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Targeting workflow overview

Kimberly Meechan edited this page Jun 2, 2022 · 7 revisions

Here we give an overview of the entire targeting workflow using the motorised ultramicrotome system, pointing to the more detailed Crosshair tutorials at the relevant steps. Note that there are some differences between using the motorised system from the Crick (built on an RMC ultramicrotome) and the system from EMBL (built on a Leica UC7 ultramicrotome). These differences are highlighted throughout the workflow.

There is also a video summary of the whole process (demonstrated on the Leica system) available here: https://www.youtube.com/watch?v=OM712ECthGg

Summary diagram

Diagram of steps colour coded by whether they are using the ultramicrotome, X-ray system, or Crosshair software. All steps are covered in detail in the next section.

Targeting Steps

Trim block

Trim the block at the ultramicrotome with a diamond knife. A flat surface must be made that has 4 corners and straight lines in between. Make sure the sides of the block face are trimmed deep enough, so the knife will always touch part of this face first, and not other parts of the block (even if the solution requires a more extreme set of angles). We usually trim about 40-100 microns, depending on the sample.

Ideally, the block surface should be within one ultramicrotome feed length of the target for the greatest accuracy (i.e. 200 microns or less for both the Leica and RMC systems). Every time the cutting feed is reset, the approach of the knife to the block must be repeated, and some accuracy will be lost.

X-ray

X-ray the block at the highest resolution possible (this will determine how accurate measures made from the X-ray images can be). Ensure the sample and the entirety of the block surface are visible.

Convert the X-ray image to 8-bit (if it isn't already) e.g. in Fiji with Image - Type - 8-bit. The image can also be cropped at this stage if there is a lot of empty area outside the resin block. Open the X-ray in Crosshair by following the tutorials here and here. Compare the 3D view/image stack and the sample. If it appears as a mirror image of the sample, then flip the image stack. This can be done e.g. in Fiji with Image - Transform - Flip Vertically, or Image - Transform - Flip Z.

Define block + target plane

Open the X-ray image in Crosshair and set the block and target planes. The target plane can be set using the 'TRACK' button and navigating in the 2D viewer. The block face can be set by placing a series of points on the block surface and fitting a plane to them. Follow the tutorial here for this step.

Define block vertices + orientation

Place one point (vertex) on each of the 4 corners of the block face. Then, decide on the orientation the block will have in the ultramicrotome i.e. which side of the block will face up? Mark the points accordingly as 'Top Left', 'Top Right', 'Bottom Left' and 'Bottom Right' in Crosshair. Follow the tutorial here for this step.

Once this is complete, it is good practice to 'Save Settings' in Crosshair to make a .json file as a record of the planes and points.

Set sample tilt 0

Set the sample tilt to zero by eye (just by looking at the scale on the arc piece and trying to get it as close to 0 as possible). For the Leica system, press 'Set Zero' in the 'sample tilt' page of the ultramicrotome user interface. For the RMC system, make a note of the current sample tilt angle.

Set knife tilt 0

For the knife zero, there is a slightly more complicated procedure than for the sample tilt. We assumed that since the knife is removed and replaced every time, there may be more variation in its position and therefore an extra step may be required to accurately find its proper zero.

Put a blank block into the ultramicrotome sample holder (i.e. just resin with no sample). Set the knife tilt to zero by eye (just by looking at the scale on the knife holder and trying to get it as close to zero as possible). Cut into the blank block at this orientation to make a flat face. Back the knife away, and rotate the sample holder exactly 90 degrees (using the motorised ultramicrotome controls). Align the knife to this face again (only adjusting the knife tilt!). For the Leica system, press 'Set Zero' in the 'knife tilt' page of the ultramicrotome user interface. For the RMC system, make a note of the current knife tilt angle.

Align knife and block face

Replace the blank block with the resin-embedded sample. Make sure it is placed the same way up as indicated in Crosshair.

Align the knife and block face, ensuring the bottom edge of the block is also aligned along the cutting edge of the knife. Any axis can be freely changed for this step, just as in normal ultramicrotome usage. For the Leica system, the motors can be disabled for this step, and the alignment done manually as with normal ultramicrotome usage. For the RMC system, the motors cannot be disabled, and the motors must be used for this alignment.

Set sample rotation 0

For the Leica system, set the sample rotation to zero by pressing 'Set Zero' in the 'sample rotation' page of the ultramicrotome user interface. For the RMC system, make a note of the current sample rotation angle.

Enter Θ_IK and Θ_IT

Enter the current knife angle and sample tilt angle into Crosshair. For the RMC system, make sure you adjust for the recorded 'zero' position e.g. if the current angle is 10 degrees, and the zero was 2 degrees - then you would enter 8 degrees into Crosshair. Follow the tutorial here for this step.

Choose solution

Use the Crosshair 'Microtome Mode' to cycle through all the possible solutions, and choose one to use. The target plane will turn red in the 3D view when there is a valid solution. Follow the tutorial here for this step.

Once this is complete, it is good practice to 'Save Solution' in Crosshair to make a .json file as a record of the solution.

Set solution angles

Back the knife away from the block, to ensure there is no risk of hitting it. Using the motors, move each axis to the angles stated in the solution from Crosshair. For the RMC system, make sure you adjust for the recorded 'zero' position of each axis e.g. if the solution angle is 10 degrees, and the zero was 2 degrees - then you would move the ultramicrotome to 12 degrees for that axis.

Approach block surface + cut solution distance

Manually approach the block and get as close as possible to its surface. Any error in this distance (i.e. starting before touching the block, or once it has already been cut into) will contribute to the final error. A good solution is to get as close as possible with the ultramicrotome's NS movement, then set the cutting thickness to some small value (e.g. 70-100nm). Then cutting can be started slowly, carefully watching the knife edge through the ultramicrotome's binocular at high magnification, and stopped as soon as any debris is visible on the knife / any pieces being cut from the block. This ensures the knife is as close as possible to the true surface.

Once complete, cut the distance specified in the Crosshair solution.

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