Targeting workflow overview
Here we give an overview of the entire targeting workflow, 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), the motorised system from EMBL (built on a Leica UC7 ultramicrotome) or a standard (non-motorised) ultramicrotome. These differences are highlighted throughout the workflow.
There is also a video summary of the whole process (demonstrated on the motorised Leica system) available to watch on YouTube.
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.
Before you start the targeting process, check that your arc piece (the curved part of the sample holder) is as vertical as possible. In some systems (e.g. the motorised Leica system) this will be fixed in position via a pin in the back of the sample holder. If not, you will have to adjust it manually by eye. (Note that this being far from vertical will affect the achievable accuracy!)
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 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 'Opening an image' tutorial and 'Interface basics' tutorial. 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.
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 'Setting up planes' tutorial for this step.
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 'Assigning vertices' tutorial 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 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). Then:
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For the motorised Leica system: press
Set Zeroin thesample tiltpage of the ultramicrotome user interface. -
For the motorised RMC system: make a note of the current sample tilt angle (read from the motorised system's display)
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For a standard (non-motorised) ultramicrotome: no action needed!
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. This is described in the Setting the knife zero with a blank block section below. If you don't require the highest possible precision, you can skip this step and follow the instructions in the Setting the knife zero without a blank block section below.
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 - if you have them). Align the knife to this face again (only adjusting the knife tilt!).
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For the motorised Leica system: press
Set Zeroin theknife tiltpage of the ultramicrotome user interface. -
For the motorised RMC system: make a note of the current knife tilt angle (read from the motorised system's display)
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For a standard (non-motorised) ultramicrotome: make a note of the current knife tilt angle (read from the knife holder scale). Make sure it has the appropriate sign (positive or negative) depending on the direction of rotation.
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 0 as possible). Then:
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For the motorised Leica system: press
Set Zeroin theknife tiltpage of the ultramicrotome user interface. -
For the motorised RMC system: make a note of the current knife tilt angle (read from the motorised system's display)
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For a standard (non-motorised) ultramicrotome: no action needed!
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.
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For the motorised Leica system: the motors can be disabled for this step, and the alignment done manually as with normal ultramicrotome usage.
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For the motorised RMC system: the motors cannot be disabled, and the motors must be used for this alignment.
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For a standard (non-motorised) ultramicrotome: align the knife and block face as you normally would.
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For the motorised Leica system: set the sample rotation to zero by pressing
Set Zeroin thesample rotationpage of the ultramicrotome user interface. -
For the motorised RMC system: make a note of the current sample rotation angle (read from the motorised system's display)
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For a standard (non-motorised) ultramicrotome: no action needed!
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For the motorised Leica system: Enter the current knife angle and sample tilt angle into Crosshair (read both angles from the motorised system's display)
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For the motorised RMC system: Read the current knife and sample tilt angle from the motorised system's display. Adjust each with respect to their recorded 'zero' position (from steps Set knife tilt 0 and Set sample tilt 0 above) e.g. if the current angle is 10 degrees, and the zero was 2 degrees - then you would enter 8 degrees into Crosshair.
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For a standard (non-motorised) ultramicrotome: Enter the current knife and sample tilt angle into Crosshair (read from the knife holder and arc piece scales respectively). Make sure you use the appropriate sign (positive or negative) depending on the direction of rotation.
Note - if you used a blank block to set the knife zero, then you will have to adjust the current knife angle with respect to the 'zero' value you recorded e.g. if the current angle is 10 degrees, and the zero was 2 degrees - then you would enter 8 degrees into Crosshair. Again, be careful with the direction of rotation!
For all systems, follow the 'Microtome mode' tutorial for this step.
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 'Microtome mode' tutorial 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.
Back the knife away from the block, to ensure there is no risk of hitting it.
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For the motorised Leica system: Using the motors, move each axis to the angles stated in the solution from Crosshair.
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For the motorised RMC system: Adjust each solution angle from Crosshair (knife tilt, sample tilt and sample rotation) with respect to their recorded 'zero' position 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.
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For a standard (non-motorised) ultramicrotome: Manually set the knife tilt and sample tilt to the angles stated in the solution from Crosshair. Make sure you rotate in the right direction depending on whether the values are positive or negative. Note that the final rotation angle (sample rotation) works differently! Rather than setting it directly to that angle (as we did for the knife and sample tilt), we instead move relative to its current position. For example, if the solution angle is 20 degrees, then rotate the sample holder 20 degrees further from its current position in the right direction.
Note - if you used a blank block to set the knife zero, then you will have to adjust the knife angle relative to your recorded zero position e.g. if the solution angle is 10 degrees, and the zero was 2 degrees - then you would move the ultramicrotome's knife to 12 degrees. Again, be careful with the direction of rotation!
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.
Crosshair uses the standard below to define a positive vs a negative rotation. If you're unsure, you can always manually move the Knife, Tilt and Rotation sliders in the Microtome Controls panel and compare to movements of the 3D model (details in the 'Microtome mode' tutorial)
