It is very important that the embedding media be homogeneous. This may be a problem with LRWhite which keeps soft areas after curing. A good adhesion between sample and media as well as similar hardness are diesired.
One technique for embedding of small samplesis the following (i.e., fibers):
2) TRIMMING THE BLOCK The block needs to be trimmed before microtoming to present a minimum area to the knife. This maximizes the cutting force per unit length of material. The harder the material, the smaller the facet needs to be. In addition to not producing thin slices, a big chunk of hard material will destroy a diamond knife very fast.
The facet may be trimmed wiht a razor blade, viewing the operation with the microscope. If you are not good at that, you may use a glass knife to cut the facet by rotating the specimen and changing the angle of the blade.
A suggested standard shape is a trapezoid:
This shape maximizes adherence between subsequent sections, to give "ribbons"
that are easy to pick up with a TEM grid.
The facet size depends on the material/resin ratio.
REMEMBER THAT MORE MATERIAL MEANS MORE GREATER RESISTANCE
TO THE CUTTING ACTION OF THE KNIFE.
3) SPECIMEN / KNIFE ALIGNMENT
Advance the knife base towards the sample, with the sample above the level of the knife so that violent movements do not damage the knife. Then clamp the holder to the microtome platform.
MAKE SURE THAT EVERYTHING IS CLAMPED PROPERLY: knife on holder, knife holder on platform, and sample in sample holder.
Approach the knife to the sample and look at the reflection of the knife on the sample to evaluate the distance between both, and to align the specimen with the knife.
WHEN YOU ADJUST THE SPECIMEN TO KNIFE DISTANCE AND ALIGNMENT, THE SAMPLE MUST BE IN THE FORWARD POSITION.
It is a common mistake; if the position is set while the sample is retracted,
on the forward movement, it will damage or break the knife. There is an annoying
beep to remind you that the specimen holder is in the retracted postion.
4) MICROTOMING Set the clearance angle of the knife to between 4 and 7 degrees, fill the boat with DI water, and set the water level to wet the edge and provide a reflection off of the surface to identify sections.
Adjust the path of the arm so that its movement slows for at least 1 s before
the specimen touches the knife and so that the specimen moves slowly through
the entire cut. This is done by advancing the hand wheel until the specimen
is in the forward position, pressing the lever up, rotating the wheel until
it hits a stop, and moving the wheel further to set the cutting window. 
BEFORE MICROTOMING, MAKE SURE MANUALLY THAT EVERYTHING
IS ADJUSTED PROPERLY, OPERATING VERY SLOWLY.
Set to coarse cut until sections come off correctly. NEVER CUT SECTIONS THICKER THAN 50 MICRONS WITH A DIAMOND KNIFE.
Switch to slow ultra thin sections of 50-60 nm. Typical speeds are indicated by the blue (diamond) or red (glass) dots on the speed setting.
While microtoming you may remove excess sections using an 'eyelash' brush. Never use a loop or gridwhile microtoming because it might get caught between sample and knife on a cycle. Let the microtome stabilize, any vibration or irregularity in the sample face decreases the chances of getting good sections.
Pick up samples directly with a TEM grid or pock up a drop of water containing the sample with a loop, then deposit it on a grid.
If you stop microtoming to pick up slices, do not assume that the alignment is still good. The sample / knife distance will be off if you stop cutting for even a few minutes. You will have to reset the distance.
DO NOT ALLOW A DIAMOND KNIFE EDGE TO GET DRY UNLESS IT IS CLEAN.
When you pick up sections, make sure the edge is wetted.
NEVER TOUCH A DIAMOND CUTTING EDGE EXCEPT WITH AN EYELASH. CLEAN FIRST WITH WATER, THEN ETHANOL.
Place a glass bar in the knife maker with the scored edge down. Push the bar all the way to the left under the scriber mechanism until it bumps against the metal stop. Secure the bar in place by loosening the front knob on the slide and the pushing the slide against the glass bar. Lower the scriber menchainm with the handle until the fit is firm. With the scriber cutting choice knob set for lateral cut, pull the white knob out towards you to scribe the edge of the bar. Now lift up on the front handle until the sample breaks. Lift up the scriber machanism, and return the white knob back to its original position.
Grab the small square and rotate it by 45 degrees counterclockwise. Place the square in the center and hold it by sliding the slide mechanism. Turn the small black handle to grab onto the end of the square. Now lower the scriber mechanism again. Change the choice knob to make a scribe only in the middle of the glass square. Pull hte white knob forward to scribe the middle of the square. Now lift up slowly on the front lever to propagate the crack across the sample.
After the sample breaks, grab the two new knoves with the tool and examine their quality. If tey are good, attach a boat to the knife with nail polish. Or use black electrical tape and paraffin to seal it. Cryomicrotomy
This technique is useful for samples that are too soft under ambient conditions. It uses liquid nitrogen to cool the sample and knife. There is a different sample clamp and holder, knife holder and chamber for the liquid Nitrogen (LN2).
The first thing to do is to turn on the heaters. This takes about half hour, and if you forget, it will delay you that much further. Fill the LN2 tank with as much as you can, refilling it after a couple hours is not fun. Remove the specimen and knife holder. Mount the knife holder plate on the stage. Place the chamber on the stage (turn the binoculars all the way to the left to get to the mounting screws) while getting the specimen C-arm aligned with the drive arm. Firmly attach all pieces (without stripping screws) and make sure all clearances are sufficient. Connect the LN2 tank hose to the back of the chamber. Set the temperatures to whatever you think you need. Mount the knife in the knife holder which should be stored in a plastic box. put the specimen block into the spec. holder and mount it in the C-arm. Align everything with the same care that should be used in ambient microtoming. Wait for the temperature to stabilize.
Remember:
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