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Step 2: Preparing the sample

Insert the sample

The sample is loaded into the microscope in a water bath. 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

Hit the "Prepare Sample" button on the main view.

Set the blade position

The goal here is to move the sample so that its top right corner (as you look at it) is next to the blade. This informs the software of the correct location to start cutting. The sample can be moved using the X/Y/Z edit boxes, the GUI arrows, or the following keyboard shortcuts:
  • 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.
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 a mm to the left of the blade and the blade tip is near the top of the sample in Z. Then hit "Set blade position" to automatically fill in the location of the blade:
BakingTray now knows where the blade is with respect to the sample. You can edit this position manually using the edit boxes (highlighted in yellow).

Take the first slice

Hit the "Slice" 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 using the parameters highlighted in yellow. Remember, however, that thickness of this first slice depends on how far below the sample surface you placed the blade. 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 Prepare GUI (The reasons for this will become clear in the next steps). It will cut for the distance listed in the main GUI (Cut Size). Ensure that the blade proceeds 2 or 3 mm beyond the edge of the agar block. This helps the slices come away cleanly and float away from the objective.

Approach the imaging depth

Likely you will need 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 ask 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.
Do not jump straight from such thick slices all the way down to the thin slices you will use for imaging. If you do this the vibratome may not cut slices at a consistent thickness: it could alternate between cutting thick and thin slices. Once you are near your target imaging depth, proceed gradually cutting increasingly thin slices by pressing the "Auto-Trim" button. This will take a series of sections culminating with three at the target (imaging) section thickness and speed.
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 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.