Collected tips for setting up or resetting a rig

Clampex software setup:

Official documentation on setting up Clampex

pClamp download page

Multiclamp Commander download page

Added notes:

New computers require individuals to login with WashU credentials. This creates some wrinkles. (Unique to Elder rig until new computers are needed elsewhere).

• Installing/updating software will likely require admin access. Requesting admin access will require the “Device name” and “Full device name”. You can find these by opening a File Explorer window, right clicking on “This PC”, clicking Properties and looking under “Device specifications”. Provide this info to IT with your WashU ID to obtain admin access.

• pClamp wasn’t designed with multiple users in mind. As a result, we need to give all users modify access to a couple of folders that would normally only be available to admins and/or the user who initially installed the software. You will need admin access to do this. The folders are:

  C:\Program Files (x86)\Molecular Devices\pClampX [where X is the pClamp version]

  C:\ProgramData\Molecular Devices\pClamp.

  For each folder, right click and choose Properties. Under Security, select the Users Group, and make sure Full Control is checked. ProgramData is, by default, a hidden folder. To find it open your C: drive (or whatever drive pClamp is installed on) with File Explorer. Click the View tab at the top of the window and select Hidden items. Secrets revealed.

Koehler illumination:

Properly aligning the condenser on your microscope can improve image quality and make finding and patching cells easier. Here is a handy guide from Scientifica.

Added tips:

• When focusing the condenser (adjusting its height relative to the recording chamber) you want the best alignment to correspond to the focal plane of the objective where a slice will sit (i.e. you want the best images when you’re finding and patching those sweet, exemplary cells). So, before adjusting the condenser, focus your 40x objective on the bottom of the chamber and then move the focus up slightly to account for the thickness of the slice. (Or use a slice harp resting in the chamber if your chamber is squeaky clean and there aren’t specks to focus on). Leave the objective in this position and then adjust the condenser.

• Be careful not to raise the condenser too high and bump your recording chamber from below.

• You may need to turn down the intensity of your light source for the blades of the iris to appear crisp (step 3 in the link above).

• On Elder Rig, the condenser height is adjusted using manual knobs on either side of the condenser arm, underneath the stage. On Big, Little, and New, the condenser arm is motorized and controlled using the Scientifica patch pad wheel normally used to control the microscope focus. To adjust the condenser, toggle the selector switch from position ‘I’ to ‘II’. Once set, make sure to move the switch back to ‘I’ to control your objective focus. It’s easy to forget to do this and move your condenser out of place - NBD, just reset, focus your objective where your slice will be, and adjust the condenser height to match, switch to position ‘I’ this time.

• This will probably feel confusing the first time you do it. Ask for help! Travis likes this stuff. You typically won’t need to do this often, but condensers tend to slowly drift downwards and things get bumped. Once you get the hang of it, you can quickly check if your condenser is aligned by closing the field iris diaphragm, seeing that it’s in focus, and then opening the diaphragm back up to make sure all sides exit the FOV at the same time.

Pressure control:

If you suspect you are losing pressure through the electrode holder, take it off of the headstage and submerge it in a clean beaker of milliQ water. Use your syringe to apply positive pressure. You may need to gently break the tip of the pipette to see bubbles expelled. You should only see bubbles from the pipette tip. Look closely where the glass pipette is inserted into the holder, where the tubing connects, and near the ground pin. If you see bubbles here or elsewhere, you’ve got some replacing to do.

The red rubber gaskets in the front wear out most rapidly with repeated pipette replacement. It’s easy to pop a new one in. Just make sure it’s the right inner diameter for your pipette glass.

You will more rarely need to replace the clear(ish) gasket that holds the silver wire in place against the gold ground pin at the back of the electrode holder. This is provided from Molecular Devices as longer tubing which needs to be cut. Use a razor to make a nice, straight cut so that the rubber is flush with each side of the holder. Cutting the right length takes a little practice. It’s more than you think, but not too much (add picture).

The tubing from your pressure control syringe to the holder may occasionally need to be replaced. Again, make careful, straight cuts of the tubing for best results. This tubing is usually fastened to the headstage or the stage with tape or a clamp to isolate the pipette holder from your movements. Remember to replace these fasteners.

Heater:

An inline heater lets you warm ACSF to physiological or near physiological temperatures. Small differences in temperatures can have big impacts on intrinsic and synaptic conductances. However, patch clamp experiments at elevated temperature are notoriously more difficult with regard to slice health and recording stability. The merits of when to go for it are fun to debate but out of scope here.

To help yourself out, you should limit the length of tubing between the heater and your slice chamber. Longer tubing means more heat loss, which therefore means the heater needs to reach higher temperatures to maintain the target temperature of the bath. Oxygen and CO2 solubilities in aqueous solutions are inversely proportional to temperature. Elevated temperatures can therefore effectively undo the carbogen bubbling of your ACSF (hypoxia and less effective pH buffering) and cause bubbles that could impede solution flow and in the worst case disrupt your recording. Small differences can have big effects here (think global warming and dying ponds). Tip the scales in your favor, make the heater’s job easy, shorten the tubing!

Heater install notes: Scientifica heaters are controlled with LinLab software and interface with the computer via a Scientifica control rack. On Elder Rig, the drivers for this rack had to be manually installed. Here is a link to download LinLab and the driver setup package should they be needed in the future. Further note: Elder rig must run version 3 of LinLab as it has a newer control box for the manipulators that is not compatible with earlier versions.

Manipulator setup:

This can take some time and a Goldilocks mindset. The PatchStar manual provides a good overview as well as tips for “Troubleshooting manipulator instability” that are worth reading proactively.

There are a few locking mechanisms or “stops” that can help with reproducibility from cell-to-cell and year-to-year. There is typically less mechanical drift when the manipulator resides in these positions. It is possible to carefully position your headstage/pipette without using these; however, it is nice to have easily found “safe” locations when exchanging pipettes in low-light conditions or with aging eyes.

You can use other rigs as a reference to place the manipulators on the stage and adjust the positions of the brackets that hold the headstages. The angle of the manipulator is less straightforward to transfer from rig to rig. We have a digital level device that makes it easy [picture]. ~25-30 degrees is a good starting point.

Test out your manipulator position by placing your harp (possibly with a spare slice) in ACSF. Arrange things in what would be a typical recording configuration. Pull a couple of pipettes.

Set your x-axis motor to be at the far end of its travel away from the chamber and your z-axis motor to be all the way up. This will be a convenient “home out” position for exchanging pipettes. You can set this by holding the “H out” button on the control cube for at least 3 seconds. Now, with one click of this button you can easily get back here.

Place a pipette in the holder. You should be able to slide the headstage holder to the end of the rail (against the stop screws) without crashing into the objective, the stage, or anything else. If this is not possible, adjust something. Small adjustments can be made by sliding the position of the headstage on the holder. Larger adjustments may require adjusting the position of the rail against the vertical rotation plate.

Pipette lengths can vary, so it’s a good idea to make sure that this home position is safe from crashes with a relatively long pipette.

Next, make sure you can position your pipette under the objective. If the angle of the pipette is too steep, the pipette will run into the objective and bend down such that it is out of focus (or break). On the other hand, if the angle of the pipette is too shallow, it may run into your harp or patching below the surface of the slice may be difficult due to tissue displacement. Setting this angle is often the most finicky step. Take care of it early in the process and make note of the angle that is compatible with your stage and objective’s working distance.

Next, make sure your pipette can be lowered to the bottom of the chamber or reach well below the surface of the slice.

Finally, make sure the pipette can travel to the far end of the chamber (or as far as you may reasonably want to patch) and similarly, make sure the travel in the y-position is sufficient to get everywhere you want to go.

Work on a rig long enough and at some point you’ll need to send in a manipulator for repair, swap out a suspicious headstage, or move a manipulator to access something in the back of the rig. To facilitate reinstallation, document your manipulator position with a photo and/or written notes.