Microtoming with Leica UC-6

Why? Basic method to prepare polymeric and biology TEM samples Has also been used in other sample preparation. –Can reduce some draw backs of ion milling. –The sample thickness should be as small as possible.

My clay in PMMA

Equipment

Basic procedure Sample embedding Sample trimming Knife preparation Sectioning Pickup Clean up

Embedding Sample Spurr’s resin is what we are using. Low viscosity epoxy resin. –Work in the hood! –Ware gloves! Consider other media if your sample reacts with epoxy components –PS, PMMA, melamine, or different epoxy Really bulky block doesn’t need embedding Diglycidyl Ether of Poly(propylene glycol) (DER 736) ERL 4221(4206) Epoxide Resin, (Carcinogen) Nonenyl Succinic Anhydride Dimethylaminoethanol (Final epoxy property very sensitive to this component) Mix well following the recipe for different properties Put in 70 degree C over night. Check EMS or Ted Pella for other epoxy resins if you need

Trimming Trimming can be done by using razor blade or glass knife –I prefer blade Make a small trapezoid tip at the top Height of the tip < 2mm Size of the trapezoid should be smaller than 0.5mm*0.25mm

Trimming cont.

Trimming setup

Prepare glass knife Put some nail polisher on the plastic boat Attach the boat to the knife Glass stripe scratch breakscratch break Clean up glasses after work!

Install knife Glass knife use 4 degree knife angle. Different diamond knives have different angle settings, read your diamond knife instruction. Fasten all screws, secure the knife.

Install knife

Fill the boat with water Diamond knife Glass knife Make a concave, the color of the reflection should be silver.

Bring the knife to the sample The black wheel on the control board can move the knife The buttons labeled STEP can move the knife stepwise Step length is set and displayed on screen

When it is really close…

Set the cutting window

Press START set the beginning of the cutting window Press END set the end of the cutting window Feeding of each cycle and sample to knife speed can be set by the black knob It uses German punctuation method. 0,60 means 0.60.

Begin cutting Press the green “RUN/STOP” button Set proper feeding, speed and return speed parameter Wait After you get section on the surface of water, read the thickness from the color of the sections and color-thickness card. Step down the thickness. 100nm is good for the JEM TEM. The color is between gold and silver

Got water? If there is water on the tip of sample, stop sectioning and dry the water with a small piece of Kimwipe. Stop for a while wait for the water at the back of the knife dry. Pull the knife back a little bit. –Because whenever you stop for a while the section expand and may damage the knife.

Picking the sections Keep the sections away from the edge during picking up.

Picking the sections cont.

Cleaning up Washing the knife with di-water and ethanol and di-water before the knife dry. Blow dry the knife if possible. Or wash the knife with the knife cleaner. –A little bit of Dial works better. Don’t soak the water in Ethanol or Acetone. Don’t use ultrasonic. Throw away the glasses.

Dry cutting Dry cutting is used if the sample dissolves in water. The sections stays on the knife is troublesome –Can be picked up with single hair brush, but a lot of practicing is required

Cryomicrotoming Cryomicrotoming can control temperature of the sample and bring the temperature below glass transition temperature of the polymer. Keep water from entering the chamber –Ice crystals break the knife. Special method is used to pick up non water soluble samples. –Dip a grid into 1mol sucrose solution. –Before the sucrose solution totally frozen, touch the sections with the solution. –Then float the grid on surface of water.

Other important things If you are angry, excited, depressed, worried, frustrated, don’t do microtoming –I don’t think you will do microtoming when you are very happy If you are hungry, drank too much coffee or tea, ate too much sugar, don’t do microtoming.

So your first microtoming will be… A group of geese on the ground is called a gaggle A group of fish is called a school A group of lions is called a pride A group of kangaroos is call a mob A group of ravens is called a murder A group of police and everyone’s first microtoming session is called -- A MESS. So begin with a glass knife and keep practicing.