Step 0: Loading the sample

If necessary, start ScanImage and BakingTray. The instructions over the following pages are summarized as a checklist.

Start a new sample

Select Sample -> New from the menu. This will reset the recipe and turn on the resonant scanner (if present).

Turn off the PMTs

The PMTs are DELICATE and will be DAMAGED PERMANENTLY if exposed to room light when they are powered on. Turn off the PMT power box before proceeding with sample set up. Make sure you always close the microscope enclosure and turn off its lights before you turn on the PMTs.

Turn on the laser

Hit the "Laser" button in the main view then press "Turn On" on the laser window. After hitting this button the text switches to "Turn Off", as shown above. The power will increase slowly and eventually the modelock indicator will turn from red to green. You can open the shutter now too: there is a second shutter on the path so there is no risk. Select the correct wavelength for this acquisition.

Remove the previous sample (if present)

If it is not already visible, open the "Prepare" GUI by clicking on "Prepare Sample", as shown below.

Enter "0" in the "Z" text entry box of the Prepare GUI to lower the water bath using the Z stage. Hose down the objective with distilled water as shown below, then clean the tip with a piece of folded lens paper. Be gentle: don't push against the tip of the objective with the lens paper. If you do not know how to do this, find an experienced person to show you.

Unscrew the blade holder with the large black thumbscrew and remove it from the vibratome. Take care not to cut yourself. Unscrew the blade retaining screw (shown below). Do not loosen the side screws! Remove the blade and dispose in the sharps bin.

Confirm whether the system requires the objective to be raised for the bath to slide out. Then slide out the bath. Dispose of brain slices as appropriate.

Clamp the new sample into the water bath

Unscrew the clamp holding the slide in and dispose of the slide in the sharps bin. Insert the new slide with the sample into the clamp. Note there is a stop on the slide clamp to keep the slide from protruding over the edge of the platform. The agar block should be placed adjacent to this stop. In the following image, the stop is a round headed screw.

Push the metal clamp over the slide and tighten it down. Do this carefully: first turn one side until it just starts to tighten and repeat on the other side. Then return to the first screw and tighten until finger tight. Repeat with the other. The slide should now be tight.

The microscope is set up such that it is not possible for any hardware elements to collide, however this does assume that the clamp is not angled upwards once secured.

Place the water bath onto the stage

Gently and carefully place the water bath onto the stage. Push it all the way back into the holder and tighten the retaining screw(s) until just finger tight.

Put in a new blade

Insert a new blade into the holder and tighten until a little beyond finger tight. Thread the blade holder thumbscrew through the holder and screw the holder to the vibratome. Whilst you tighten, push down on the blade holder with two fingers as shown below.

Pushing down on the blade holder is a critical step. This ensures the holder is secured at a reproducible angle with respect to the Y stage. Not doing this can lead to sectioning artifacts.

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