Which Alexa dye to use?

There are several Alexa dyes available and useful for our purpose: Alexa 350, 488, 546, 555, 568, 594, 633, and 647. The number indicates (roughly) the excitation wavelength (Fig. 6). The emission wavelength is typically 20-50 nm longer. If you don't use a confocal microscope, make sure it is in the visible range. If you do, make sure there is a laser with the right excitation wavelength available.

Another point to consider is auto-fluorescence. Many objects, including the Aplysia ganglion, show fluorescence. Even without dye injection, the ganglion emits some red light when put under the fluorescence microscope. While this helps outlining the ganglion, autofluorescense often is unwanted and you need to consider it when choosing the dye.You might want to avoid using dyes of similar wavelength. Aplysia tissue shows some fluorescence emiting light in the red part of the spectrum. Alexa 488 seems to have become our lab favorite. It absorbs blue light (490 nm) and emits green light (520 nm).

Figure 6: Emission wavelength of Alexa dyes. ©Invitrogen

Preparation steps for dye injection

1. Pull a glass electrode (use glass with a filament like WPI type TW100F-4 )

2. Fetch the dye from the freezer and thaw it in your hand. The dye comes either ready made or dry. The powder is mixed with KCl (KAc, KCitrate, LiCl, etc. should work, too) to make a 200 mM solution.

3. Dip the back of the electrode in the dye (Fig. 7a). The amount you need is close to nothing! The smallest drop will do, such as the ones in the lid of the dye container.

4. Put the electrode aside and wait for 1-2 minutes. You'll see the dye crawling along the glass filament and slowly filling the tip (Fig. 7b).

5. Put the dye back in the freezer.

6. Fill the upper half of the electrode with KCl (or whatever you normally fill your electrodes with). The air bubble prevents the dye from being thinned too much. However it might make the recording a bit noisier. (Fig. 7c)

7. If you usually do it: bevel your electrode tip. The resistance will be slightly higher than usual. For example ~15 MΩ instead of 7 MΩ.

Figure 7: Filling of the glass electrode.

Injecting the dye into a neuron

First, establish a stable recording of the cell you want to fill. Alexa dyes typically do not affect the recording properties of the electrode too badly. That means you can do some physiological experiments prior to filling the cell. If you believe that the electrode is stable enough to stay in the cell for the next hour, start injecting hyperpolarizing ('negative') current into the cell. For Aplysia neurons use -20 nA. Most other cells will not be able to handle this kind of current (or the sharper electrodes required will not pass it), so you might have to reduce it to 5 nA or even less.

While many use DC current, pulsed current can prevent the electrode from clogging (which is not as much of a probleems with Alexa dyes than it is with ouhter dyes). The theory is that the dye molecules have a negative net charge. By forcing (negative) electrons in the electrode, dye molecules are repelled and 'electrophoretically' forced out of it. Since the dye molecules are large, they easily jam the tip of the electrode. Pulsed current could prevent that from happening. A good starting point is a 1 Hz square wave with -20nA amplitude at a 75% dutycycle (Fig. 8). Switch off the light to minimize any bleaching of the dye!

Figure 8: Current pulses used for driving the dye out of the electrode and into the cell

Injection time depends on cell size and how much dye you want in it. About 30 minutes produces good fills of most Aplysia cells, but longer fills rarely hurt. It is a good idea to frequently switch off the current injection to check on the recording. Make sure that you are still in the cell, that the electrode still passes current and hasn't clogged. If the cell is still healthy and spikes - even better.

You might not have epi-fluorescence at the microscope on your electrophysiology setup, but still want to check if the cell is filled to your satisfaction. In our lab we successfully use a small custom-built fiber optic light. It is designed for use with Alexa 488 and costs little to build (Fig. 9).

Figure 9: A custom build fiber optic light helps to judge if a cell is sufficiently filled. It allows epi-illumination, which is necessary as Aplysia ganglia are yellow and act as filters themselves. The narrow bandwith of the LED (type BUBC333W20BA09 www.besthongkong.com) replaces the excitation filter. An emission filter (Kodak Wratten #12 yellow) improves the image, but generally is not necessary.

After you pull the electrode out of the cell, allow 'diffusion'-time for the dye to be (passively or actively) transported within the cell. Depending on the cell's morphology, you should see significant improvement in smaller branches within 30-60 minutes (you might want to store the preparation overnight at 4 °C). The preparation should be shielded from light - wrap it in aluminum (tin) foil or put it in a dark box.