Step 2: Preparing the sample

Insert the sample

The sample is loaded into the microscope in a water bath filled with low osmolarity phosphate buffer. 50 mM room temperature PB works well. PBS and/or higher concentration buffer just create a lot of annoying salt deposits.

Brains are best loaded bulb down and with the ventral side facing the blade. The blade is affixed to the blade holder (we use a new blade every 200 to 300 sections).

Open the prepare GUI

In case the prepare GUI is not already open, hit the "Prepare Sample" button on the main view to open the Prepare GUI, which is used for sample trimming and moving the stages manually.

The sample can be moved by editing the text entry boxes, using the large and small motion step arrows, or keyboard shortcuts. More on this below. Trimming of the sample is achieved using the slicing buttons at the bottom of the window.

Set the blade position

The goal here is to move the sample so that its top right edge (see below) is next to the blade. This informs the software of the correct location to start cutting. The gap between the blade tip and the agar block can be small (one mm or so is fine) if the edge of the agar on the blade side is straight and vertical. The smaller the gap, the less time is wasted cutting nothing.

The blade positioned next to the agar block, ready to take the first slice.

It is easiest to coarsely move the sample using X/Y/Z text entry boxes for stage position, before transitioning keyboard shortcuts for the fine positioning.

Keyboard shortcuts for moving the stages. Hold SHIFT for large motion steps.

• WASD for X/Y (W and S for Y; A and D for X).

• To move the stage up use either Q and E or use R and F. Q and R moves up. E and F moves down.

• Hold shift for large motion steps. Step sizes are those indicated in the GUI.

To use the keyboard shortcuts, the Prepare GUI must have mouse focus but you should not have clicked on a text edit box, or you will end up typing in it. Hold shift for large motions. You should position the sample such that it's about one mm away from the blade and the blade tip is near the top of the sample in Z. Then hit "Set blade position" to store this position:

BakingTray now knows where the blade is with respect to the sample. You can edit this position manually using the edit box (highlighted in yellow, above).

Pressing "Set blade position" button also updates the "Cut Size" (arrowed, above), which is the number of mm the blade moves through the sample whilst cutting. This number should be should be sufficient for the blade to get through sample and proceed a little further over the other side so the cut section clears the sample block. You can edit the Cut Size value at any time.

Take the first slice

Hit the "Slice once" button to take one section. The system will start cutting and the button text becomes red and says "Slicing". You may stop the procedure at any time by pressing "STOP SLICING". The slice is taken at the starting point you defined above and at the speed listed in the Prepare GUI (above). The block will be moved upwards by the thickness value in the Prepare GUI_ (not the main GUI) and cut at the speed listed in the

The thickness of the first cut depends on how far below the sample surface you placed the blade in the previous step. i.e. if the blade is 2 mm below the agar surface, the first slice will be 2 mm thick. If you think the first slice might be too thick (e.g. over about half a mm) then set the cut speed to something slow, such as 0.2 mm/s. If you cut a thick section too fast the agar block can come unstuck.

Confirm that the blade proceeds 2 or 3 mm beyond the edge of the agar block and edit the Cut Size if it does not.

User-initiated Z motions are blocked by the GUI after the first cut has been made. This is to avoid accidentally altering slice thickness. You can override this by unchecking the Z Lock checkbox in the prepare GUI.

Approach the imaging depth

You may wish to trim away some agar or maybe part of you sample before you reach a location where you want to start imaging. e.g. You may choose to slice through most of the cerebellum before starting. Here we do this by asking the Prepare GUI to take 3 slices of 350 microns (0.35 mm) each by pressing the highlighted button. You can change how many slices it will take using the edit box and you can stop at any time with the "STOP SLICING" button.

We are cutting these slices fairly slowly at 0.3 mm/s. Speeds of up to about 0.50 mm/s are safe to use for acquisition.

Even if you do not want to slice off a lot of tissue, take at least once slice that is about 100 to 200 microns thick after your initial slice. This calibrates the microscope and ensures that the slice thickness value in the Prepare GUI is close to what the system is actually doing (this will not be the case after the first slice for the reasons explained above).

Cutting thin slices

You can not jump straight from thick slices all the way down to the thin slices you will use for imaging. If you do this, the vibratome may not cut at a consistent thickness: it could alternate between cutting thick and thin slices. Once you are ready to image press the "Auto-Trim" button. This will take a series of increasingly thin sections culminating with three at the target (imaging) section thickness and speed. For more information on choosing a cutting thickness see "Choosing imaging settings".

If the vibratome is working correctly, the slices should all come away looking the same. If some (e.g. every other slice) is mangled, then something isn't right with the cutting. You may need to cut more slowly or cut thicker.

A thickness of as little as 40 microns is usually reliable. If you are seeking to cut slices thinner than 40 microns you will probably need to slice at speeds of under 0.3 mm/s to achieve consistent section thickness.