Hi,

You should first check the noise in the system with holder disconnected. The cage should be connected to signal groups of Axopatch 200B but the table and the rest of equipment within the cage to the rear gold connector of the headstage (see Axopatch manual). Do not try to add ground connections to anything that moves; start with as little as possible. Remove all cables and wires that enter the cage (such as the HBO lamp cable coming to the microscope), see if your noise is smaller. For whole-cell configuration and well-isolated cage, you get RMS of noise of 0.5-0.6 pA. Then add the cables that are absolutely necessary, one by one. If any of them causes noise, try to wrap it in foil, connect the foil to the cage ground, make sure that the foil does not touch the table (you do not want to accidentally connect the signal ground and the headstage ground, as you may get a ground loop). See if this helps...

Good luck

Nathan

Thank you professor Nathan

I connected the equipment in the cage (microscope) and micro manipulator holders to the rear gold connector of the headstage and the noise and the residual current (100~300pA) in open circuit mode disappeared. Thank you for the valuable advice. 

I cheeked the Axopacth 200B manual (https://www.autom8.com/docs/Axopatch-200B.pdf) and it mentions to use rear gold connector of the headstage to ground the preparation (at page 55).

 I used to use the rear gold connector only to ground the bath solution via an agar bridge and I did not ground the microscope via the rear gold connector. 

Now the total amplitude of noise current in recorded current at open circuit mode is limited to 4pA. 

I guess I can improve more with the procedure you mentioned. I'll be working on them too

Thank you again

Hi: I think that one of the main reasons could be "water" existing inside the tubing occurring somewhere. "Drying" of tubing is important. The noise is slowly fluctuating with about 60 cps.

Thank you Professor Sheng-Nan

I use a microperfusion system with a low perfusion rate (~200ul per min) without a vacuum suction unit to aspirate the perfusion out from the bath. (image attached)

The perfusion consists with 8 lines of tubing (Eight 50ml syringes  connected to one Manifolder). A Microfil non-metal needle (28 gauge) is mounted to the Manifolder and which release perfusion on to the cells. (image attached)

I use gravitational flow  for the perfusion. To avoid any air-bubble formation and blocking of the manifolder, I fill all the syringes (even the ones I will not use) with external bath solution. Therefore the tubing is always filled with a saline solution. 

The 50ml syringes are mounted on a plastic like (non metal ) tube holder. (image attached)

Is there a way to ground the perfusion/tubing to remove noise? 

Thank you again 

Tharaka 

Hi Tharaka!

The thing with noise is that the largest source dominates the rest (for a nice explanation of noise in e/phys  see Axon Guide), and so you might not get any improvements if you ground smaller noise sources until you take care of the largest one. And you have to be really careful not to induce any antennas or ground loops. From what I can see the perfusion system is inside the cage and is gravity-based, therefore it should not be a significant source of noise. Also, the tubing connects with the grounded recording chamber.

Bearing this in mind, if you still decide to ground the perfusion, you could insert wires into the reservoirs and connect the other ends to the Faraday cage. Or even better, like Nathan suggested, connect it like the rest of the equipment to the headstage ground.

But first make sure you really need to do this. The fewer cables you use, the better. If it ain't broke, don't fix it.

Good luck!

Hi Tharaka! Microperfusion system is most likely to be a "noise source".  In particular, you have "eight" channels. You need to use vacuum to clear any water droplets inside the tube. If one is using microperfusion, one might need to ensure that perfusion line is free of air bubble.

Thank you so much Professor Sheng-Nan and Marcin Siwiec

I will try the suggestions you proposed. I may be able to reduce any residual noise further.

Things seems to be good now with the microscope is grounded through the head stage.

Thank you all 

Hello, I wonder why the grounding of the microscope to the rear 4 mm banana socket did not work. Might be you have some old display (TV screen for video camera) creating high frequency fields that are picked up by the wire. The reference socket on the headstage is actually for voltage reference, even that in the Axonpatch the rear socket and headstage socket are connected. For many headstages, it is actually a direct voltage reference, and should have as less voltage load as possible. The microscope could add some offset there - you can measure directly by placing a voltmeter between ground and microscope chassis. Often the grounding of the equipment on a screw on the metal table top that is interconnected with a large diameter wire to the banana socket of the rear panel of the Amplifier is sufficient. Best

Thank you Professor Thomas

I use two monitors in my rig. Both of them are newer LED/LCD type. Both of them are grounded via the graound bus connected to the 4 mm banana socket.

Yes it's odd. The noise and offset in baseline (100pA current at open circuit) disappear only when I ground my Microscope to the head stage. And the noise does not appear when I use the Reference cell or when I remove the silver wire from the pipette holder. 

I cannot figure out why the the system is picking up a ~100pA current at open circuit (when pipette is in open air. not in the bath). 

Actually the voltmeter Idea is good. I will try with the lab multi-meter. 

Thank you again

Hello, sometimes an automatic objective revolver creates high frequency noise. This noise cannot be removed by chassis grounding, only by direct shielding of the revolver with foil. I am not sure about the magnetic field component, so nickel or stainless steel material might be a safe choice and needs to be placed between electrode and revolver. Other test is to put a grounded stainless steel box around electrode silver wire, with and without grounding to the main grounding socket. If no automatic microscope revolver, then of course … . Best T

The monitors with LED/LCD type should not create noise clearly, as compared with those equipped with old TV monitor or cathode ray oscilloscope. However, microscope could be the problems. Sometimes, one might use microscope with direct currents (DC), instead of using that with alternating current. Otherwise, you might need to use metallic clip to clip any screw points nearly from which the light source comes. Light source could create noise with bizarre shape.

You mention that you see the offset and noise "even when the patch pipette was not touching the bath solution". Perhaps you don't need to eliminate noise in that condition. Often simply putting the pipette in the bath, and advancing close a cell, will cause such noise to disappear. So, do you still see this noise and offset when you have a pipette in the bath, approaching a cell?

To answer your question: no, you should probably not ground the preamplifier's reference to the common ground. See for example the section "Electrical Isolation Methods" in the Axon Guide.

To test whether it is radiative noise, you can take a large piece of aluminum foil, ground this (with e.g. alligator clips and a wire) and then hold it in front of your rig, in different places, to see how this affects the noise you see.

Since you mention that the noise only appears after ~40 minutes: do you use a pump in your setup? Does unplugging the pump reduce the noise?

Could there be a "salt bridge" developing over time? This is when your bath solution leaks and generates a (sometimes transient) electrical connection between e.g. the bath and some grounded part of your system (like the stage)? If you have a slow leak of bath solution, this could explain why you don't see the noise for the first 40 minutes.

Hope this helps!

Dear Benjamin

Thank you so much for the valuable advice. 

1. So, do you still see this noise and offset when you have a pipette in the bath, approaching a cell?

No I do not see a noise when patch pipette touches the bath solution. However, the noise reappears when I make the Giga seal. It looks like the this noise is present when the current flow through the pipette/head stage is restricted. Noise generated in both these conditions (Before pipette touches the bath & after giga seal formation) is eliminated when the  head stage is grounded via the main ground. 

2. Yes I tried unplugging the pump and all possible equipment. Unfortunately, unplugging the pump did not work.

3. "salt bridge" developing is an interesting theory. I would definitely check on that. And ill update my results here

Thank you again Dr Benjamin.