Back with another chapter in our Fundamentals Series – please comment… We would be happy to add to this, for it to become a great resource for all those plagued by electrical noise on electrophysiology rigs.
1. First step – Plan your hunt
After a couple of recent noise-hunting expeditions, I thought it might be useful to put a few ideas down which are better organized than my recent handwritten notes and text message exchanges with colleagues. It’s easy to find noise troubleshooting advice online, but I don’t find any of these particularly helpful. In my opinion, these are either too technical (all of us don’t do electrical engineering in our spare time), not comprehensive enough (notes from manufacturers or online forum exchanges), not applicable to electrophysiology, or based on patch clamper superstitions (not that some of these superstitious recommendations have not proven helpful!).
So, here’s my attempt – I’ll start out with a bit of background to the concept in general. We’ll then look at general good practice when setting up your rig and how to maintain it. These sections should help with the conclusive remarks, where I offer a few tips for noise troubleshooting. An important part of this is to address the mindset I think is useful in approaching noise hunting. Much of this approach should be intuitive for scientists, but often the frustration accompanied with noise troubleshooting pushes this intuition to the background. I’ll therefore list some questions worth asking and how to approach the hunt more methodically.
“The cage made a drastic change to the amount of noise we were picking up. …We are happy with the large reduction that the cage offers. Thank you for helping me sort this out. And a big thank you to your engineers for getting it set up for us.”
– Jake Diggins, Aston University
1.1. General concepts – Motivation behind taking on the expedition
As far as electrophysiological recordings are concerned, electrical noise is interference which degrades or masks the recorded signal. Since we’re working in a forest of minuscule signals and high electrode impedance, combined with a complex environment (wet stuff, lots of electronics), the electrophysiology experiment has its unique set of challenges to address. This can become very time-consuming and frustrating to resolve, even if you know what you’re doing.
Let’s categorize what we’re dealing with…Some noise types are inherent to the measuring instrument and the technique. When using the instruments in the prescribed way, nothing more can be done to improve this (The Axon Guide: Chapter 11 offers a good summary on this). We’ll call this “inherent” noise – still unwanted, but not much you can do about it. External noise is the kind of non-optimized interference you get, typically from interaction between devices and the environment.
You can get away with less than optimum noise levels, depending on what you’re trying to measure. Your traces will usually have some noise presence (inherent plus some external). With your electrode in the bath, looking at your noise baseline, evaluate the peak-to-peak amplitude and RMS noise level readouts. Decide based on this whether it’s acceptable, or if you’ll have too much signal masked by the noise values (typically, you can multiply the RMS with six to get a snapshot of what 99% of your trace peak-to-peak values will fall within. For example, an RMS value of 3pA means 18pA peak-to-peak values on your traces not an ideal situation!).
1.2. The hunting grounds
To think systematically through the problem, it’s helpful to distinguish between the three actors when troubleshooting noise:
- the source (quite often several of them)
- the receiver [in our case usually the sample, electrode(s), preamplifier/ headstage, cable running to the amplifier, amplifier itself, cable to the Data Acquisition System or analogue/digital converter (referred to the DAQ hereafter), or the DAQ itself]
- the coupling mechanism (so how does the noise get from the source to the receiver)
Remember that no system can be quieter than its noisiest link. This is evident from the following RMS formula:
Noise Total = [(Source 1)2 + (Source 2)2 + (Source 3)2]1/2
Take for example [(30 pA)2+(14 pA)2+(10 pA)2]1/2 = 34.52 pA, which demonstrates the overwhelming impact of the noisiest link. One of the implications of this is that you may run into situations where the biggest noise source hides multiple other sources below its RMS behaviour. Once the biggest issue is resolved, you may only get drop a few pA or mV in peak-to-peak noise, with the next biggest source now being the main culprit. Knowing this will help you to manage expectations when troubleshooting – you may need to dig deep into your patience reserves!
1.3. The ecosystem – typical coupling mechanisms
There are four ways noisy interference typically gets coupled from the source to the receiver:
- Electromagnetic interference – typically via free-space air; can be over great distances (e.g. radio frequencies)
- Conducted interference – also called “galvanic coupling”; this is when the interference is conducted via cable, chemical compound (e.g. salt residue) or liquid (e.g. pipette solution overflow into pipette holder). A special case here is “common impedance coupling”, where conductive cables are shared between two circuits, and activity on the one circuit affects performance on the other circuit. Common impedance coupling in the electrophysiology context is more typically an issue within instruments instead of between instruments
- Magnetic coupling – also called “inductive coupling”; typically, near-field activity, and often exacerbated by coiled cables in and around the setup
- Electric coupling – also called “capacative coupling” or “electrostatic coupling”. Also has an impact, but only over short distance. Stray potential differences cause electric fields to build up and interfere with nearby conductors
All four of these mechanisms are contributing factors to noise in electrophysiological setups, with electrical coupling probably being the most common.
1.4. Identity of the beast you’re hunting – Noise categories affecting electrophysiology recordings
How does noise typically present itself in an electrophysiology setup?
Once you have your pipette in the bath, your traces (displayed in gap-free mode) will often display a consistent noisy pattern. These patterns offer clues about the source and sometimes also the coupling mechanism. It’s therefore important to try to characterize the noise on your trace. Of course, noise sources tend to mix (remember the RMS implication above), but there will often be a single dominant pattern visible. The most common types are:
- Cycle noise – also called “line frequency noise” or “hum” (e.g. power supplies or light sources); This type of noise is probably the most common of all external noise sources and is picked up from mains power. Therefore, it has the characteristic 50Hz or 60Hz cycle (corresponding to the frequency of your mains AC power), or a multiple thereof.
- Digital noise – also called “white noise”; common devices on rigs like computers, some digital-processor amplifiers, microprocessor-based devices, and digital or analogue cameras have high frequency clocks or oscillators – these are often not completely electrically isolated, which may generate stray signals. Because these are high frequency (often in the MHz range), they can be hard to troubleshoot, and pin-pointing the source may be more difficult
- On/Off switch noise – this is when you see spikes on your traces when devices switch on or off (or activate/deactivate). Switching devices on or off happens rarely mid-experiment but using TTLs for activating devices or changing the speed on perfusion pumps or valves is of course a common occurrence. Additionally, air conditioners and fridges/freezers in the lab often cause these on/off switch artifacts
- Other oscillations – Might be high or low frequency, and often caused by mechanical movements, pumps and interference between devices may cause oscillations. When troubleshooting, it’s important to look at your traces over short and longer time frames in order to identify these oscillations
Once you have identified the type of noise, you can start to build your elimination strategy. A good start would be to identify potential issues in the way your setup is arranged. Let’s have a look at general good practice for low-noise recordings.
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2. Good hunting ethics – what a low-noise rig looks like
Remember, your primary weapons for sorting the noise are repositioning, swapping or shielding cables and equipment. But first consider some good practices for setting up a low-noise rig:
2.1. The hunting grounds – your lab space
Awareness of where you set up your rig is critically important. I’ve been in labs where the rig was set up on the other side of a wall attached to a hospital’s main transformer and electric breakout box. Plus, positioning directly under air conditioners, or near fridges or laser power supplies is common error.
2.2. Setting up camp – Shielded space
You want to have the environment as hassle-free as possible. If other factors are playing along, you can get away without using a Faraday cage. But the moment you have issues, you’ll wish you had a cage… It is better to invest in one from the start, in my opinion. You can do a self-build, or order a custom and cost-effective solution from MCI Neuroscience.
Faraday cages perform optimally when it is made from a continual high conductivity material. Therefore, any cable ports or openings should be kept to a minimum as the length of any opening will determine the cut-off frequency for the cage. Also try to keep doors shut during recording. If you can’t close the doors during the experiment, it may be worthwhile investing in a shielding screen positioned between the recording area and the operator. Additionally, a computer monitor/screen EMF shield might be useful in some instances.
2.3. Everything in its place – Equipment positioning
The more complicated your setup, the more aware you’ll have to be of where to position your devices. Of course, you’ll need to consider available space and ergonomics, but do think about where your amplifier and DAQ are relative to other devices.
Try to position these devices away from typical noise culprits like computers, monitors, supplies for illumination sources and other noisy power supplies. It will be even better if you could install it in a separate rack. If you don’t have enough space, try to position them as far apart as possible or get a thick metal shielding plate between the potential source and the receiver.
For more complicated rigs, you’ll need plenty of sockets for all the devices around your rig. Ensure your socket outlets have protective earth contacts. It’s good practice to not use a power strip for your amp and DAQ, and to plug them into an individual socket set separate from one another and other devices it could impact on.
MCI Optical Table Shelves are a great option when you have limited lab space. These overhead shelving units not only provide extra space for your equipment, but noise culprits such as amplifiers and data acquisition systems can be placed overhead and farther away from your recording chamber. Click on the banner above to learn more on our custom Optical Table Shelves for peace of mind.
2.4. Connected to earth – Grounding configuration
Use a central grounding point. Most people prefer a grounding bus (conductive metal block, often copper, with holes to plug grounding cables into). You can buy something more sophisticated from NPI Electronic.
The grounding bus is then connected to the breadboard of the antivibration table (acting as a common ground) and, if available, to the “signal ground” socket on the amplifier (Axon amps and some other models have this on the back). We’ll discuss grounding in greater detail in the troubleshooting section below. Only ground devices and components when necessary.
2.5. Your primary weapons – Cable management
With some setups looking like proper birds’ nests, the many cables around your setup could very easily become sources (generating electric or magnetic fields) or receivers. Cables can also act as antennae, picking up signals from nearby radio stations, mobile phones or computers or wireless devices in the 2-5GHz range (even short cables can pick up noise from these). Some general comments about cable management:
- Keep cables short – longer cables invite issues. They’re often coiled up (which invite magnetic field generation or pick-up) and longer cables increase likelihood of impedance coupling. When you must use long cables, make sure to use (or add) good quality, heavy gauge shielding.
- BNC cables – buy BNC cables with consistent impedance. 50 Ohm cables (instead of 75 Ohm) are recommended.
- When signals of more than 100kHz are problematic, you may want to use some ferrite toroids or beads around the cables (if not already integrated by the manufacturer)
- Extension leads and power strips should not be draped near power supplies and should be kept as far away from your workstation components as possible
- Keep signal/data cables and power cables far apart, as running them in parallel and close to each other increase chances of noise pick-up. If you can’t keep them apart, try to position them at right angles relative to each other at the point where they cross.
- Run headstage/preamplifier cables in bundles – this may help with noise voltages cancelling each other and keeps them separate from power cables. Try to keep as much of the headstage cables inside the Faraday cage.
- Also make sure you have only one bath reference electrode when you have multiple headstages.
- Bundling AC power cords – as far as possible, bundle these together. This may help with averaging electrostatic and magnetic fields and reduces net radiation as a result.
- And then, a more general principle – placing your cables too delicately in order to prevent noise is not a good strategy – this strategy may work for competing sources of hum to cancel each other out, but in a working lab, it’s just a matter of time before something in the setup changes, a cable gets moved, which may cost you a lot of time in troubleshooting. Develop a robust strategy which won’t develop constant issues.
2.6. The watering hole – around the chamber
Give special attention to the area around the recording chamber – use the shortest pipette and grounding electrode possible (extending 5mm beyond the cap of the electrode holder will be enough). Remember, longer cables invite trouble. Related to this, a short-barrel electrode holder is good practice, if it doesn’t restrict access too much.
Position your perfusion inlet/outlet in a way which will most likely give you laminar perfusion. Turbulent perfusion is more likely to dislodge a slice and could potentially cause electrical issues.
2.7. Avoid the booby traps – on ground loops
When adding grounds to your setup, you want to drain the noise away in one direction towards the ground (which, in this case, will be the ground bus on your table).
A ground loop is when you have a return loop connected to the origin of the ground cable. This can act like an antenna or source – a changing magnetic field around the loop causes emitting fields to build up around the loop. Ground loops are a common cause of cycle noise and can be picked up despite the cables being shielded.
Some pointers for avoiding this:
- Only use grounding cables when necessary
- Avoid having more than one grounding bus. Maintain as best as possible a “star” ground configuration for all equipment. In this configuration, there is one central reference point (one single grounding bus) and all common connections go to that point. It makes it easier to spot errors
- Never daisy-chain your grounding cables
If you were to remove the orange cables depicted in the figure below, you won’t have ground loops in the setup anymore:
Figure 1: Depicting the difference between correct grounding and creating ineffective grounding loops
2.8. Stay fit for the hunt – Good rig maintenance
Of course, this prevents all kinds of other issues as well. As far as noise development is concerned, this revolves around three main areas:
- Keep your setup clean – spills should be carefully cleaned up. Dried salt residue (especially around the recording chamber) may act as a conductor. Spray some water followed by 70% alcohol and then wipe it dry. It’s good practice to treat areas often exposed to spills with a thin layer of grease or some other rust preventative
- Check connectors from time to time – connectors which get moved often may wear out more quickly. Connectors which have been plugged into the same socket for years may oxidize. You’ll be able to identify problematic connectors by wiggling it whilst monitoring your trace
- Keep a logbook of rig maintenance and noise levels at different time-points and under a fixed set of conditions. This becomes especially useful when transferring use of a rig from one user to the next
3. How to approach the stalk – some thoughts on strategy
Your approach will be partly determined by whether it’s a new rig, or whether it’s a working setup which develops noise suddenly. But as a general principle, avoid the temptation to barge in and make changes… Gather as much information as possible before you start ringing the changes and WRITE EVERYTHING DOWN. Make notes, take pictures, keep record of traces. Approach it like you’d approach a new and complex experiment. Be very methodical!
3.1. Know your prey – define the problem properly
Ask yourself some questions, depending on the setup’s history –
- What exactly are you looking at on the trace? Are there other ways of interpreting it?
- What has changed in the environment around your setup? Has anyone else been using the same rig?
- Are there any other unusual/concerning/suspicious issues on the rig? Has something that presented itself just before the noise surfaced?
3.2. Being obsessed with buffalo when you could easily go after rabbits – What would an easy solution look like?
For this, my suggestions would be to look out for some basic user-errors resulting in noisy traces – these could be:
- Bad recording electrode – your electrode may not be coated properly or the connection with the pin may be unstable
- Bad connection on the reference electrode, or the reference electrode not being coated properly
- Bodged pipette solution – not enough chloride for example
- Wet or dirty pipette holder – Basic error, but someone using your rig (not you, of course not!) may be overfilling the pipette, resulting in the pipette solution being pushed out into the pipette holder. You only need 2-4mm of the electrode length to be immersed in pipette solution. If wet, dry it with clean air. If dirty, you’ll have to disassemble the pipette holder, remove the silver electrode and the rubber o-rings, rinse with ethanol and then distilled water several times and allow to dry. If you have a sonicator it may be useful to put the components through the process to remove small particles and salt residue
- Could it be that someone’s changed some of the cabling and/or the settings on your software? Incorrect scaling or gain settings may lead you to think you have more noise when you immerse the pipette in the bath
- Spillages and dried salt around the bath may lead to noise from an electrochemical junction. This appears as an unstable, fluctuating noise on a time scale of milliseconds to seconds. It may be caused by salt solution that is spilled, for example near the chamber, that creates a battery between dissimilar metals, or that bridges different ground connections. Daily rinsing is the bare minimum you can do for the system. A regular thorough cleanse should be marked on your calendar and parts that are in contact with solutions should be exchanged for new ones regularly
- Have you recently changed the levels of your perfusate in the bath? Perfusate levels which are too high or too low may lead to increases in noise
- Are all your electrical rig components up to date? An outdated mercury lamp for example, can produce noise on top of being hazardous
- Headstage pin maintenance – make sure the pin is clean. You may need alcohol on a cotton swab for this, but be careful not to have alcohol flow into the sensitive headstage electronics
- Is your pneumatic antivibration table floating properly? An unusual one, but I’ve seen this before
- Make sure the Faraday cage is also properly grounded to the central ground
Help the Electrophysiology community by participating in our survey on Electrical Noise in the Lab. We regularly post some survey feedback on social media
“In the new building we uncoupled the electricity in our lab from the mains using isolating transformers (got rid of big peaks from lasers in the physics department). With a good central/consistent grounding system, we never had any noise; screening/Faraday cages were not necessary for classical whole-cell patch clamping!”
– feedback from the MCI Survey: Electrical Noise in the Lab
4. Your weapons arsenal – useful tools
If none of these basic errors seem to be the cause of the issue, you’ll need to systematically go through the rest of the setup. Here’s a list of useful tools for the troubleshooting process. These are inexpensive, and useful to have in any electrophysiology lab toolkit:
- A good multimeter with audio output signal – for testing whether cables are intact and looking for voltage differences between points
- Ground bus – always useful. You can use a brass or copper bar and drill and tap some holes yourself. Mount it onto the airtable. Connect to the signal ground of the amp
- Grounding cables of varying lengths – remember, you don’t want any cables to be unnecessarily long
- Connectors – Crocodile clips, banana plugs and terminal rings
- Aluminium foil – the ultimate in sophistication!
- Conductive tape – for wrapping cables and sealing gaps in equipment housing where noisy signals may escape from
- Model cell for checking the amplifier – a model cell is usually included with an amplifier, but some manufacturers sell them separately as well
- Mini Faraday cages – Cardboard sheets covered in foil can be useful to shield potential fields from the headstage. Same with a coffee or soft drink can. Of course, this should always be connected to the ground with a grounding
The following tools may be useful to invest in if you don’t manage to solve the issue quickly. Still, if you don’t have all of it at hand, you’re likely to find some if you were to ask around. A neighbouring lab may have it stashed away in a drawer:
- An adjustable clean lab power supply unit. Try this as an alternative to some supplies on the setup. This will require quite a bit of soldering to have the correct connectors, and you’ll need to be careful not to overload your expensive equipment with too much current or switch polarity
- Test an alternative headstage or amplifier – this is when you’re quite desperate and unable to find the solution elsewhere
- Try finding the source using an oscilloscope with open-ended probe. It’s useful if your oscilloscope has an audio output channel. The “clinical” version of this approach is demonstrated nicely in a video by Neuralynx Inc. (skip in to approximately 1:30)
- Use a notch filter – sometimes these are built into the amplifier, but they’re available as separate units. If you don’t have one, NPI Electronic might be able to help
- Use a cancellation device, like the Humbug – of course, with both the Humbug and the notch filter, you’re running a risk of filtering or cancelling out some relevant part of your signal. Bear this in mind, but these devices are sometimes essential to enable you to continue with your experiments
5. Looking through the scope – Further pointers of where you stand a good chance of finding noise sources
Once you’ve eliminated the easy errors, you want to methodically look for the cause by the process of systematic elimination. Remember, the remedy tends to be in repositioning, swapping or shielding cables and equipment.
Knowing the most likely causes, play around with it by methodically evaluating and ticking off the most likely causes on your rig. This may require many switch off-unplug-test reiterations.
Plan your approach carefully. With the knowledge you already have, you can trust your intuition – but remember to do it systematically, with notes, pictures and screenshots. Decide beforehand what your test approach will be following each change, and keep to it: make the change, make notes describing this, take the necessary pictures and screenshots, and make sure you investigate the effect properly. For example, change the time axis on your trace to detect slow cycles induced by the change. Also zoom in to look for a change in the waveform shape, which may give you some clues.
So, let’s take stock: At this stage you have probably investigated all the suggestions and best practice solutions for getting rid of unwanted noise in your rig. Having also integrated the best practice suggestions above into your approach, some further priorities for your troubleshooting protocol should include other common causes (some of these have been alluded before):
- The lab’s air conditioning system
- Cheap power supplies, especially switching power supplies (e.g. AC/DC converters)
- Make sure you only have DC cables inside the cage – no AC
- Keep mobile phones away from the headstage – switch it off
- Be suspicious of electromechanical devices, including manipulators, valves and mechanical shutters
- Devices with system clocks and oscillators are likely sources
- Old/Damaged cables, that either lack conductivity or exclude shielding (this is especially important to investigate when any old power/data/ground cables have been repaired with improper DIY solutions
- Cables acting as antennae, electric or magnetic field sources and receivers more extensively discussed under Section 2 – “Good hunting ethics – what a low-noise rig looks like”. Be particularly suspicious of very long cables, coiled up cables and cables carrying heavy loads. Try repositioning cables and/or shield it with grounded shielding plates, conductive tape or foil
- Electrostatic noise from devices with high loads, very often electrical stimulators and high-current power supplies for light sources
- Hairline cracks in thermistors, resulting in electrical current leaking into perfusate
- Any anodized components near the chamber are likely to be electrically disconnected from ground and may, therefore, need separate grounding. This may include:
- Some microscope components. Microscopes often have dedicated grounding sockets – These may be useful, but some components are not bonded electrically to the grounding point.
- The experimenter’s body – this is easy to see if you touch the table and the noise drops…wear an earthing strap to resolve this
- If you don’t see the noise before you start recordings, a bad seal is most likely the cause. If this is the case, also consider an unstable recording electrode or a tissue slice which is moving around because of perfusion inlet/outlet which is not positioned properly
6. On target – or not…
You’ve planned well, applied your intuition and logic and asked the right questions. Your approach has been systematic, and you’ve managed to interrupt the coupling and protect the receiver. Congratulations!
But what if the process of switching, re-positioning and unplugging has not delivered? You may want to approach it from the other side…switch everything in the room off. Unplug everything except the amplifier, DAQ and computer. Start from this skeleton setup and reconnect step by step, doing a reverse hunt.