Electron Microscopy (EM) is the method used to obtain high-resolution images of plant and animal tissue. There are two basic types of electron microscopy: scanning electron microscopy, (SEM), and transmission electron microscopy, (TEM). SEM yields high-resolution surface morphology from a secondary electron beam, which ‘bounces off’ the sample, is collected in a scintillator, and displayed onto a monitor as a relief image showing the surface features of the sample. TEM yields high-resolution ultrastructural detail from a transmitted electron beam through the tissue, allowing morphometric analyses of the internal detail of sectioned cells or direct preps of macromolecules, localizing where exogenous substances reside within a cell, or immunologically marking specific antigens in a cell.
The TEM can be broken down into a few main components, which are aligned in a column under vacuum (Fig. 1a): the gun, which produces electrons; the condenser system, which forms the probe; the sample (sample preparation is important and time-consuming); image formation; projection of the image (magnification); and viewing and recording the image. The image is projected onto a fluorescent screen, which can be directly viewed through a port at the base of the column; or it can be collected by a digital camera inserted into the column, either above or below the fluorescent screen, and imaged, as well as stored, by means of a monitor connected to a computer. The UF CoM EM Core uses a Hitachi H7600 TEM with an AMT digital imaging system (Fig. 1b)
When you come to the EM Lab to use the Hitachi TEM at a pre-determined appointment time, the microscope will be turned on, aligned and ready for you to use, as follows:
1. Log your PI’s name and your name, the start time, and the ‘Fil. count’ displayed on the right-hand monitor (Fig. 2, green arrow) onto the 3 x 5 card provided on the corner desk.
2. On the right side of the column, approximately at eye level, is the specimen chamber and the specimen holder (Fig. 3) where the specimen grid will be inserted into the vacuum in the column. Pull the specimen holder straight out, until it stops. Turn the holder counter-clockwise until it stops. Do not try to take the holder out yet. There will be clicking noises. On the bottom of the specimen chamber is a toggle switch (Fig. 3, red arrow). Move the toggle switch down to the “air” position to admit air into the specimen chamber. The light should turn red.
3. When the red light on the specimen chamber turns off (about 30 seconds), carefully remove the specimen holder from the specimen chamber by pulling it straight out.
4. Place the specimen holder on the plastic stand on the corner table (Fig. 4). Try not to touch any part of the holder except the plastic handle.
5. Using the provided curved forceps, carefully remove the gold planchet from the specimen holder by first pushing the screw holding the spring (Fig. 5, blue arrow) towards the plastic handle, then lifting out the planchet (Fig. 5, red arrow) and placing it on the lens paper on the stand. (Try to avoid scratching the holder or the planchet, as the gold coating is thin and expensive.)
6. Using the sharp forceps, #3 or #5, carefully place the grid (Fig. 6a, black arrow), specimen-side up, on the specimen holder (Fig. 6b).
7. Replace the planchet (Figs. 6a, 6b, red arrows), using the curved forceps, and gently release the spring. Try to hold the planchet in place with one forceps’ prong, while releasing the corner of the spring with the other (Fig. 7).
8. Turn the specimen holder over, and gently tap with the forceps to verify that the planchet and the grid will not fall out.
9. Carefully insert the specimen holder into the specimen chamber: note the prong on the holder and the slot on the chamber for correct alignment (Fig. 8, red arrows).
10. Gently but firmly push the specimen holder into the specimen chamber until it stops; while holding it in place, push the toggle switch on the specimen chamber (Fig. 3, red arrow) to the “up” position to evacuate the chamber. The light will be red while it is evacuating.
11. When the light turns green (about 30 seconds), quickly (within 10 sec.) rotate the specimen holder clockwise (toward the back of the column) until it stops, then release it. It will be sucked into the column to a ‘rest’ position.
12. When the specimen holder is in the ‘rest’ position, the sample is under vacuum, but not in line with the beam; rotate the specimen holder counter-clockwise (toward the front), and it will be sucked the rest of the way into the column.
13. On the right-side monitor (for microscope control), use the mouse to turn on the ‘Beam’ (Fig. 2, blue arrow).
14. On the left-side monitor (for imaging control), use the mouse to insert the camera into the column (Fig. 9b, ‘camera control’, fat red arrow,).
15. Use knobs on the panel at the left of the column (Fig. 10) to (a) move specimen (also on the right panel, Fig. 11), (b) increase or decrease magnification, (c) adjust brightness or (d) switch from Low Mag to Zoom 1.
16. The focus knobs are on the panel at the right of the column (Fig. 11), in the center.
17. To set up a file for your images, on the left monitor (Fig. 9a) open File – open Case Study – select ‘Create New Case) or ‘Resume Previous Case’ (Fig. 9b); type in or select a name.
18. To collect an image, first focus by using the ‘focus’ button on the left monitor (Fig. 9b, blue arrow), or the wob. button on the right panel, and the focus knob on the right panel (Fig.11b). The image is parfocal for all mags below 30,000, and for all mags from 200,000 down to 40,000, so focusing at a higher mag produces better images. When you are satisfied with the focus, return the button on the monitor to the ‘survey’ mode (Fig. 9b, red arrow), and select the cabinet icon on the left edge of the monitor. The collection will take a few seconds, and a box will appear on the monitor allowing you to type in descriptions, including sample identification and conditions, which will be included in the displayed image. Follow the instructions to save or cancel, and return to your sample display.
19. When you have finished imaging your sample, you may change to another sample or remove your sample and place the TEM on standby for the next person, as follows:
- Select the ‘camera out’ button on the left monitor (Fig. 9b, fat red arrow)
- Press the ‘lens’ reset button on the left panel; mag will return to 2,000x (Fig. 10b).
- Return to steps 2 – 5 to remove the specimen from the column.
- Replace your grid with another, or replace the planchet without a grid.
- Continue your session starting at step 6.
20. If you are finished, continue to step 11; turn off the ‘Beam’ using the mouse on the right-side monitor (Fig. 2, blue arrow).
21. On the 3 x 5 card, record the time and the ‘Fil. count’ from the right monitor (Fig. 2, green arrow) to log out.
22. You may download your images onto your flash drive or a CD, using the computer on the left. Look for your file in the ‘images’ folder on the C drive.