3. If the block has already been started, line up the knife with the
cryo-planed surface by rotating the knife holder until the thin
light sheet emerging below is of homogenous thickness.
4. Introduce the cryostat into the chamber and set to 25–30%.
2.4.3 Trimming Domes
from the 200 μm Carrier
1. Manually advance the block for a few microns and then set it on
automatic mode with a feed <100 nm and a cutting speed
between 80 and 100 mm/s until reaching a depth of approximately 10 μm. The circular cryo-planed area should have a very
smooth, reflective surface without any irregularities or holes. If
not, vitrification is not good, and the sample should be
discarded.
2. Cut 50–100 μm more on the left and right sides of the cryoplaned circle to generate a prominent thin vertical rectangle.
3. Rotate the specimen holder by 90
in order to make a square
block-face.
4. Finish block-face by repeating step 2 above.
2.4.4 Trimming the Type
a 100 μm Deep Carrier
This method entails cutting through metal and sample, two materials with different cutting properties. Trimming steps should favor
as much as possible cutting through only one material at a time.
When cutting through both cannot be avoided, the cutting speed
should be reduced.
1. Orient the specimen holder so that the carrier is presented
vertically to the knife.
2. Bring the knife to the carrier.
3. Trim through the metal at 70 nm feed and 60–80 mm/s speed.
It will take approximately 800 μm of trimming to reach the
sample.
4. When the sample is reached, reduce the trimming speed to
50 mm/s. At this point, you will be cutting through the
dextran and the sample will look like a clear circular structure.
5. Continue cutting until the sample enlarges horizontally to fill
the full 100 μm depth of the carrier. At this point, there should
be a vertical separation between the sample and the bottom of
the carrier.
6. Shift the knife in order to cut only through the vertical metal
and not the sample.
7. Trim 200 μm deep, gradually approaching the sample at
100 mm/s (see Note 16).
8. When both the sample and metal are being trimmed, lower the
speed to 50 mm/s.
9. When the vertically oriented sample is well cleared on both
sides, rotate 90
.
Methods in Cryo-Electron Tomography
97
cryo-planed surface by rotating the knife holder until the thin
light sheet emerging below is of homogenous thickness.
4. Introduce the cryostat into the chamber and set to 25–30%.
2.4.3 Trimming Domes
from the 200 μm Carrier
1. Manually advance the block for a few microns and then set it on
automatic mode with a feed <100 nm and a cutting speed
between 80 and 100 mm/s until reaching a depth of approximately 10 μm. The circular cryo-planed area should have a very
smooth, reflective surface without any irregularities or holes. If
not, vitrification is not good, and the sample should be
discarded.
2. Cut 50–100 μm more on the left and right sides of the cryoplaned circle to generate a prominent thin vertical rectangle.
3. Rotate the specimen holder by 90
in order to make a square
block-face.
4. Finish block-face by repeating step 2 above.
2.4.4 Trimming the Type
a 100 μm Deep Carrier
This method entails cutting through metal and sample, two materials with different cutting properties. Trimming steps should favor
as much as possible cutting through only one material at a time.
When cutting through both cannot be avoided, the cutting speed
should be reduced.
1. Orient the specimen holder so that the carrier is presented
vertically to the knife.
2. Bring the knife to the carrier.
3. Trim through the metal at 70 nm feed and 60–80 mm/s speed.
It will take approximately 800 μm of trimming to reach the
sample.
4. When the sample is reached, reduce the trimming speed to
50 mm/s. At this point, you will be cutting through the
dextran and the sample will look like a clear circular structure.
5. Continue cutting until the sample enlarges horizontally to fill
the full 100 μm depth of the carrier. At this point, there should
be a vertical separation between the sample and the bottom of
the carrier.
6. Shift the knife in order to cut only through the vertical metal
and not the sample.
7. Trim 200 μm deep, gradually approaching the sample at
100 mm/s (see Note 16).
8. When both the sample and metal are being trimmed, lower the
speed to 50 mm/s.
9. When the vertically oriented sample is well cleared on both
sides, rotate 90
.
Methods in Cryo-Electron Tomography
97