Coating grids
Materials: Any type of grid that needs film coating but in this
lab we use Synaptek slot grids (Ted Pella, Inc, cat # 4514), an Effa film
caster (Electron Microscopy Sciences, Cat# 7130501), Plexiglas box stage, glass
bowl of at least 7” diameter, black paper to cover the glass bowl, curved
forceps, some powdered dishwasher detergent e.g., Alconox, 2 glass Petri dishes
approx. 90-mm diameter, 70% ethanol, Ross Lens tissues, plain glass microscope
slides (VWR Scientific Products, cat # 48300-036), a 1 to 1.2% Pioloform (Ted
Pella, Inc., cat # 19244) chloroform solution, chloroform (American
Bioanalytical, cat # ab350), a timer, a small jar containing filter paper
(Whatman) and partly filled with silica gel #6-16 mesh dessicant beads (VWR
Scientific Products, cat # 26668-109), single edged razor blades, Parafilm, plastic
Petri dish sets 90-mm diameter, Whatman filter paper 90 mm diameter, gooseneck
lamp, pen, safety goggles, gloves, lab coat and/or apron, fume hood.
- Select a dry, non-humid day, to coat grids. High
humidity seems to diminish film quality.
See diagram of set up of the Effa film caster in a fume hood for
coating glass microscope slides:
The
rest of the set up can be outside of the fume hood for casting the film on
water. See this diagram:
- Before setting anything up, dissolve about ½
teaspoon of concentrated dishwasher detergent, e.g., Alconox, in double
distilled water in each glass Petri dish. It seems to take at least a half
an hour to partially dissolve. Or try mixing or sonicating to speed up
things up. The Pioloform solution
can be made up the day before (see Special Procedures at end of this
section for recipe) or can be up to several months old. An old Pioloform
solution may not be effective.
- Wear safety goggles, gloves, and lab coat while
working in fume hood with the Effa film caster. Assemble the Effa film caster by inserting the upper glass
cylindrical tubing into the round, volumetric base. Then, rinse the Effa
film caster once with chloroform. Discard this chloroform in appropriate
waste container. Next, add about 150 to 200 ml of the Pioloform solution
to the Effa film caster. Secure
the Effa with its little metal springs and glass cap. The rubber tubing
with squeeze bulb can always stay attached to the side arm of the glass
base.
- The bottom and sides of the glass water bowl
should be covered in black plastic or paper to shut out reflective
light. Fill this bowl to the brim
with double distilled water and place it in the plexi-glass box stage as
diagrammed above. Set up the gooseneck lamp to illuminate the water
surface through the roof of the stage as diagrammed above.
- Once the soapy, dish detergent solution is ready,
prepare to wash the glass slides. To do this, first rinse each slide with
70% ethanol and scrub it clean with Ross Lens tissues.
- Rinse each slide in running double distilled
water to remove all ethanol.
- Dry each slide well with Ross Lens tissues and
place each slide in the soapy solution. Prepare a number of them in this
way. It only takes a minute of soaking to soap-coat a slide. Or the slides
can sit in the soapy water for several hours, if necessary.
- Take one slide out of the soapy water and scrub
it dry with Ross Lens tissues. Hold one end of the slide with a lens
tissue while drying the rest of slide. Avoid putting any fingerprints on
the slide.
- Transfer this slide to the upper chamber of the
Effa film caster and place the glass cap on the top of the chamber as
shown in the first diagram.
- Turn the stopcock so it is in the “T” position,
to only allow pumped air into the bottom chamber.
- Squeeze the bulb rapidly to force Pioloform
solution to gravitate up into the upper chamber to coat the glass slide.
Try to only coat the lower ¾ of the slide and leave the top ¼ dry for
handling, if possible.
- As soon as the upper chamber is ¾ filled with
Pioloform solution, stop squeezing the rubber bulb. Then slowly turn the
stopcock clockwise until the solution begins to drain down back to lower
chamber. Do not turn any further as it is draining, unless you are trying
to control the rate of draining.
Allow the upper chamber to drain undisturbed. It is the rate of
draining that controls the thickness of the film. Too fast a drain rate
results in a thicker film (gold to purple interference color). Too slow a
drain rate results in a thin film (gray interference color). It is
personal preference that guides film thickness. A moderate rate of
solution drain seems to result in a medium to heavy silver interference
color, indicating a certain film thickness. This medium to heavy silver
film color is preferred for serial sections, as this thickness is rugged
enough to sustain all the handling involved; yet, the resolution under the
electron beam is not compromised.
Once the rate of drain is controlled for, then note if the film
continues to come out too thin or too thick. If this is the case, then the
Pioloform solution can be made more concentrated (add more Pioloform
powder) or dilute (add more chloroform).
- As soon as the Pioloform solution is drained from
the upper chamber of the Effa film caster, then quickly remove the slide
and transfer it into the small jar that contains desiccant. See diagram:
Allow
the slide to dry in this jar for one minute exactly. Carry the jar/slide over
to the next station, that is, the Plexiglas box stage area.
- Remove the slide from the jar and prepare to cut
the film on the glass slide. To do this, hold the glass slide by its
handling end and brace it at an angle against a clean area of a counter
top, for example.
- Use the corner of a new razor blade to make cuts
along the edges of both sides of the glass slide. See diagram:
- Next, position fingers on the handling end of
slide. Then, submerse the slide into the bowl of water, about ¾ deep –
just a quick dip to wet the slide. Avoid touching the water with your
hands, as the water must stay clean. See diagram:
- Then, slowly re-dip the length of the slide into
the water. The film from each side of the slide should release onto the
surface of the water. Shine the
lamp from above to reveal the interference color of the film. Note any
mars or blemishes in the films. Discard any unusable films, by wiping the
surface of the water with a Ross Lens tissue. On the other hand, if the
film doesn’t release at all, then suspect that there wasn’t enough of a
soapy film on the slide, or that the soap solution was contaminated.
Alternatively, one can try re-cutting the glass slide with razor, as
described above, or try re-dipping the slide more slowly into the
water. There may be the possibility
that the glass slide itself is a poor releaser. In that case, try another
brand of glass microscope slides.
- Once the film(s) is floating on the water’s
surface, look for the best areas on it to drop grids. Position the lamp so
it bests illuminates the floating films.
If using notched Synaptek slot grids, pick up the grid, notch side
up, with the curved forceps as diagrammed:
- Drop the grid onto the good areas of the film.
Place as many grids as desired onto the good areas of the floating film.
When finished, return to each grid and gently press one edge of the grid
lightly, with a closed forceps. This will help to attach the grid to the
film.
- Prepare to pick up the load of grids. To do this,
first stretch about a 2 inch length piece of Parafilm over a clean, dry
glass microscope slide. Do not touch the “good” surface of the Parafilm,
as this side will be in contact with the “film of grids”.
- Position the end of the Parafilm-covered slide
above the “free” end of a floating film. Then dip slide into the water to
catch this free end and continue to plunge the slide deeper straight into
the water in order drape the entire film of grids onto the glass slide.
Then pull the slide straight up out of the water. Invert the slide for a
minute to drain any water out of the slide. See diagram:
- Have ready the plastic Petri dish sets (90-mm
diameter) with filter paper (also 90-mm diameter) lining the bottom dish.
Label the dish or filter paper with today’s date and with anything else
appropriate.
- Place the microscope slide containing the grids,
grid side up, into one of these dish sets for storage. The grids can be
used on the next day. These grids should be ultimately inspected in the
electron microscope before using with thin sections. Select one or two
grids from each glass slide and examine under 5000X to 10,000X
magnification. The film should look blank. If it shows many holes (several
small holes are acceptable) or many streaks or tracks, then discard this
batch of grids. You can recycle the Synaptek grids, however. Many streaks
indicate that the glass slide itself, used in film coating, is casting its
own impression. Try another brand of glass microscope slides.
- Coat as many such grids as needed for up to 6
months or more supply.
- When finished for the day, pour the Pioloform
solution back into its original container and seal the cover with
Parafilm.
- Rinse the Effa film caster apparatus four times
with chloroform. Discard the rinse chloroform into an appropriate
container.
- Separate the upper chamber from the bottom
chamber and allow these parts to dry overnight in the fume hood. On the
next day, store the film caster in an appropriate area.
Special Procedures
Pioloform-chloroform
recipe:
Materials: Pioloform powder
(Ted Pella, Inc., cat # 19244), ultra-pure chloroform (American Bioanalytical,
cat # ab350), a 250 ml capacity (approx.) amber glass jar with lid, Parafilm,
stir bar, a 250 ml graduated cylinder, stir plate, top loading balance with
draft shield, weigh boat, fume hood, lab coat and/or apron, gloves, goggles,
surgical mask (optional).
1. Pre-rinse the amber bottle, graduated cylinder and
stir bar with chloroform in the fume hood. Discard the rinse chloroform into an
appropriate container.
2. Next, add 200 ml of chloroform into the amber bottle
and add the stir bar. Cover this bottle and place on stir plate, also located
in the fume hood.
3. Weigh out 2.4 g of Pioloform powder (for a 1.2%
solution) in the fume hood.
4. Slowly add the 2.4 g of powder to the 200 ml of
chloroform while it stirs.
5. Continue to stir the solution until all the powder is
dissolved.
6. When complete, wrap some Parafilm around the cover of
amber jar. Store in an appropriate area at room temperature.
Repairing a hole in a coated grid
1. If a hole or tear is present in a coated grid, distant
to a stained ribbon of serial sections, for example, then, it is possible to
repair it. See diagram:
2. To do this, use the same equipment used in coating
grids. The exception is that the Pioloform solution has to be more dilute (0.6
to 1%) or the film has to be very thin so the final grid film thickness does
not compromise resolution under the electron beam.
3. Prepare a thin film (coating a clean but soap-filmed
glass slide in Effa film caster) and release it on the water surface.
4. Choose an area that is gray in interference color.
Carefully place the slotted grid that has the hole onto a good area of the
film, Epon section side down. Then gently press the grid onto the film a bit.
5. Pick up the film with a Parafilm covered glass slide,
as previously described.
6. Store the slide in a clean Petri dish set. Allow the grid to dry at least
overnight. This should do the trick for
grid repair!
