Photographing across the depth of a thin section

 

One way to assess biological tissue quality is to take photographs across the width or length of a thin section. The photographs are taken of every field or every other field, for example, in a straight, line direction. The ability to rotate the grid, via a rotating holder, orients the grid for this type of photography. See diagrams below depicting Epon thin sections with either 400 or 100 um tissue slices. Each thin section is oriented for the straight, line photography.

The photography should begin at the top or to one side of the thin section. By moving the X or Y stage controls on the electron microscope, every field in a straight line can be photographed.  On the JEOL 1200 EX and 2010 electron microscopes, it is the field within the outer brackets, marked on the phosphorescent screen, which is included in each photo/negative. See diagram below. Note that the photographic field is rectangular. In addition, the label projected onto each negative is usually located on the right side of this field. The photographic field of the same area will naturally encompass more or less when decreasing or increasing magnification, respectively. For example, at approximately 6000X, the photographic field will include an area 12.5 um x 16.2 um.  At approximately 10,000x, the photographic field will include an area 7 um x 9 um. Consequently, this rectangular field is used as a ruler to measure distances.

The scale bar adjacent to the brackets on the screen is equal to 5 cm. At approximately 10,000X, this scale bar is equivalent to 5 um. On the JEOL 2010, moving the X, Y stage controls shows a corresponding readout on the CRT of the distance in um traveled. These are all the tools that are available to take photographs in a straight-line fashion.

            In our laboratory, we photograph a thin section of the rat hippocampus, across the length or width, from one surface to the other. In some studies, it is of interest to study the tissue near the vicinity of electrode placements. This latter interest will serve as an example of such photography.

 

 

Procedure:

 

1. At LOW MAG mode, e.g. 60X, orient the grid by rotation so that the air versus net will be in the north/south orientation, respectively. Study the appearance of the tissue/section so that different areas will still be recognizable even though the magnification will be increased, e.g., to 6000X, for photography. If the electrode hole is obvious, position the central black dot on screen near it.

 

2. Put the objective aperture back in the beam path and increase the magnification to 6000X. The area near the electrode hole should be in view. Rotate the grid as needed to re-orient the tissue.

 

3. Align the length or width of the rectangular photo field, depending on preference, to the top of the tissue. Rotate the grid as needed. See diagram:

4. To determine if the sequence of photography can be maintained in a relatively straight line, from “air” surface to “net” surface, turn either the X or Y stage control, whatever is applicable. Continue turning, until the entire field, from top to bottom in this case, is traversed.

 

5. Switch to LOW MAG mode again, remove objective aperture, and observe where the central black dot (on the phosphorescent screen) ended up. If it is located directly across from the starting point, then the grid is oriented properly. If it is not, then try rotating the grid in clockwise or counterclockwise until the orientation is acceptable.

 

6. Put the objective aperture back in. Go back to the air surface at 6000X, and prepare to photograph across the depth of the tissue.

 

7. The first step is to align the rectangular field (either with its length or width) to the “air” surface and rotate the image as needed. Include a bit of Epon interface to indicate the “air” surface in the first photograph, if so desired.

 

8. To take an electron micrograph, first, focus the image using the wobbler as needed.

 

9. Once in focus, one can under-focus 2 clicks on the fine focus knob on the JEOL 1200 EX and on the JEOL 2010, one can set the auto-under-focus to 2. These values are arbitrary. You should first take a series of photos at all the under-focus settings and determine which works the best for your research. Leave the small screen in place during the photo shoot.

 

10. Set the brightness or illumination to 0.5 seconds exposure. This short exposure seems to work best on sections attached to coated slot grids because there is less time for image drift to occur. If there is image drift present, then wait a few minutes before taking a photo. Hopefully, with time, the drift becomes minimal.

 

11. Check that the photo setting is on AUTO and not MANUAL. (The MANUAL setting is good for taking photos under LOW MAG conditions where there is a range of exposure times to choose from).

 

12. Make sure that the all white incident lights are off in the room. Lock the room door to avoid someone walking in during a photo shoot.

 

13. Press the photo button once or twice, as needed, to shoot the picture. Avoid touching the scope during the 0.5 seconds of photography to avoid vibrations. Once the photo is shot, prepare to move on to the next field.

 

14. Turn the X or Y stage control (whatever is appropriate) to move to the adjacent field. Alternatively, one may choose to shoot every other field at pre-calculated depths. Remember that the rectangular photo field is used as a counting device of um traversed. See diagram:

15. Once all photographs have been taken, it is useful to return to LOW MAG mode and observe the path of photography traversed, indicated by the beam “burn”. If all the photos followed a relatively straight path, the area burnt in by the beam is obvious. Take a photograph of this image under LOW MAG.

 

16. To take a photo under LOW MAG, focus on the image as usual, using the wobbler assistance.

 

17. Set the photo to MANUAL.

 

18. For 60X to 100X, set the exposure toggle to either .35 sec or .5 sec exposure (the second setting will result in a darker negative). For 100X to 200X, set the exposure to .5 sec or .7 sec.

 

19. Leave the small screen in place.

 

20. Press photo button once or twice to photograph the image.

 

21. Now you have a record of the depth profile photographed. This low magnification image of the electron beam “burn” markings is also useful when isolated photos are taken at a distance from the tissue surface. For example, if only several photos are taken sequentially in a straight path across a tissue, then the positive print or negative of the low magnification of the beam burn markings is a useful tool. One can measure distances in um on the print or negative itself! See diagram: