0. Safety precautions
This procedure involves the use of hazardous chemicals (including embryotoxins). Review MSDS for information on exposure limit, health risks, first aid, handling, etc.
Where indicated, perform this procedure under a chemical fume hood.
Wear appropriate Personal Protective Equipment for this procedure:
Lab coat
Nitrile gloves (double-layer; regularly check for holes)
Eye goggles
Mask
Place a piece of absorbent sheet on the work surface before starting the procedure.
Have all waste containers ready (see Clean-up).
1. Reagents and supplies:
TEM Grids (SynapTek Be-Cu Notch-Dot 1 × 2 mm slot; Ted Pella 4516)
20-ml scintillation vial
sonicator (stored under fume hood in NHB 3.360E)
disposable pipets
Ethanol (100%; this can be excess from epoxy embedding)
Chloroform for cleaning grids and glassware (Fisher C298-500)
90-mm glass Petri dish lined with filter paper
forceps
extra dry chloroform to dissolve Pioloform F in (Fisher AC36432-1000)
Pioloform F (Ted Pella 19244)
250-ml glass bottle
glass slides (Gold Seal Microslides, Fisher 12-518-101)
coplin jars
Ethanol (70%)
Purified water (double-distilled or ASTM Type I equivalent)
Alconox detergent (powder; VWR 21835-032)
lens tissue (EMS 71700)
film casting device (EMS 71305-01)
glass funnel
glass jar containing desiccant (e.g., Ted Pella 19961), lined with filter paper
Plexiglas box stage
lamp
large glass container (e.g., VWR 89090-700), covered with matte black paper
RO water
Single-edge razor blades
Parafilm (2-in. squares)
150-mm plastic petri dish, lined with filter paper
2. Cleaning grids (should be done the day before use, or earlier)
Perform this procedure in a fume hood. Wear two layers of nitrile gloves, lab coat, and eye protection.
Follow this procedure to (1) prepare new grids, or (2) reuse grids previously coated with Pioloform.
Place grids into a 20-ml scintillation vial.
Add ~10 ml of 100% EtOH into vial.
Loosely cap the vial and place it into sonicator filled with RO water (water level should be about the same as EtOH in the vial).
Sonicate for 5-10 min.
Use a disposable pipet to remove EtOH. Collect the waste EtOH into "Flammable" waste bottle.
Add ~10 ml of chloroform into vial and sonicate for 5-10 min. Repeat one more time. Collect waste chloroform into "chloroform-Pioloform" waste bottle.
After removing chloroform, tap out the grids onto a glass Petri dish lined with filter paper. As the grids dry out, spread them into a single layer.
Keep lid of the dish ajar and let the grids air dry overnight in fume hood.
3. 0.7% Pioloform solution (should be made the day before use, or earlier)
Perform this procedure in a fume hood. Wear two layers of nitrile gloves, lab coat, mask, and eye protection.
In a 250-ml glass bottle, add 1.4 g of Pioloform into 200 ml of extra dry chloroform. Use a stir bar to dissolve Pioloform.
Store in the flammable cabinet.
Note 1: It may be necessary to adjust the concentration of Pioloform, depending on thickness of the resulting film.
Note 2: If your film is getting too may holes, you have too much water in your Pioloform solution. Time to make a new batch.
4. Saturated Alconox solution (should be done the day before use, or earlier)
Add saturating amount of alconox to 100 ml of purified water. Stir overnight.
Use a vacuum filter unit (0.22 micron pore) to clear the Alconox solution. This can take a while.
Store in a glass bottle at RT.
5. Cleaning glassware (should be done the day before use, or earlier)
Perform this procedure in a fume hood. Wear two layers of nitrile gloves, lab coat, and eye protection.
In a fume hood, add a small amount (~100 ml) of chloroform into flask of the film casting device through glass funnel.
Insert casting cylinder of the device and secure it with two holding springs.
Using the hand pump, circulate chloroform back and forth between the flask and casting cylinder several times.
Remove the holding springs and casting cylinder from the flask, and rinse out chloroform into "chloroform-Pioloform" waste bottle.
Repeat Steps 1-4 at least once more.
Leave the disassembled device and glass funnel in fume hood to air dry overnight.
6. Coating
Part 1: Preparing Glass Slides
Prepare 3 coplin jars containing the following:
70% EtOH (stored in flammable cabinet)
Purified water
10 times dilution of the saturated Alconox solution: add 5 ml of the Alconox solution into 45 ml of purified water.
Place glass slides into the jar containing 70% EtOH for ~5 min. One coplin jar can accommodate up to 7 glass slides.
Scrub the slides clean with lens tissue. Avoid putting any fingerprints on the slide.
Place the cleaned slides into the jar containing purified water for ~5 min. Then Scrub the slides dry with lens tissue.
Place the cleaned slides into the jar containing Alconox solution for ~5 min. Then Scrub the slides dry and clean with lens tissue. Store the Alconox-coated slides in a clean container until use.
Part 2: Casting Pioloform Film
This part must be performed in the fume hood. Wear personal protective equipment (minimum required: two layers of nitrile gloves, lab coat, and eye protection)
Add ~200 ml of 0.7% Pioloform solution into the flask of clean film casting device in fume hood.
Assemble the film casting device.
Transfer one of the Alconox-coated slides to the casting cylinder of film casting device and place the glass cap on top.
Close the air-out valve (black) and open the air-in stopcok (red) to allow pumped air into the flask.
Squeeze the bulb to force Pioloform solution up into the casting cylinder to soak the lower 3/4 of the glass slide.
Close the air-in stopcok (red).
Slowly open the air-out valve (black) to drain Pioloform solution from the casting cylinder at a uniform rate. (The drainage rate can be controlled to some extent by changing how fast/slowly the air-out valve is opened. Fast drainage will result in thicker film.)
As soon as the Pioloform solution is drained from the casting cylinder, quickly remove the slide and transfer it into the small jar containing desiccant. Allow the slide to dry in this jar for at least 1 min.
Disassemble the film casting device and pour the Pioloform solution back into its original container.
Rinse the film casting device and glass funnel with chloroform as described above. Discard the rinse chloroform into "chloroform-Pioloform" waste bottle. Leave the disassembled device and glass funnel in the fume hood to air dry overnight.
Part 3: Coating Grids
Carry the jar/slide over to a lab bench where the Plexiglas box stage is set up.
Fill a large glass container (covered with matte black paper) with RO water. Avoid touching the water with your hands, as the water must stay clean.
Turn on the lamp and adjust its angle/location such that the reflection on water surface can be seen easily.
Remove the slide from the jar by holding the frosted end. Brace the slide at an angle against a few layers of lens tissue on bench top.
Use the corner of a new razor blade to make cuts along the edges of both sides of the glass slide.
Holding the frosted end, briefly submerge the tip of slide (about 1/2 in.) into the bowl of water (just to wet the slide).
Slowly submerge the length of the slide into the water. The film from each side of the slide should release onto the surface of the water. Adjust the lamp from above to reveal the interference color of the film.
Note any wrinkles and blemishes in the films. Discard any unusable films, by wiping the surface of the water with a lens tissue.
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
Drop the grid onto the good areas of the film. Place as many grids as desired onto the good areas of the floating film.
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.
Wrap a clean dry glass microscope slide with a 2-in. square piece of Parafilm while keeping the paper backing. Press onto Parafilm or lightly scratch on the paper backing to get Parafilm stick onto the slide. On frosted end of the slide, write down the batch information (this usually consists of today's date and which side of which slide the film was casted on; e.g., "20130126-3b", for a film casted on Jan. 26, 2013 on the backside of slide #3.). Do not remove the paper backing until ready to pick up the Pioloform film containing grids.
Have ready the plastic Petri dish sets (150-mm diameter) with filter paper lining the bottom dish. Label the dish or filter paper with today’s date and with anything else appropriate. One dish set can accommodate up to 5 slides.
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. You can also flick excess water off the slide.
Place the microscope slide containing the grids, grid side up, into the 150-mm plastic Petri dish for storage. Allow the grids to air dry overnight. For best results, the grids should be used on the next day after quality check.
7. Clean-up
Waste containers:
Hazardous Liquid Waste: Pour all waste into the proper waste collection bottles available in flammable cabinet.
Chloroform-Pioloform (chloroform from cleaning grids and glassware; old Pioloform solution)
Flammable Solvents (100% Ethanol used to clean grids; this ethanol should NOT contain anything else)
Hazardous Solid Waste: Place all contaminated solid waste (e.g., gloves, weighing dishes, etc.) into hazardous waste bags in fume hood.
Sharps: Place all used razor blades and contaminated broken glassware into a sharps container.
Uncontaminated glass: Place all used intact glass slides into the uncontaminated glass disposal container.
Glassware and equipment:
Rinse the film casting device and glass funnel with chloroform as described above . Discard the rinse chloroform into "chloroform-Pioloform" waste bottle. Leave the disassembled device and glass funnel in the fume hood to air dry overnight. On the next day, store the device in an appropriate area.
All other glassware (e.g., coplin jars) should be rinsed with RO water and air-dried.
8. Quality control
Pick one grid from each batch of Pioloform film.
Load the grids into tSEM, and examine at ~5000x 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 (They can be reused; see above ). See below for troubleshooting.
9. Tips and Troubleshooting
Select a dry, non-humid day, to coat grids. High humidity seems to diminish film quality
Alternative technique for draining Pioloform solution in the film casting device: As soon as the casting cylinder is 3/4 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.
Film thickness: It is personal preference that guides film thickness, which is determined by the rate of draining. 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). 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).
The films do not release from the slide: 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.
Film quality: Many streaks indicate that the glass slide itself, used in film coating, is casting its own impression. Try another brand of glass microscope slides.
