Mounting the preparation for viewing

For a quick look you might be able to fit the whole prep-dish under the microscope. However, for taking pictures and detailed viewing you will have to transfer the ganglion to a microscope slide and cover it with a coverslip.

'Live whole mount'

The most simple method is to place a drop of saline on a slide, drop the ganglion in it (use a cut-off pipette), and put a coverslip on top of it (first touch slide with one edge and than slowly lower coverslip to avoid air bubbles, holding the other edge with forceps, see Fig. 10). This methods works best e.g. if you are interested in which nerves carry the axon of the cell you filled. It will give you the brightest picture of your cell, but be aware that it will also distort and damage the ganglion.

to coverslip

Figure 10: Touch the slide with one edge of the cover slip and than slowly lower the opposite edge, holding it with forceps. A depression slide or spacer avoids crushing the ganglion.

To avoid damage to the ganglion you can use depression slides (generally 0.8 mm deep) or spacers under the coverslip (Fig. 10). Traditionally pieces of broken coverslip or small amounts of modeling clay are used. Make sure the modelling clay is non-toxic. A self-adhesive 'CoverWell' gasket (0.5 or 1 mm thick) does a great job, too. Aplysia ganglia are fairly thick and might be happier with a spacer. However, with a regular fluorescence microscope, it will be impossible to see fine branches in an uncompressed ganglion.

'Fixed whole mount'

If you intend to keep the preparation for longer than just a day, you need to fix it (follow the protocol on the left). You can then clear it, that is, make the unfilled parts transparent. Clearing works by embedding the tissue in a medium with a similar refractive index.
SVEN VILIM'S ISH FIX BUFFER
5 M NaCl
1 M MOPS buffer
0.05 M NaOH
4% Paraformaldehyde
fix for 3 hours at RT or over night at +4°C
wash 3 times 15 minute with 70% EtOH
store at -20°C

Methylsalicylate smells pretty strongly but works very well for this purpose. Fix the tissue, then dehydrate it completely with an ethanol series to 100%. Some use Acetone as a last step. Put a drop of Methylsalicylate on a slide and drop the tissue in it (using forceps). The tissue should become transparent within the next couple of minutes - depending on its size. Careful: once the preparation is transparent it is very easy to lose. You will not be the first one to do so.

Glycerol is a less smelly but also a less effective alternative. The tissue doesn't have to be dried as thoroughly as Methylsalicylate requires. Glycerol is typically mixed with some ethanol (e.g. 70/30 %), depending on how transparent you want your tissue to be.

Viewing the filled cell

It's time to view the filled cell. You will now need either a fluorescent microscope, like our Labophot (Fig. 11), or a confocal microscope. I will give brief intructions for the LaboPhot here. For practical instructions on how to use a confocal microscope read Dirk Bucher's guide.

You will also find a lot of information on various aspects of microscopy on the manufacturers website. Check out Olympus, Nikon, Zeiss ('How to', 'Tutorials & Calculations' ), and Leica ('Company'>'Downloads'>'Scientific and Technical Information').

Nikon LaboPhot

Figure 11: Nikon Labophot with fluorescence attachment and digital camera.

First, you need to start the lamp for epi-fluorescence illumination. It is a 'high pressure mercury short arc' lamp which has a power supply living in a separate box close to the microscope (Fig. 12 left). Switch it on, press the start button and the 'lamp stable' light should come on within 20 seconds or so. It will take several minutes for the bulb to reach maximum brightness. This is the right time to write your name and time in the log book.

The mercury arc bulb has a lifetime of only a couple hundred of hours. After that, it darkens, heats up, and explodes. To maximize its life time:

1. once it is switched on, leave it on for 20 minutes so that the mercury can fully vaporize

2. once it is switched off, let the bulb cool down for 20 minutes so that the mercury can fully
condense

3. don't switch it on and off more than necessary

Figure 12: Left: The power supply for the mercury arc lamp. Right: The lamp housing with a shutter and three neutral density (ND) filters mounted in front of it. The shutter is used to switch the epi-illumination on and off while viewing an object. The ND filters are used to decrease the intensity of the light.

Make sure the epi-light shutter (Fig. 12 right) is closed to block the light path.

Lower the stage by turning the focus knob, select the 4x objective (Fig. 13 left), and switch on the (trans-) illumination build into the 'foot' of the microscope (Fig. 13 right).

Figure 13: Left: The turret with 2x and 4x objectives in visible. Right: The microscope has a trans-illumination build into its 'foot'. The switch turns it on, the slider varies intensity. Use cardboard to cover it while switching back and forth between the epi-illumination.

Next, put your slide on the stage and position it so that your 'region of interest' is in the center of the illuminated field. Shut off the room light! Raise the stage just under the objective. Now look through the eye pieces and lower the stage again until your preparation is in focus. This methods avoids bumping the objective into the slide. If it's hard to focus on the tissue, try focusing on the edge of the coverslip first.

Cover the trans-illumination with a piece of cardboard. Select the correct filter block using the slider above the turret (Fig. 14). The setting depends on the dye you used. Next to each slider position you find the wavelengths of excitation filter (EX), dichromatic mirror (DM), and emission filter (BA). For Alexa 488 use the setting 'B-2A', but also try out the other settings to see what looks best.

filter-block slider

Figure 14: The slider lets you select between four different filter blocks. Each filter block has a certain combination of excitation filter, dichromatic mirror, and emission filter.

Open the epi-illumination shutter and look through the eye-piece.

You should now see the filled cell shining bright and colorful on a black background. Keep the epi-shutter open for as short a time as possible. Dyes bleach, so minimize the time they are exposed to bright light.