Critical Specimen Focus in Biological Pathology and Research Applications
*Kenneth L. Tiekotter, Dept. of Biology, University of Portland, Portland, Oregon *97203 USA
*tiekotte@up.edu

Anyone who has learned to use a transmission electron microscope in the past 40 years can attest: one of the most difficult aspects to master is that of specimen focus. Everyone has gone through the process of finding a hole in the substrate or imaging a small piece of precipitate ‘pepper’ to view the contrast of the Fresnel fringe to set under- or over-focus conditions. This procedure is done at moderately high magnifications using the focusing screen and the binocular and wobbler. Furthermore, under-focus is necessary to create additional contrast in an otherwise flat image.

The use of CCD digital imaging does not require the use of the TEM fluorescent focusing screen or the binocular. In addition, focus can be adjusted for critical resolution instead of adjusting for improvement to image contrast. When you consider the importance of full resolution in a digital image, the concept of contrast is of somewhat less importance, i.e., provided you have sufficient dynamic range, you can easily adjust for gamma, brightness, and contrast in the final digital image using software.

The procedure of critical resolution focus is very easy to follow when using Gatan DigitalMicrograph software for image selection and acquisition. The Camera View palette is one of the central areas for selection of Search, Exposure Mode, Focus Loupe, Auto Survey, and the Camera Insert selection. However, for now we will only be concerned with the Search set-up and Focus Loupe (Fig. 1).

Figure 1. The Camera View palette is central to starting specimen search and focus. Search mode is selected as is the Focus Loupe.

The basic procedure contains these easy steps:

1) Locate the field of interest in your specimen using the Search mode of Camera View. This is analogous to searching for a particular area of your specimen on the fluorescent screen (Fig. 2).

2) Select the Focus Loupe by clicking the check box. A red, square box appears in the image. This is analogous to using the focus screen (Fig.3).

3) Move the Focus Loupe (the square box) to an area of interest anywhere in the field of view. This feature is unique in that it provides the ability to see the difference in the focus loupe area in contrast to what is seen in the area surrounding it and can be moved anywhere in the field-of-view (Fig. 3).

4) If necessary, change the shape and/or the size of the Focus Loupe area by dragging the ROI handles (green corners). This is a unique feature, allowing complete flexibility in how you select your area of focus interest.

5) Activate the Zoom function by pressing the letter key “Z” on the computer keyboard. This unique feature is analogous to the combined magnification of fluorescent focusing screen and binocular (Fig. 4).

6) Next, critically focus the image for better resolution by changing the focus setting on the TEM and observing the change of fine details in the image.

7) Click the button for Start Acquire, found in the Camera Acquire palette, and the process is complete, resulting in an optimally focused image with the best resolution (Fig. 5).

Figure 2. Locate the field of interest in your specimen using the search mode of Camera View.

Figure 3. Select the Focus Loupe. Move the Focus Loupe to an area of interest anywhere in the field of view area.

Figure 4. Activate the Zoom function by pressing the letter key “Z” on the computer keyboard. Critically focus for resolution.

Figure 5. Full resolution image acquired after selecting Start Acquire in the Camera Acquire palette, resulting in an optimally focused image with the best resolution. Note image selection in the red area with the red arrow.

Figure 6. Actual 4x enlarged image from Figure 5 (red box). Each arrow is equal to 1.0 micrometer. Note clear periodicity between the major dense line and the narrower, intraperiod line.

The 4x enlargement of the myelinated sheath (Fig. 6) from the original image of Figure 5 clearly demonstrates the advantage of critical focusing with the use of the DigitalMicrograph Focus Loupe/Zoom tool. The major dense line and narrower intraperiod line are clearly visible as are cytoplasmic RER and axonal neurotubules, and neurofilaments. The advantage of critical focus for resolution and not for contrast makes this feature a valuable tool for the production of consistent quality, high-resolution images.