Lucifer Yellow blocks my micropipettes

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tapioka_brown's picture
Lucifer Yellow blocks my micropipettes

Hello! For my diploma thesis I am trying to photoinactivate neurons in the cricket's auditory system. Therefor I have to fill the neuron iontophoretically with lucifer yellow. Unfortunately, lucifer yellow blocks my micropipettes. I'm using 5% LuYe in 0.5m Lithiumchloride and 1m Lithiumchloride for backfilling the pipettes. If I fill the pipettes just with LiCl, I get nice intracellular recordings, but with LuYe they block almost instantly. Do you have any tips or ideas?

Moonshoon's picture
I had the same problem as you

I had the same problem as you with stomatogastric neurons in the lobster. I had to change my electrode (sharp electrode, 20-30 megohm) several time before having a proper filling of the neuron. Instead of LY, you should try carboxyfluoresceine which is soluble in acetate potassium (and probably in the pipette solution you use) and does not clog electrodes. However be careful not to light too long when you photoinactivate : it seems carboxyfluoresceine releases a lot of free radicals that leak from the cell (i killed several prep). I usually lit for 30 min max (but surely you can monitore the death of the neuron and shorten the duration of photoinactivation).

pbm's picture
I haven't used LY much

I haven't used LY much recently, but we used to use it all the time. The formation of crystals (or failure to dissolve) is not uncommon.
1. which salt of LY are you using ? You will want the lithium salt, as it has much greater solubility than most of the other forms available. 
2. The maximum solubility for LY-Li is about 9% in water, so you should be ok with 5%. 
3. It may help to filter (0.22 um filter) the solution before filling the pipette. Another approach is to briefly sonicate, vortex, and then centrifuge the solution. Then only draw your filling solution from the top of the vial. 
4. One common cause of precipitation is incorrect pH. Normally these high salt solutions are not buffered, but you could drop the concentration from 1M to something lower (150-250 mM), and buffer the pH with HEPES. 

I would guess that you are using sharp electrodes, but how fine are the tips? If they are more than about 0.2 um in diameter, you might consider that the bath solution could mix in the tip with your pipette solution a little. The K and Na salts of LY are much less soluble than the Li salt, and so this might cause some precipitation right at the tip. Perhaps a little positive pressure on the pipette (by little, I mean about 0.05 cc of push with a tuberculin syringe), similar to patch recording, might help prevent mixing. Just a thought.