Electrophysiologists: Are we superstitious?

Small changes go a long way

This commonly used phrase holds a special meaning in electrophysiology and not necessarily in a positive way. Minimal changes can massively alter the performance of experiments, and here we describe a few examples.

Pipettes

Having a good pipette is essential. The size and shape of the tip will influence the resistance and stability of a recording and, in the case of patch-clamping, even the sealing to a cell. However, having a good pipette is not as simple as it may seem. After settling on the material and thickness of your glass, the choice of pipette puller is also important and therein lies the rub. Adjusting all the parameters to get the ideal shape is a process that can take from hours to weeks. If you are not convinced, look at the Sutter Pipette Cookbook; a hundred pages dedicated to obtaining the desired pipette. So, when a careless user burns the puller’s filament or erases a pulling program, you can expect all hell to break loose.

Perfusion

Perfusion is very important for oxygenation and drug application. Especially when working with tissue slices, bicarbonate-buffered artificial cerebrospinal fluid (aCSF) needs to be constantly oxygenated and applied. A constant flow is also important for drug administration and solution changes, which are important for certain experiments. Getting a constant flow involves a controlled height at which solutions are placed, the precise length and width of the tubing and the right amount of suction to remove the solution from the bath. Overflow or dry up events are not rare and they need to be avoided. Changes in the perfusion system not only affect the speed at which drugs are applied or washed away, they can also create undesired baseline changes and alter the stability of a seal. Therefore, once a good constant perfusion is achieved, any change is undesirable, even if this means never changing the tubing ever again.

Cables

Even the most basic electrophysiology rig is equipped with the electronics required for amplifying and recording the electrical activity of cells. But, depending on the nature of the experiment, you may also have a camera for cell visualisation, a micromanipulator controller, a stimulus isolation unit, a peristaltic pump for recirculating the bath solution, and more. That means that you have a lot of equipment connected, each of which is a potential source of electrical noise which can affect your recordings. Seemingly inoffensive movements of cables can introduce noise to the system, as well as connections which seem not to be related at all, such as cell phone chargers. This also means that once you have a noise-free setup you don’t want anybody lingering close by, thus the fixed gaze of vigilance you may have experienced. Finding and solving such problems can take time, which brings us to our next section.

Aluminium foil, our best friend?

As stated above, it is important to keep noise at minimum and people have already discussed many ways to detect the source of this noise and fix it. Knowing your system, which includes all the connections and how they are grounded, should be enough to solve this problem. However, it is not that simple, and an easy solution for a sudden disturbance during an experiment can be by isolating the problematic area with aluminium foil. Shielding certain connections can create a Faraday Cage effect blocking the electromagnetic fields.

The problem comes when this “temporary” solution becomes permanent. It is because that solution fixed the original problem, that no one is ever allowed to remove that piece of foil. Some people even use it as a preventive measure. But if removing a piece of aluminium brings back a source of noise why remove it from the setup? Some of the shields become legendary!

Remember, nobody can touch a fine-tuned setup, and that includes a bunch of aluminium foil covering useless cables.

Preferably Far Away (PFA)

Electrophysiologists work with live tissue. The lab may also use paraformaldehyde (PFA) to fix tissue for post-processing by immunohistochemistry techniques. PFA exerts its function by creating covalent bonds between proteins. This substance is volatile and usually handled in a hood, wearing a mask and gloves. So, it is more than understandable that people working with live tissue want to make clear distinctions between all the tools in a lab that are in contact with PFA and the ones that are not. But even if electrophysiologists are not the only ones working with live tissue in a laboratory, at least in our personal experience, we are the most neurotic about keeping PFA preferably far away. We don’t want a closed bottle of PFA in the fridge close to our solutions, or a plate with some PFA close to our setups, or people weighing some PFA powder in the same room.

Less evident but still present is the problem with soap. Soap is a detergent, and detergents solubilise the lipid membranes of cells making patch clamping impossible. So, electrophysiologists don’t like to wash up with soap.

It doesn’t matter if everything was rinsed 3 or 10 times, if something doesn’t work after contact with PFA or soap, we will blame the PFA or soap.

Solutions to every problem

The solution may be a good intracellular solution. Everyone has their own recipes and tricks. Some people like to make it relatively fresh, while others try to keep aliquots of a good solution forever. And the point is that, chemistry looks great in papers, but the same recipe doesn’t lead to the exact same solution (it is possible to get different osmolarities and pH levels). These parameters influence the many aspects of the experiment from seal formation to cell survival. The origin of the differences is not well understood, so that’s why having a very good solution is a bit of a lottery, and once you have one, you don’t want to share it.

As far as external solutions go, some electrophysiologists can also be a little picky. Even insuring the Milli-Q water always comes from the same source may be fundamental.

Electrophysiologists are made, not born, and in the process of learning the tricks of the trade they realise how small changes within an experimental setup can have huge consequences, that may take weeks to fix. This is the reason that once they believe their setup is optimised they become precious about letting others near it.

Do you think electrophysiologists display paranoid behaviour? Or, do you think their actions are justified. Let us know your opinions, personal obsessions or experiences of encountering an electrophysiologist.