Procedures (Click on each thumbnail for a larger image. Click on the large image to close.):
- Pre-clean copper grids by sonicating them for 10 sec. in acetone followed by 10 sec. in ethyl alcohol. Rinse in
deionized water and pour them over filter paper in a dust free environment to dry (Fig. 1).
- Prepare Formvar solution. Add 0.17g of Formvar powder to 50 ml of chloroform and mix well on a
magnetic stirrer until dissolved (Fig. 2). Pour the solution into a clean Coplin jar (Fig. 3). This solution is hygroscopic
but will store well for several months if kept in a brown, sealed bottle.
- Clean a glass slide with water and detergent. Rinse well with water to remove the detergent then
rinse again with de-ionized water and dry with paper towels or KimWipes. Make sure that the slide is
free of dust and lint. Blow off any lint with compressed air. Keep the slide in a dry, dust-free
environment until use (e.g. on filter paper under an upturned beaker). If the plastic film adheres to the slide and cannot be
floated off (Step 6), several different methods of cleaning the slide -- or even NOT cleaning the slide should be tried. Recent
attempts (Sept. 2005) have shown that higher-quality, pre-cleaned slides work the best. There have even been reports that a
little bit of grease, such as that obtained by rubbing one's finger against the side of the nose, helps to prevent the film
from sticking.
- Add about 50 drops of a 50% glycerol/water solution to the surface of the Formvar solution. Place the
tip of a probe sonicator onto the surface of the solution and sonicate until mixed (Fig. 4). Sonication intensity should
be great enough to cause the solution to bubble vigorously. Mixing often requires no more than about 5 seconds. The solution
should now appear cloudy or milky. This should produce numerous holes that are 1-2 μm in diameter and suitable for use
with frozen-hydrated samples. Sonicating for longer periods of time produces smaller holes in the film; shorter times
produce larger holes.
- Immediately after sonicating, coat the clean slide with Formvar by dipping it into the solution. Touch the edge of the
slide to filter paper to wick off the excess fluid. The film will be milky when wet but it will clear somewhat as it dries. Dry the slide upright in a dust-free environment (Fig. 5).
- The next step is to remove the Formvar film from the slide. Fill the rectangular,
black dish with de-ionized water until a meniscus is formed on top of the dish. Then drag the plastic rod (See Fig. 7)
across the surface of the water to remove any floating debris. Score the edges of the Formvar film with an acetone-cleaned
razor blade (Fig. 6). Thoroughly breathe over the length of the slide to loosen the film, and float the film off onto a clean
water surface by slowly immersing the slide into the water at a ~15° angle (Fig. 7). If the film is difficult to
see, a bright desk lamp can be used to reflect light off the surface of the water and the film.
- Place grids, shiny/bright surface down, onto good (uniform, gray color, un-wrinkled, etc.) areas of the film (Fig. 8). Place a small piece of clean, white office paper onto the surface of the grids and film and allow it to soak up water. The grids will show
up through the paper when the paper is fully soaked (Fig. 9). Pick up the paper, grids and film with forceps and place on dry filter
paper, grid side up, in a covered Petri dish to dry. (Fig. 10).
- When the paper is completely dry, place the paper with the film and grids onto a piece of filter paper that is just saturated with methanol. The glycerol in the Formvar solution often produces bubbles in the film not open holes. The methanol treatment will break the thin film
over these "pseudo-holes". Soak in a covered petri dish for about 40-50
minutes. Allow the film to dry before carbon coating.
- Carbon coat to desired thickness. See the instructions for
doing this elsewhere on this web site. Holey films need the added stability of thicker carbon. These should be
coated to a gray/black color which takes about 400 milliseconds on the Emitech carbon coater (Fig. 11). For large particles or complexes (>800 Å), it may be beneficial to carbon coat
very heavily to increase the depth of the holes.
- At this point, the grids may be examined in a light microscope with phase contrast illumination to determine the
quality of the films.
- Remove the Formvar film by placing the white paper and grids onto a piece of filter paper saturated
with ethylene dichloride. The level of ethylene dichloride should be sufficient to completely soak the backing paper
without submersing the tops of the grids. One half hour should be sufficient time to dissolve the Formvar film and not
damage the carbon support. Remove the paper and allow it and the grids to dry.
- Tape the grids and paper to a glass slide with "Scotch" tape to prevent the grids from being
jostled. Place the slide in a covered petri dish. Care must still be used when handling the grids to prevent them
from being scattered.
Fig. 8. Cleaned grids on the Formvar film. |
Fig.9. White paper placed over grids in order to pick them up. |
Fig. 10. Formvar-coated grids drying in petri dish. |
Fig. 11. Comparison of carbon coating thickness of normal carbon films and holey or perforated carbon films. |
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Fig. 1. Grids drying in a petri dish.
Fig. 2. Formvar solution on stirring plate.
Fig. 3. Formvar solution in a Coplin jar.
Fig. 4. Sonicating the solution. The probe of the sonicator is placed right at the
surface of the solution and the current is set high enough to produce vigorous bubbling.
Fig. 5. Slides drying under beaker.
Fig. 6. Scoring the Formvar film with a razor blade. The film is thicker on the end of the slide and appears milky.
Fig. 7. Floating the film on the surface of water.
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