Making a gigaohm seal - help please!!!!

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Bubli
Bubli's picture
Making a gigaohm seal - help please!!!!

Hi, I've just a masters in electrophysiology and I'm having making a giga-ohm seal in tissue slices.  My cells under IR-DIC look fine, I sterilze filter my recording solution and internal recording solution but I still can't make a seal.  Any suggestions? 

I've tried making the electrode smaller, and using a KCl based recording solution . . .any suggestion would be immensely appreciated!!!

The FFM
The FFM's picture
what are  the molecular

what are  the molecular compositions of your extracellular and pipette solutions and what are the measured osmolarities of each solution when they are prepared?

If you have too large a difference in osmolarity between the two solutions you will have difficulty keeping a seal.  However you will need a slight difference between the osmolarities of the two solutions

Most physiological solutions are between 296-330 mOsm

You can check the osmolarity of your solutions using a vapor pressure osmometer - these devices are made by companies such a Wescor.  Pipette solutions should normally have an osmolarity about 5% less than the extracellular solution.

E.g pipette soln 295 mOsm

 Extracellular solution 310 mOsm

you can adjust the osmolarity of your solutions by adding an appropriate amount of sucrose.  If the difference between the osmolarities of your solutions is significantly more than 5% you may well have problems keeping a good seal.

Bubli
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Thank you for your help.  I

Thank you for your help.  I will look into the osmolarity of the solutions.  :)

Moonshoon
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You can also check that your

You can also check that your glassware is washed only with distilled water and not soap. Any traces of soap will alter your cell membrane without you necessarily seeing it under DIC

Moonshoon
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If you want to test if you

If you want to test if you have a problem with your pipette solution problem try to patch your cells with external solution instead of pipette solution. You should have a gigaseal easily

hypa_dude
hypa_dude's picture
Solution osmolarities are

Solution osmolarities are important, but so is your approach. Here are a few suggestions:

1. Use borosilicate glass electrodes with tip diameters of 1-2 microns and resistances of 4-8Mohm.  Some electrophysiologists are more particular and only use 1-micron tips and 6Mohm resistances (example). 

2. Use a microfil to add solution to the electrode and make sure that there are no bubbles.  A trick I learned recently was to hold the large end of the electrode up and slide the threads of a machine screw across the glass. This shakes free any small bubbles. Large bubbles will not be removed with this method.  Keep bubbles out of the syringe to prevent large bubbles.

3.  Use a new, clean pipette for every approach.  If your electrode tip has been sitting in your slice for more than a couple minutes, it is probably already too clogged to use.  You can usually see debris clinging to the electrode tip with IR-DIC.  Your tip has to be completely clear.

4. It is absolutely critical that your pipette holder seals are in tact and are maintaining positive pressure inside the pipette.  I attach a 1CC syringe with a short section of flexible tubing and use 0.1-0.2 CCs of pressure.

5.  Approach the cell slowly and gently while not taking up too much time. The tissue should move away from your tip slightly if pressure is maintained.  Once your tip is just above the plane of focus of your cell and aligned laterally with the cell, relieve tension in the slice by backing out the electrode and pushing it straight back in (diagonally) towards the cell.  Depending on how your maniuplator is oriented, this might not be practical for you.  When your tip is right above the cell, move forward and down very, very slowly until you see a small depression form on your cell and start to expand. As soon as this happens, open your pressure valve and watch as the dperession collapses around your tip.  If the tissue or depression does not collapse around your tip, you have lost positive pressure and need a new electrode.

6. Often, this is enough to get a seal.  If not, apply gentle, steady suction with the same 1CC syringe.  Usually less than 0.1CC is enough.

I have found that all of the steps are absolutely critical and missing any of them will make it close to impossible to get a good seal.  Good Luck!