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8.7 Bath Electrode

51

The body of the electrode is a 1 ml plastic syringe body that has been heated and pulled to form a small, bent
tip. The electrode proper is a chlorinated Ag wire that is first inserted with the plunger into the fluid-filled body;
then hot agar is sucked into the tip by withdrawing the plunger partially. The filling solution can be a typical bath
solution or something similar, such as 150 mM NaCl. More concentrated salt solutions are not necessary, and they
can leak out, changing the composition of the bath solution.

http://www.heka.com

Содержание EPC 10 USB

Страница 1: ...Hardware Manual Version 2 8 EPC 10 USB Computer controlled Patch Clamp Amplifier...

Страница 2: ...eka com D 67466 Lambrecht Pfalz Email sales heka com Germany support heka com HEKA Electronic Phone 1 800 597 0580 84 October Hill Road Fax 1 508 429 5732 01746 Holliston Web site www heka com Massach...

Страница 3: ...Software only 11 3 2 Installing Hardware and Software 11 3 3 Conventions 11 3 4 Connecting the Hardware 12 3 5 Protection Key Dongle Installation 12 3 5 1 USB Dongle Installation 12 3 5 1 1 MS Windows...

Страница 4: ...rch Mode 40 7 Compensation Procedures 43 7 1 Series Resistance Compensation 43 7 2 Capacitance Compensation 45 7 3 Offset Compensation 46 8 Patch Clamp Setup 49 8 1 Mounting the Probe 49 8 2 Ground Wi...

Страница 5: ...2 Appendix I Digital Inputs Outputs 61 12 1 Digital I O Connector 61 12 2 Digital In Connector 62 12 3 Digital Out Connector 62 12 4 Breakout Cable 63 12 5 Connecting Cables 63 12 5 1 DG 4 5 Lambda 10...

Страница 6: ......

Страница 7: ...the digitally controlled EPC 10 USB patch clamp amplifier has no knobs switches or dials The acquisition software Patchmaster replaces the analog controls of conventional amplifiers by using a compute...

Страница 8: ...ced and complete discussions of individual topics which can be found in original articles and in the books Single Channel Recording B Sakmann E Neher eds Plenum Press New York 1995 and Methods in Enzy...

Страница 9: ...periments In Methods in Enzymology 207 123 131 Academic Press New York 1 3 Naming Conventions EPC 10 USB Single EPC 10 USB Double EPC 10 USB Triple and EPC 10 USB Quadro Throughout the present manual...

Страница 10: ...4 Introduction http www heka com...

Страница 11: ...nd directly attached to the recording micropipette It contains the sensitive amplifier that constitutes the current to voltage converter as well as components for injecting signals into that amplifier...

Страница 12: ...ms however allow you to re initialize the amplifier in case you forgot to turn it on first Note Since the calibration settings of the amplifier have been determined for a warmed up amplifier switch on...

Страница 13: ...tep change are available in the software 2 s which is the minimum required to avoid nonlinearities in the internal circuitry and 20 s which is preferable for all but the fastest measurements to reduce...

Страница 14: ...lifier EPC 10 USB only or interface LIH 8 8 only system AUX DAC Telegraphing Inputs These ports are for inspection purposes only Sound A 3 5 mm jack for connecting phones or speakers The volume and re...

Страница 15: ...ng of the AD DA channels Figure 2 4 Double Staggered Acquisition Double Staggered Acquisition EPC 10 USB using the LIH 8 8 The EPC 10 USB uses the double staggered acquisition mode To support cophasic...

Страница 16: ...10 Description of the Hardware http www heka com...

Страница 17: ...h buttons Signal display is provided by an oscilloscope like display The software provides the capability of data acquisition and analysis but can also be used for calibration and self testing the har...

Страница 18: ...em ory device Therefore only standard operating system files are required This provides ease of installation and flexibility for moving the Hardware from one computer system to another 5 It is possibl...

Страница 19: ...S Windows Before installing the Software on computers running MS Windows 7 you should make the following precautions 1 Log in as an administrator 2 Lower the threshold of the User Account Control Sett...

Страница 20: ...on procedure is exemplary for the EPC 10 The Software will warn you that this procedure may take up to 10 minutes depending on the speed of your computer Go ahead by clicking the Yes button Then you a...

Страница 21: ...LIH 8 8 Your amplifier if applicable EPC 10 USB PG 310 USB The series number and version of your Hardware if applicable e g EPC 10 USB Single serial number 520552 revision letter R The questions prob...

Страница 22: ...16 Installation http www heka com...

Страница 23: ...seal formation In this setting only a capacitance of 6 pF is left over corresponding to the fast capacitance of a pipette sealed to the cell membrane This mode allows you to test the C fast compensat...

Страница 24: ...d 5 where you should read a value close to 10 M A possible voltage offset can be automatically canceled by clicking on the Auto V0 button 6 After doing so the V0 control 7 displays the offset potentia...

Страница 25: ...tems can be output into the Notebook via the Help menu of Patchmaster The predefined protocols are stored in a text file called pro and can be edited with any text editor For this tutorial it is not n...

Страница 26: ...oaching a value close to 6 pF you should see the spikes become smaller You may have to adjust fast 3 in the same way As soon as you are overcompensating you will see the spikes going into the opposite...

Страница 27: ...sition of the model circuit This will simulate a model cell with 22 pF membrane capacitance 500 M membrane resistance and 5 1 M input resistance in the whole cell configuration This mode can be used t...

Страница 28: ...f compensating C slow by hand you can also press the Auto button 8 in the C slow section of the amplifier control panel for an automatic compensation of C slow and R series If the compensation fails t...

Страница 29: ...o different amplitudes for VC and CC modes therefore the Test Pulse is set to 0 pA initially Now you need to inject current into the circuitry 100 pA should be a reasonable value The current injection...

Страница 30: ...05 to 0 2 the medium Gain range from 0 5 to 20 and the high Gain range from 50 to 2000 mV pA Note Because of poor dielectric properties in the internal switch the model circuit introduces excess rando...

Страница 31: ...ier dialog 1 Amplifier 2 Amplifier or 3 Amplifier Now select the menu item Full Test from the EPC 10 USB menu You will be told to remove everything from the probe and shield its input Please make sure...

Страница 32: ...26 Verifying and Testing the EPC 10 USB http www heka com...

Страница 33: ...Amplifier window is very similar in all our acquisition programs It provides the amplifier control functions such as Gain and Filter settings and it gives access to the automatic compensation routines...

Страница 34: ...igh Gain range but the full 100 kHz bandwidth is available Currents of up to about 20 nA can be recorded This range is used mainly for whole cell recordings and for this purpose the special features o...

Страница 35: ...e Figure 5 5 Input ADC Imon1 Current monitor 1 after Filter 1 Imon2 Current monitor 2 after Filter 2 Vmon Voltage monitor output AD 0 4 Any of the other AD channels Recording Mode Sets the Recording M...

Страница 36: ...will be changed to 10 mV in Whole Cell and Out Out Recording Modes or 10 mV for On Cell and In Out Recording Modes which corresponds to the true zero current potential V0 Pipette Offset V0 displays th...

Страница 37: ...the whole cell con figuration The 30 100 and 1000 pF ranges actually allow capacitance values to be compensated in the ranges of 0 12 30 pF 0 4 100 pF and 4 1000 pF respectively The adjustment range i...

Страница 38: ...ed beforehand in the cell attached configuration The compen sation may be improved by alternating cycles of Auto C fast and Auto C slow If the compensation fails the small box in the Auto button E wil...

Страница 39: ...use an external filter rather than Filter 2 you can proceed as follows Connect current monitor 1 Imon1 to the external filter input feed the filter output back into one of the AD channels AD 0 AD 4 an...

Страница 40: ...ill reset the EPC 10 USB to its initial default configuration It will reset the DA channels to zero which is useful to cancel the output of C slow and R series set by Cap Track 5 1 2 Hidden Controls S...

Страница 41: ...ed to keep the membrane voltage constant while switching to current clamp mode Thus V mon will be identical to the original V membrane commanded under voltage clamp In analogy upon returning to voltag...

Страница 42: ...display scaling for the Test Pulse The value of 1 no display gain corresponds to full scale 10 24 V of the built in AD DA converter Thus without display gain one can easily see when the input signal...

Страница 43: ...st please contact HEKA to find out whether or not it needs to be serviced External Gain Calibration This option allows to use an external precision resistor to improve the internal automatic Gain cali...

Страница 44: ...38 The control software http www heka com...

Страница 45: ...signal Before being applied to the pipette a further variable offset is added from V0 which includes the value of LJ see chapter 7 on page 43 6 2 Current Clamp Mode The current clamp mode can be used...

Страница 46: ...ce neutralization adjustment in the current clamp mode However like capacitance neutralization settings on conventional microelectrode amplifiers excessive capacitance neutralization can result in osc...

Страница 47: ...6 4 Search Mode 41 of Patchmaster which will adjust the offset potential automatically in order to zero the current Thus the search mode is simply a repetitive update of V0 http www heka com...

Страница 48: ...42 Operating Modes http www heka com...

Страница 49: ...ew value after a step change The second detrimental effect of series resistance is that it yields errors in membrane potential when large membrane currents flow In the case of Rs 5 M a current of 2 nA...

Страница 50: ...e current causes the membrane capacitance to charge quickly to its final value of 100 mV In general when a voltage step of size V is commanded the pipette potential actually receives an initial transi...

Страница 51: ...l guess of what the time constant of the slow transient might be Therefore it is a good idea to set typical values for C fast fast C slow and R series in advance they may be saved in the protocol for...

Страница 52: ...olarity and magnitude at all times These are V0 An adjustable hardware offset voltage The actual potential applied to the pipette is the sum of V membrane and V0 Auto V0 An automatic operation which s...

Страница 53: ...ction potential between the KCl containing pipette and the NaCl based bath solution Example 3 An episode with low chloride bath solution during the experiment of example 2 It is assumed that the refer...

Страница 54: ...48 Compensation Procedures http www heka com...

Страница 55: ...Faraday cage etc It is best to have the high quality ground wire run parallel to the probe s cable in order to avoid magnetic pickup and ground loop effects This pickup becomes visible only when the...

Страница 56: ...older should be of a low loss material and should have a hydrophobic surface to prevent the formation of conducting water films Polycarbonate fulfills these criteria better than any material we have t...

Страница 57: ...wire that is first inserted with the plunger into the fluid filled body then hot agar is sucked into the tip by withdrawing the plunger partially The filling solution can be a typical bath solution o...

Страница 58: ...52 Patch Clamp Setup http www heka com...

Страница 59: ...nd to have better noise and relaxation properties however the important parameter here is the dielectric loss parameter which describes the AC conductivity of the glass Although the DC conductivity of...

Страница 60: ...kening of the tip You can tell whether you have melted the tip closed and also get an idea of the tip diameter by blowing air bubbles in methanol with air pressure supplied by a small syringe 9 5 Use...

Страница 61: ...e current trace may go off scale check clipping in that case click on the Auto V0 button or reduce the Gain until the trace reappears From the size of the current response to the Test Pulses the pipet...

Страница 62: ...tte glass with low dielectric loss e g aluminosilicate glass or if you are careful to coat the pipette with a thick coating and near to the tip the relaxations will be smaller You can cancel part of t...

Страница 63: ...clamp response For smaller values of u the comp setting should be kept below the point where ringing appears in the current trace As in the case of patch recording there is rarely need to use the ful...

Страница 64: ...58 Using the Patch Clamp http www heka com...

Страница 65: ...n your technique Under the best conditions i e with a clean holder an aluminosilicate pipette etc we have observed the noise reading increase to about 130 fA when the holder and pipette are present an...

Страница 66: ...ane vapor caution nasty stuff into the back of the pipette before or after filling it Lower noise is obtained by immersing the pipette a shorter distance into the bath this reduces the coupling of noi...

Страница 67: ...IN 3 8 IN 11 9 IN 4 10 IN 12 11 IN 5 12 IN 13 13 IN 6 14 IN 14 15 IN 7 16 IN 9 17 IN 1 18 NC 19 GND 20 GND 21 GND 22 GND 23 NC 24 STROBE 25 OUT 0 26 OUT 8 27 OUT 1 28 OUT 9 29 OUT 2 30 OUT 10 31 OUT 3...

Страница 68: ...ine 12 3 Digital Out Connector The Digital Out connector at the rear panel of the EPC 10 USB USB amplifiers can be used to trigger external devices which require TTL inputs The signals of pin 13 12 an...

Страница 69: ...rs Figure 12 4 DSub 25 female to 8x BNC breakout cable Please contact sales heka com to get further information on this cable 12 5 Connecting Cables 12 5 1 DG 4 5 Lambda 10 2 Lambda 10 3 to EPC 10 USB...

Страница 70: ...UT Digital Output Line GND Ground Line NC Not Connected EPC 10 USB Lambda 10 2 PIN Signal PIN Signal 13 OUT 0 2 Command Bit 0 Filter Position 12 OUT 1 3 Command Bit 1 Filter Position 2 OUT 2 4 Command...

Страница 71: ...and Bit 6 Speed 17 OUT 7 14 Command Bit 7 Shutter A 18 OUT 8 9 Command Bit 8 Wheel Select Bit 19 OUT 9 16 Command Bit 9 Shutter B 4 11 GND 24 GND Table 12 8 Pin assignments of Digital Out Connector of...

Страница 72: ...Position 1 OUT 3 5 Command Bit 3 Filter Position 14 OUT 4 6 NC 15 OUT 5 7 NC 16 OUT 6 8 NC 17 OUT 7 14 NC 18 OUT 8 9 NC 19 OUT 9 16 NC 4 11 GND 24 GND Table 12 9 Pin assignments of Digital Out Connec...

Страница 73: ...he probe is mounted on a 38 mm x 90 mm x 4 mm plate which has 4 wholes with 3 mm diameter Since the plate is wider than the probe there is room for custom mounting wholes on both sides of the probe Fi...

Страница 74: ...68 Appendix II Probe Adapters http www heka com...

Страница 75: ...ction which gives it the opportunity to operate in the 3 Electrode Mode Figure 14 2 S Probe with pipette holder cable and connector The headstage has a thread connector to provide a safer electrical c...

Страница 76: ...70 Appendix III S Probe http www heka com...

Страница 77: ...ferent outer diameters of the glass electrodes the following pipette holders are available Pipette Holder BNC Type 1 0 mm Order number 895227 Pipette Holder BNC Type 1 3 mm Order number 895228 Pipette...

Страница 78: ...Holder SMA Type 1 3 mm Order number 895149 Pipette Holder SMA Type 1 5 mm Order number 895150 Pipette Holder SMA Type 1 7 mm Order number 895151 Pipette Holder SMA Type 2 0 mm Order number 895152 Ple...

Страница 79: ...ble 63 Installation 11 Calibrating the Hardware 14 Connecting the Hardware 12 Conventions 11 Hardware 11 MacOS versions 11 MS Windows versions 11 Software 11 Dongle 12 MacOS 13 MS Windows 13 USB Dongl...

Страница 80: ...Recording Mode 29 Reset 34 Rmemb Rpip 34 Rs Comp 32 Sound 34 36 Stimulus 33 Test Pulse 29 Track 33 Tracking 30 V mon 29 V mon Gain 35 V scale 36 Zap 34 Pipette Holder 69 BNC Type 69 SMA Type 70 Probe...

Страница 81: ......

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