Cryosectioning notes from Chyongere Hirsch (Wadsworth Center Albany,NY)24 Mar 05

Grids

200 mesh Quantifoil for tomography Glow discharge Carbon coat Gold markers Microtome Set-up and Sample loading If it takes more than 30 min to cool down

tank is dirty

pump is frozen
Use a 5mL beaker of LN2 in well to pour samples into and catch samples if dropped when loading. After chamber is cooled load sample Position arm at bottom when loading sample Insert knives Cool knives down with LN2 Clean knives of frost with LN2 before gathering sections. Ionizer If samples collect at edge of knife TURN UP If samples curl up and fly away TURN DOWN Clean knives with 50% ETOH
Trim/Section
500-1000nm sections at 100mm/s for Al. Brass hats are harder trim carefully. Trim metal into block one day and section the next. Trim 700 microns minimum into hat. Trim 1. sample side >] 2. metal > ⌐< 3. long metal side last >[
Section long way [
Block face size 250 microns or less for 50 nm sections- smaller block face lessens effects of compression.
Cover sectioning well whenever possible
Can section 6 ribbons of 5-6 sections before collecting samples. Space ribbons at least on blockface apart form each other. Expect to lose 50% of ribbons when collecting samples.
Place samples in the center of the grid for tomography

Section Manipulation
Turn off ionizer when collecting samples.
Manipulate sample with dog hair. It is more durable than human hair.
Dog hair cools down quickly. Can cool with LN2 well or place in chamber for 1 minute. Do not need to cool down unless collecting samples.
Place a 3x9mm piece of indium foil on the brass sample platform. Place a grid on the foil. Retract knife to protect it by moving knife to platform. Manipulate indium foil and grid to knife edge. Tweezers do not need to be cold for this.
Touch dog hair to knife edge to release ribbons.
Drag ribbons to grid and place in center of grid. Critical for tomography samples.
Move grid to edge of indium for easy pick up.
If you lose all of the samples do not reuse the grid unless it has been glow discharged.
Set best dog hair aside for sample manipulation only.
Make sure you know methodically which side of the grid has the sample on it in the box.
Indium is used for folding clam shell grids and for frost protection.
Press grids again before scoping if they have been stored in liquid nitrogen.
Store sections only 1-2 days or sample may float off grid.
Put grid box into 5mL beaker with LN2 to transfer to 50mL storage tube.
Best to look at sections right away.
Theory
Hats better frozen at center next to metal 25-40% humidity Warmer samples theoretically section better Less crevices Can get dangerously close to devitrification temp 20% dextran preferred cryoprotectant
Tools
Section flattening tool- for folding clam shell grids within indium foil Section press- flattening samples unto grids Can store samples while waiting for microscope if viewed the same day
Humidity gauge
Tweezers Long hook EMS 78351-66 Straight EMS 78354-1192 LN2 cup for tweezers
Cryoglue Like honey -140 Frozen -160 Cryoglue can be scary because temp needed to affix sample is very close to the devitrification temp

-- KdDerr - 08 Jun 2005