer fringe shows an over focused image.
