1. Sample Preparation (Polymers)

a. Important parameters: Tg, Tm, ODT’s
b. Prepare a sample that is at least 1mm x 1mm x 1mm by temperature or solvent annealing that is bubble-free. It is important that you are confident that the sample contains a morphology at room temperature that is the thermodynamically (or kinetically trapped) structure of interest. Use your knowledge of the ODTs, Tg’s, Tm’s to properly anneal. Bubbles in the sample will make microtoming and imaging very difficult or impossible.

2. Microtome startup

a. Turn on the surge protector.
b. Turn on the microtome controller.
c. Attach the necessary stage (cryo or room temperature). Make sure the appropriate cables are attached (back light for rm temp and back light and temp control for the cryo stage).
d. Attach the specimen holders for the appropriate sample stage. Make sure that if you are using the cryo-stage then you also have the weight for the microtome arm appropriately placed.

3. Cryo-temperature stage startup (skip for rm temp work)

a. Fill the LN2 reservoir.
b. Put the LN2 transfer line in place very carefully and slowly (this is the most fragile component of the microtome). One end goes in the reservoir and the other in the cryo-temp stage – these must be put in simultaneously.
c. Turn on the temperature controller.
d. Set the temperature for the sample and the knife (approximately -60 °C below the lowest Tg or Tm). A good starting point for most polymers is -100 °C for both the knife and the sample. Most of the time it is best to set the same temperature for both the sample and the knife.
e. Turn on the LN2 pump.

4. Sample mounting for cryo-microtoming (skip for rm temp work)

a. Using a razor blade, cut a wedge of the polymer that is approximately an equilateral triangle.
b. Place a metal post (2, 3 or 6.5mm heads) on the black disk like post holder.
c. Place a drop of the Tissue-Tek freezing medium on the top of the post.
d. Place your wedge of polymer, sharp side facing up, on the drop of Tissue-Tek.
e. Place the disk holder (and post with sample on it) inside the microtome chamber on top of the perforated aluminum stage.
f. Wait until the Tissue-Tek medium is frozen and your sample is fixed in place (the Tissue-Tek medium will turn from clear to white).
g. Once the sample is frozen, put the post in the specimen holder and secure it using the hex with the white label on it.

5. Sample mounting for rm temp work (skip for cryo)

a. Glue your sample using super-glue onto a post. Make sure the sharp side of the wedge is facing straight up from the post.
b. Once the glue dries, place your sample into the specimen holder.

6. Trimming

a. Always use a glass knife or the diamond trimming knife for trimming (never the diamond knife).
b. For cryo, mount the knife on the knife holder and tighten the holding screw by using the blue and white hex key. Screw in the black knife holder handle.
c. For cryo, place the knife holder (and knife) in the temperature stage.
d. For rm temp, place the glass knife spacer and the glass knife in the rm temp stage.
e. Make sure the knife is secured by tightening the appropriate screw.
f. The final size of the trapezoid facing you should be relatively small (how else can I describe this?), and you must remember that the larger the face, the more knife you will be ultimately using. A face that is too small will give you sample too small to collect. Err on the side of small, since the sample should get larger the more sections you cut out it if you have trimmed the sample properly.
g. To trim, you may use the automatic cutting mechanism or spin the cutting wheel manually. If using the automatic setting, I recommend to set the cutting window appropriately, use a sectioning thickness of about 200nm, and use a cutting speed of about 1-2 mm/sec.
h. You can move the knife up to the sample by using the stage advancement knob on the left side of the stage. It will help to determine how close the sample is to the knife by focusing on the knife edge and then moving the sample up and down using the cutting arm until it is also in focus.
i. Using the glass knife, trim away at your sample until you have made trapezoid with the larger of the parallel sides facing down. A pyramid-like shape with a flat face works best. Achieve this by loosening the specimen holder hex screw using the white-labeled key and rotating the sample holder (you have three choices for what to rotate, pick whatever works best for you). You can also rotate the knife to cut an angle using the gray knob on the right side of the cryo temperature stage, or the knife rotation knob on the rm temp stage. The exact procedure will depend on how you prepared your sample using the razor blade and in what orientation you glued your sample to the post.
j. Remove the glass knife and discard or save for another use. Glass must be disposed off in the proper container in the wet lab.

7. Sectioning with a diamond knife for room temperature work

a. Place a few grids (as many as you want to collect) on a piece of filter paper, preferably shiny-side up.
b. Place a piece of filter paper on a small plastic Petri dish (you will place your grids with sections on this). Label it with the name of your sample and the cutting thickness.
c. Load the diamond knife into the knife holder.
d. Fill the knife boat with water.
e. Select what region to use on the diamond knife, and use the black lateral stage translation knob to move the knife to the appropriate region. I like to start using a knife on the right edge and slowly move my way down to the left. A diamond knife should last at least two years, assuming heavy use.
f. Bring the sample as close to the knife as you can without allowing the knife to make contact. You can move the knife up to the sample by using the stage advancement knob on the left side of the stage. You can also use the manual arm advance button. It will help to determine how close the sample is to the knife by focusing on the knife edge and then moving the sample up and down using the cutting arm until it is also in focus. Practice using the glass knife if necessary!
g. Set the cutting window generously – allow for plenty of space above and below the sample.
h. Set the cutting speed to a value between 0.4 and 1 mm/sec.
i. Set the cutting thickness to 50-100nm.
j. Turn the cutting arm on (press “Cut”). It is best not to stare at the sectioning process while cutting takes place since your body heat/breath will affect the sample and knife temperatures, and cause fluctuations in sample thickness.
k. Once enough acceptable sections appear on the knife edge stop the cutting process by pressing the “Cut” button again. Make sure the cutting arm is below the knife.
l. You can rearrange the sections if necessary using a hair tool. I discourage this unless you have lots of experience and a steady hand since you can damage the diamond knife even with a hair tool.

8. Sectioning with a diamond knife for cryo work

a. Place a few grids (as many as you want to collect) on a piece of filter paper, preferably shiny-side up.
b. Place a piece of filter paper on a small plastic Petri dish (you will place your grids with sections on this). Label it with the name of your sample and the cutting thickness.
c. If you are using the section pick up method described in the next section, make sure you warm-up your sugar solution before you get going on sectioning.
d. Load the diamond knife into the cryo knife holder (using the blue and white hex key and the black knife holder handle) or directly into the rm temp stage.
e. Tighten the appropriate screw to make sure the knife is secure.
f. Make sure the sample and knife are at the temperature of interest, and then wait about 5 minutes after it has equilibrated.
g. Select what region to use on the diamond knife, and use the black lateral stage translation knob to move the knife to the appropriate region. I like to start using a knife on the right edge and slowly move my way down to the left. A diamond knife should last at least two years, assuming heavy use.
h. Bring the sample as close to the knife as you can without allowing the knife to make contact. You can move the knife up to the sample by using the stage advancement knob on the left side of the stage. You can also use the manual arm advance button. It will help to determine how close the sample is to the knife by focusing on the knife edge and then moving the sample up and down using the cutting arm until it is also in focus. Practice using the glass knife if necessary!
i. Set the cutting window generously – allow for plenty of space above and below the sample.
j. Set the cutting speed to a value between 0.4 and 1 mm/sec.
k. Set the cutting thickness to 50-100nm.
l. Turn the cutting arm on (press “Cut”). It is best not to stare at the sectioning process while cutting takes place since your body heat/breath will affect the sample and knife temperatures, and cause fluctuations in sample thickness.
m. Once enough acceptable sections appear on the water surface stop the cutting process by pressing the “Cut” button again. Make sure the cutting arm is below the knife.
n. Step back 10-15 times using the manual arm advance button so that the cutting arm has moved away from the sample back 10-15 x section thickness.
o. You can rearrange the sections if necessary using a hair tool.

9. Collecting sections for cryo work

a. There are a few ways to collect sections. The method below is the least likely to damage the diamond knife. However, if your sample is soluble in water or if the structure is very sensitive to water you cannot use this method.
b. Warm a saturated sugar solution up to room temperature.
c. Prepare four small plastic Petri dishes filled with DI water.
d. Once there are sections on the knife edge, use one of the smaller platinum loops to pick up a drop of the saturated sugar solution. Do not use pure water, it will freeze too quickly.
e. Before the sugar solution freezes on your loop, touch the sample using an outward (away from you) motion with the droplet (never allow the platinum loop to touch the diamond knife) to pick up the sample. Be very careful not to touch the knife edge, especially once the liquid freezes.
f. Touch (lightly) the droplet on the shiny side of a grid that is on the filter paper. You should now be able to pick up the droplet and the grip using your loop. If the solution runs into the filter paper then you pressed down to hard – cut more sections if necessary and collect new sections.
g. Pick up the grid using the droplet and place face down onto the DI water bath (such that the section is face down on the water).
h. After 5-15 min, transfer the grid, face down, to the next water bath using the larger loop. Make sure that for the next grid you keep the order the same (DI water bath stage 1, 2, 3, 4).
i. After the last water bath, pick up the grid from the water using the larger loop and place, face up, on a piece of filter paper. Allow the water droplet to touch the filter paper so that the grid will gently fall off the loop and onto the filter paper. Make sure the section is facing up, or else you might lose your section.
j. Repeat steps 8l through 9i for as many grids as you want.

10. Collecting sections for room temp work

a. Pick up a grid using tweezers and dip the grid halfway down the water. Pick up the sections using the shiny side of the grid.
b. Put the grid, section side up, on filter paper to dry, preferably overnight.

11. Shutting down

a. Once you are finished, press the red reset button on the microtome controller to retract the cutting arm back to its original position.
b. Using a hair tool, remove all stray sections that are left on the knife edge. Use a motion that is away from you. This is very important, because any left over sections may adhere to the knife edge making that part of the knife unusable.
c. Put the cutting arm in its lowest position to prevent the diamond knife from hitting the sample inadvertently while removing the knife.
d. Using the stage advance knob to retract your sample back away from the knife as much as possible. Never force the stage advance/retract knob.
e. Turn off the LN2 pump.
f. Remove the LN2 transfer line very, very carefully and slowly and place in the LN2 transfer line holder. This is the most likely time that you will break the transfer line since it is now brittle.
g. Remove the diamond knife and allow it to warm up to room temperature.
h. Remove your sample and save for later use (by putting it in a freezer) or dissolve the Tissue Tek medium in DI water to recover the posts. If you used super glue for rm temp work, you will have to discard the post.
i. Allow the cryo stage to warm up to room temperature. If someone is going to use the microtome the next day, make sure you hit the warm button and allow the microtome to be at 35 °C for about an hour. Leave overnight uncovered to allow any water to evaporate. Return the next day to cover the microtome to prevent dust accumulation.
j. Once the diamond knife has warmed up to room temp, rinse using DI water only. Never use any organic solvents, since it will dissolve the diamond knife mounting,
k. Leave the diamond knife to dry (by evaporation) overnight. Put a “do not disturb” sign with your name and the date. Make sure you come back the next day to put the knife away.
l. Clean your loop tools and the plastic Petri dishes using DI water. You can use the Petri dishes that were water baths next time for holding the filter paper with grids on them. Place tools back in the tool box; leave the Petri dishes to dry overnight.
m. Turn off the temp and microtome controller, and turn off the surge protector.

12. The day after shutdown

a. Put away the diamond knife and the Petri dishes.
b. Cover the microtome.