Our Setups

Amplifiers

• For patch clamp recordings we use an Axopatch-1D and an Axopatch 200 B from Axon Instruments. These devices offer a sophisticated electronic design and are well-suited for single channel and whole cell recordings. They provide a lot of features, some of which are a little tricky to use, however. You should make sure the amplifier really does what you think you told it to, e.g. if you use Track together with an external pulse generator or a software seal test.



• Tip recordings generally could be performed using every intracellular recording amplifier. Most of these devices do not provide a sufficiently high amplification (about 200 fold), however. We use a relatively old device, which once was custom-built by an electronic workshop, probably in Munich. Similar amplifiers are used in the Max-Planck-Institut für Verhaltensphysiologie in Seewiesen, and can be obtained from Peter Heinecke.

Conditioning and Storing the Data

• Although we try to do as much online analysis as possible, we generally store all data on tape additionally. It is important to be aware that most recorders designed for audio purposes apply a high pass filter to the recorded signals. In many DAT recorders this filter appears to be located in the digital part of the circuitry, and therefore is not so easy to disable. So you should make sure the irresistible offer from the discounter can be customized accordingly. We use a two-channel DAT recorder from Bio-Logic and an elderly 4-channel FM tape recorder from RACAL. In order to record more experimental parameters, we are currently trying an 8-channel DAT recorder (DSR 8/3) from O-5.
  
  
• To get a quick overview over the recorded data, we use to write the recordings on strip chart recorders. For each rig we use one unit with four input channels each, a Gould EasyGraf, and a WindoGraf. These chart recorders must be equipped with separate signal condtioners for each input channel. Since the signals are conditioned elsewhere in the pathway, the basic signal conditioner in the series (13-6615-11) meets our requirements best.
  To deal with the inverted current polarity in cell-attached- and inside-out recordings, one of the signal conditioners was customized accordingly.



• Before digitizing the signals must be passed through an anti-aliasing filter. 4- or 8-pole bessel filters are common for this purpose. The Axopatch amplifier is equipped with an internal filter, which can also be applied to external signals, as from the tape. We additionally use two stand-alone filters. Frequency Devices offers a variety of filters for many applications. We use a 900C/9L8L, which provides many additional features. The other filter was custom-built by E.S.F. electronic and does no more than filtering.



• For A/D conversion we use Axon Instruments Digidata 1200 and 1322A digitizers. The Digidata 1200 is no longer produced, and is outperformed by the 1322A in all categories.



• Data acquisition and evaluation is done using Axon Instruments software. AxoScope 8 comes together with the digitizer and allows basic data aquisition we use for the extracellular recordings. Patch clamp experiments require a more sophisticated acquisition software. We use Clampex 8 from the recently upgraded software package pCLAMP.



• See the Links and Addresses page for other manufacturers of patch clamp hard- and software.

Drug Application and Bath Solution Switchers

• Small drug quantities are applied to the cells or patches using a General Valve Picospritzer. The flow of the drug solution is visualized by adding 0.1 - 0.5 % food dye from the supermarket. For special purposes we use theta capillaries from Sutter (BT-150-10).

Mechanics and Micromanipulators

• The extracellular recordings are done with old mechanical Leitz manipulators. They are the kind of device that either stand anywhere in the lab, or else cannot be obtained anywhere. Provided they are halfway maintained, these very solidly designed manipulators should work fine for another 30 years.



• For positioning the patch clamp headstage and drug application pipettes, we employ three XYZ units of Luigs & Neumann Mini 25 manipulators. These electrical manipulators combine a minimum of drift with the possibility to store, and therefore quickly find, different pipette positions. The manipulator units are attached to aluminium profiles (X-95) using custom clamp mounts. Profiles, mounts, and other mechanical equipment can be obtained from Luigs & Neumann, Newport, or Spindler & Hoyer.



• Other patch clampers prefer hydraulic micromanipulators (e.g. Narishige) attached to the microscope's object table. This can also lead to satisfying results, provided the manipulators are properly maintained. But even with a lot of experience, bringing a drug application pipette in place cannot be as fast as with a microprocessor-controlled device.



• All devices within the (custom-built) faraday cage are screwed to a steel plate with M6 thread wholes, which rests on a thin layer of cork on top of a concrete plate. Vibration isolation is achieved with inflatable tubes of the size used for wheelbarrows. Sheets of cardboard or thick paper separate the tubes from the concrete plate and the table the entire construction rests on, respectively, which prolongs their lifetime considerably. Nevertheless, they must occasionally be replaced. So it is not a bad idea to put small wooden blocks under the corners of the concrete plate. And remember, Murphy's Law is always with you! So have a few spare tubes ready, they are hard to get in the late afternoon, right during the recording of the week.

Optics

• Patch clamp recordings on cultured cells require an inverted microscope. We use a Zeiss Axiovert 35 and a 135, both equipped with phase contrast optics and additional heat filters (KG 1). The condensors are used without front lenses, to allow easier access to the preparation. This makes it impossible to precisely adjust the optics according to Köhler. However, since the recording pipette distorts the surface of the bath solution, and therefore prevents ideal conditions anyway, one soon gets used to working with sub-optimal optics. The lamp power supplies were customized to avoid electrical interference. Any stabilized DC power supply placed outside the Faraday cage should serve this purpose.

Pipette Pullers and Electrode Glass

• Sutter P-97 Puller.
• Science Products GB 150T-8P capillaries for patch clamp, and
• Clark Capillaries GC 150-10 for extracellular recordings.

Those tiny things, one can spend weeks to find

• Certain compounds of electrophysiological setups use to lie in a hidden corner of a hidden drawer in every lab. But, due to Murphy's Law, you always need one more item than there is left. And no one remembers, where that thing was bought many many years ago. Small Parts Inc. offers a broad variety of this kind of things, including tubing, tube connectors, syringe needles (even blunt ones and ones made of teflon), tools, and so on...



• Cell cultures are said to be very sensitive towards metal ions. Therefore we use quartz syringe needles (WPI) for filling the recording pipettes. Polyethylene tubing pulled over an alcohol flame may serve the same purpose.