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Using the Patch Clamp

EPC9 Manual

78

the current level. Additional suction sometimes lowers the access resistance, causing
the capacitive transients to become larger in amplitude but shorter. Low values of
the access (series) resistance are desirable and, when R

s

-compensation is in use, it is

important that the resistance be stable as well. A high level of Ca

2+

 buffering

capacity in the pipette solution (e.g., with 10 mM EGTA) helps prevent spontaneous
increases in the access resistance due to partial resealing of the patch membrane.

Capacitive Transient Cancellation

If the fast capacitance cancellation was adjusted (as described above) before breaking
the patch, then all of the additional capacitance transient will be due to the cell
capacitance. Cancelling this transient using the 

C-slow

 and 

R-series

 controls will then

give estimates of the membrane capacitance and the series resistance. The easiest
way of cancellation is provided by the 

Auto C-slow

 function which may be activated

by selecting the 

Auto

 button or the 

Whole Cell

 mode (if included in the macro

function). In cases where the 

C-slow

 transient has a short time constant (100 µs or

smaller) , some improvement of the overall compensation may be achieved by
alternating cycles of 

Auto C-fast

 and 

Auto C-slow

 (if this doesn't work satisfactorily

you may fine-tune the compensation controls manually). After an iteration or two, it
should be possible to reduce the transient to only a few percent of its original
amplitude. However, if the cell has an unfavorable shape (for example, a long
cylindrical cell or one with long processes), the cell capacitance transient will not be a
single exponential, and the cancellation will not be as complete.

Series Resistance Compensation

Series resistance (R

s

) compensation is important when the membrane capacitance is

large or when the ionic currents are large enough to introduce voltage errors. To use
R

s

-compensation, you first adjust the transient-cancellation controls (including 

C-fast

and 

τ

-fast

 if necessary) to provide the best cancellation. Then you select 

R

s

-comp

 by

turning up the 

%-comp

 control to provide the desired degree of compensation.

Note: The “R-series” control determines (along with the “%-comp” control) the amount of
positive feedback being applied for compensation. It should be adjusted with some care, since
too high a setting causes overcompensation (the EPC9 will think that R

s

 is larger than it is);

this can cause oscillation and possible damage to the cell under observation.

How you should set the R

s

-compensation controls depends on the approximate

value of the uncompensated membrane-charging time constant 

τ

u

, which you can

calculate as the product of the 

C-slow

 and 

R-series

 settings (for example, suppose 

C-

slow

 is 20 pF and 

R-series

 is 10 M

Ω

; 

τ

u

 is then 20 pF · 10 M

Ω

 = 200 µs). If 

τ

u

 is smaller

than about 500 µs, you should use the 2 µs setting of the R

s

-compensation switch to

Содержание EPC 9

Страница 1: ...8 52 EPC 9 Manual HEKA Elektronik Dr Schulze GmbH Wiesenstra e 71 D 67466 Lambrecht Germany Tel 49 0 6325 9553 0 Fax 49 0 6325 9553 50 Web Site http www heka com E mail sales heka com support heka com...

Страница 2: ...Triple __________________________________________ 15 3 Installation 16 Installation Procedure ____________________________________________ 16 Calibrating the EPC 9 ____________________________________...

Страница 3: ...e _____________________________________________ 54 Test Mode ____________________________________________________ 57 Search Mode __________________________________________________ 58 7 Compensation Pr...

Страница 4: ...Compensation 78 Current Clamp Recording__________________________________________ 79 11 Low Noise Recording 80 Appendix I Controlling E9SCREEN 83 Communication between E9Screen and other Programs ___...

Страница 5: ...EPC9 patch clamp amplifier has no knobs switches or dials The Pulse software replaces the analog controls of conventional amplifiers by using Macintosh computers and a built in ITC 16 interface The c...

Страница 6: ...nciples o f the EPC9 s functions and patch clamp techniques It is assumed that the reader has some familiarity with patch clamp techniques should you be a newcomer to the field perhaps the best place...

Страница 7: ...r 2 Tight seal whole cell recording In Single Channel Recording B Sakmann E Neher eds Plenum Press New York Heinemann S H 1995 Chapter 3 Guide to data acquisition and analysis In Single Channel Record...

Страница 8: ...will explicitly mention the particular amplifiers where it is required Windows versions The EPC9 is supported on Windows 3 1 Windows 95 Windows 98 Windows NT 3 51 Windows NT 4 0 and Windows 2000 Throu...

Страница 9: ...ws NT 4 0 etc Your type of computer Mac PPC 8500 Pentium II 300 MHz etc Your acquisition hardware if applicable EPC9 ITC 16 ITC 18 Your amplifier if applicable EPC9 EPC9 Double Axon 200B etc The seria...

Страница 10: ...obe or head stage of the EPC9 is contained in a small enclosure designed to be mounted on a micromanipulator and directly attached to the recording micropipette It contains the sensitive amplifier tha...

Страница 11: ...de by digital switches in the main unit including those which depend on the properties of components in the probe The calibration parameters are preset by the manufacturer and contained in the softwar...

Страница 12: ...ies from slew limiting amplifiers in the command processing circuitry and also reduces the amplitude of the current transients from rapid charging of the pipette Two degrees of filtering specified as...

Страница 13: ...easons However channels 0 5 are freely available for application programs For example the Pulse program can use these channels to monitor temperature pressure or outputs from other sensors DA Outputs...

Страница 14: ...e EPC9 Digital I O TTL level digital input and output lines are available here for the control and monitoring of digital signals See pin assignments in Appendix II Technical Data VR10 100 This digital...

Страница 15: ...BUS light The green LED light of the active amplifier flickers when the digital lines are alive during command transmission The notion active amplifier does not imply that the other amplifiers are not...

Страница 16: ...ny probe with any amplifier and replace a broken probe without having to send the amplifier in for recalibration Before starting the calibration make sure that the amplifier has reached its operationa...

Страница 17: ...want to save the new calibration file and generates the proper name for the calibration file It is not advicable to save the file under a different name because in that case you would have to manually...

Страница 18: ...ill be displayed and will instruct you to remove anything from the probe and shield its input Again please make sure that nothing is connected to the probe except for the metallic cap that came with y...

Страница 19: ...e probe input via a BNC adapter and the plug goes to the black Gnd connector on the probe The model cell MC9 The model circuit provides a switch with three positions simulating the following condition...

Страница 20: ...the EPC9 you could use that program instead The figures shown were taken from E9SCREEN Step 1 Applying the Test Pulse First connect the model circuit to the probe input via a BNC adapter and plug the...

Страница 21: ...or V mon 11 and the current monitor I mon 12 should be close to zero You could also do the offset potential cancellation in a more classical way by clicking into the V0 control 9 and dragging the mous...

Страница 22: ...e voltage offsets E SoundOn TRUE beep E SoundOn FALSE Note E9SCREEN has a built in macro interpreter that executes command lines of the form Window Control parameter comment E g the line E Gain 10 wou...

Страница 23: ...citor in the model circuit Activate the C fast compensation by clicking into the C fast field 2 and dragging the mouse upwards While you are approaching a value close to 6 pF you should see the spikes...

Страница 24: ...rding After compensating C fast well you can now switch into the 0 5 GOhm position of the model circuit This will simulate a model cell with 22 pF membrane capacitance 500 M membrane resistance and 5...

Страница 25: ...the Delay field 10 With a delay of 1 ms and a contemporary computer Pentium II 300 MHz this feature allows you to measure the membrane capacitance at a rate of 15 Hz You can output the results of the...

Страница 26: ...th voltage clamp conditions it takes much longer to reach Vmax therefore you should increase the duration of the test pulse to a more appropriate value of 100 ms 4 Note In contrast to voltage clamp co...

Страница 27: ...constant of 11 ms which the amplifier can easily follow If you want to have a better estimation of the amplifier s speed under current clamp conditions you should do the same test as above with the 10...

Страница 28: ...28 The following figure shows a recorded action potential from H Taschenberger H von Germsdorff normal current clamp 250 s 20 mV 0 mV 60 mV Presynaptic Action Potential of the Calyx of Held in the Rat...

Страница 29: ...r 3 Note The three different gain ranges of the EPC9 are separated by lines in the gain popup menu The low gain range goes from 0 005 to 0 2 the medium gain range from 0 5 to 20 and the high gain rang...

Страница 30: ...ead a noise value between 90 and 110 fA 2 Tip If you wish to ground your setup you should now attach the pipette holder to the probe insert a glass pipette bring the pipette tip into the recording pos...

Страница 31: ...p delivered with the EPC9 a 10 M resistor e g the model circuit and 5 short BNC cables Start the E9SCREEN program In case you have an EPC9 Double or Triple select the amplifier you want to test from t...

Страница 32: ...ier DAC 1 DAC 3 not tested not tested ADC 6 If the connection test fails you will get an error message and the chance to repeat the test an additional time After the connection test you will have to r...

Страница 33: ...KA first and supply us the error protocol before sending the amplifier in You might save yourself valuable time and effort Status of EPC9 Amplifier OK Probe NOT ok Connections OK Please print now the...

Страница 34: ...ude of about 1 volt is appropriate Note The probe input usually needs to be shielded from low frequency noise A simple way to do this is to cover the input connector with a cap made from aluminum foil...

Страница 35: ...e Value Last V memb CC Fast Speed Sound Volume Hz M Rmem Sound 100 100 Scope 1 Amplifier CC range 1pA mV Test Pulse Ampl itude 6 0 mV Dur ati on 4 0 ms Imon2 Gain 1 02 x Clear Reset Overlay Close Stim...

Страница 36: ...art macro recording click on the Record button Then perform all desired actions the Notebook window will print a protocol of the macro actions While recording a macro you have the option of actually e...

Страница 37: ...o cancel the output of C slow and R series set by Cap Track Also Reset is very useful to define the initial state of the EPC9 when recording a macro V membrane Displays the actually measured pipette v...

Страница 38: ...high gain range is intended for single channel recording It has a very low noise level but this is obtained at the expense of a maximum current limit of about 200 pA The maximum available bandwidth is...

Страница 39: ...voltage which is added to V membrane to obtain the pipette command voltage It can be set either by the Auto V0 operation or by manually dragging the mouse after clicking into the item Furthermore V0...

Страница 40: ...onal offset corrections LJ should be 0 mV when using identical pipette and bath solutions It may be changed to any desired value within 200 mV in case asymmetrical solutions are used or the bath solut...

Страница 41: ...rrent clamp mode Last V membrane can then be used to conveniently restore the original V membrane value This button is normally hidden behind the oscilloscope window it can be seen when closing the os...

Страница 42: ...citive currents that charge the pipette and other stray capacitances range 0 15 pF With nothing connected to the probe input cancellation is typically obtained at a setting of 1 1 5 pF due to the resi...

Страница 43: ...the option Make C fast If the file created by this procedure CFast epc is found in the default directory E9Screen the table will be used Otherwise when starting E9Screen a message will appear that the...

Страница 44: ...temporarily Rs comp is turned off the stimulation is interrupted and the gain is internally set to an appropriate value 0 2 2 or 50 mV pA depending on the effective gain range If the C slow Range is s...

Страница 45: ...nsation 2 s Very fast compensation The choice of speed depends on the recording time constant and the degree of compensation desired as described in Chapter 7 Compensation Procedures on page 59 Fast R...

Страница 46: ...illoscope display With the oscilloscope closed the controls for sound encoding of R membrane and Current Clamp Speed EPC9 version C and later become accessible There will be no test pulse while the os...

Страница 47: ...0 pA when switching to current clamp mode Last V membrane Quite often the membrane potential under current clamp will have changed and when returning to voltage clamp V membrane will possibly differ...

Страница 48: ...other text file One can add further information to the text file or delete messages that should not be stored While in the Notebook window you can copy to and paste from the clipboard Thus you can exc...

Страница 49: ...content All entries are normally disabled unless the Notebook is selected The menu entries conform to the standard Macintosh functions The Cut Copy and Paste commands copy the text selection to and f...

Страница 50: ...recording Execute while recording If checked the actions will be executed Otherwise the actions will be logged to the macro but not change any settings EPC9 Help Enters the online Help mode for EPC9...

Страница 51: ...ude The amplitude of the voltage pulses used to compute C slow and G series C slow Cycles The number of voltage pulses used to compute C slow and G series Update R membrane Delay This option sets the...

Страница 52: ...parentheses Note Large notebook buffers require a lot of CPU time and RAM If execution time during acquisition is an issue the buffer size should be kept small or the Buffered Output should be turned...

Страница 53: ...oaded automatically if it has the name CFAST EPC and is found in the E9Screen folder Full Test Performs a hardware test This generates a list of diagnostic values This may be used to inform the manufa...

Страница 54: ...d from the signal applied to Stim In with a variable offset added from the V membrane control The sum of these two sources is displayed and monitored as the V mon signal Before being applied to the pi...

Страница 55: ...the Pulse Generator or from the I membrane control that would give rise to 1 mV in the Voltage Clamp mode instead gives rise to 1 pA in the Current Clamp mode This relationship of 1 and 10 pA mV holds...

Страница 56: ...and potentially the destruction of the cell membrane The best way to use the C fast control is to first adjust it in the voltage clamp mode e g by using the Auto button it is then automatically adjust...

Страница 57: ...30 ms in duration and sine waves at 1 Hz are reproduced faithfully Note The test integrator inverts the externally supplied signal For accurate frequency response and to avoid degrading the sensitivi...

Страница 58: ...er to ease the adjustment of the zero current potential V0 The EPC9 does not require special analog circuitry as the software provides for an easy way of doing this The Search mode of the EPC9 simply...

Страница 59: ...step is applied to the pipette electrode The time constant of charging is given by u Rs x Cm where Cm is the membrane capacitance For typical values of Rs 5 M and Cm 20 pF the time constant is 100 s T...

Страница 60: ...ld Corr Loop Pred Loop Together the two parts of the EPC9 Rs compensation circuitry cancel the effects of a fraction of the series resistance This means that the charging of the membrane capacitance i...

Страница 61: ...nly up to about 120 mV in amplitude Overload of amplifiers obvious in practical use due to the loss of proper transient cancel lation will occur if larger pulses are applied unless the comp setting is...

Страница 62: ...ieving an accurate update of R series The procedure will be described in more detail later Chapter 10 Using the Patch Clamp on page 75 Capacitance Compensation The EPC9 provides automatic procedures f...

Страница 63: ...arameters on the screen may not represent the hardware settings The relationship between the values of the compensation network C slow and R series and those of the pipette cell assembly Cm and Rs is...

Страница 64: ...evant Recording Mode which may or may not result in inversion of holding and commanded voltages In addition it offers three features which interact in a way to provide for simple online handling of of...

Страница 65: ...line main salt 140 mM NaCl is listed Polarity is that of physiological saline with respect to the given solution according to the convention of Barry Lynch Note When applying the above rules for calcu...

Страница 66: ...um will be the sum of this liquid junction potential and the correction of Example 2 3 mV Taking the value for a low Cl solution e g sulfate Ringer see table we arrive at a value of LJ 3 6 3 mV which...

Страница 67: ...which can easily arise from a ground loop can result in significant 50 or 60 Hz noise A high quality ground is available at the Gnd terminal of the probe this is internally connected through the probe...

Страница 68: ...help Connections to other Instruments Depending on the needs of the particular user the EPC9 software can substitute to a great extent for many analog instruments e g variable filter oscilloscope puls...

Страница 69: ...ed in more detail later Chapter 10 Using the Patch Clamp on page 75 The pipette electrode is simply a thin silver wire that is soldered onto the pin that plugs into the probe s BNC connector The chlor...

Страница 70: ...rode proper is a chlorided 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...

Страница 71: ...lasses borosilicate aluminosilicate Some sources of glass pipettes Soft Glass Supplier OD Non heparinized hematocrit tubing any scientific supplier 1 3 mm Drummond Microcaps Drummond Scientific Blooma...

Страница 72: ...m region and the second to pull the two halves apart leaving clean symmetrical breaks Both halves can be used The length of the first pull and the heat of the second pull are the main determinants of...

Страница 73: ...even if stored in a covered container small dust particles from the air stick readily to the glass and can prevent sealing However with some easy sealing cells we have made the experience that pipett...

Страница 74: ...C9 Manual 74 check the noise level of the empty holder using the Noise Test function if it increases solution has probably spilled inside the holder in this case the holder should be cleaned again and...

Страница 75: ...t Up mode Voltage Clamp and V membrane 0 Filter 1 is set to 10 kHz and Filter 2 to 3 kHz Rs comp to Off C slow Range to Off and Stimulus Rise Time to 20 s Before the pipette is inserted into the bath...

Страница 76: ...he Gain to perhaps 50 mV pA the trace should still appear essentially flat except for capacitive spikes at the start and end of the voltage pulse Cell Attached Recording You can now use the On Cell mo...

Страница 77: ...sients also reflects dielectric relaxation in the plastic of the pipette holder and in the pipette glass These relaxations are not simple exponentials but occur on time scales of about 1 ms If you use...

Страница 78: ...n controls manually After an iteration or two it should be possible to reduce the transient to only a few percent of its original amplitude However if the cell has an unfavorable shape for example a l...

Страница 79: ...clamp to current clamp recording just select the Current Clamp mode This provides a recording of the cell membrane potential which can be monitored at the V mon output of the EPC9 When you switch to...

Страница 80: ...ontrol is 50 mV pA With the probe placed in a shielded enclosure and with nothing connected to the input the Noise reading is usually 80 100 fA If you get a reading higher than this try varying the C...

Страница 81: ...e pipette holder be kept clean and dry Noise can be coupled into the pipette from the thermal motion of ions in films of aqueous solution especially on the inside of the pipette A good practice for lo...

Страница 82: ...Judicious use of filtering can improve the time resolution of your analysis For example if you are using the 50 threshold crossing analysis technique to analyze channel open and closed times the best...

Страница 83: ...the option Enable Background in the EPC9 drop down menu is de selected E9Screen then resumes acquisition when the user switches back and the AD DA board driver is not reserved by another process Thus...

Страница 84: ...meaning of the parameters in the following lines are The first number is the index of the described AD channel The letter in the square brackets gives the logical unit of that channel i e V for the vo...

Страница 85: ...r a network even one with different platforms such as Windows Macintosh Unix OS 2 or workstations Thus it is now possible to control the EPC9 from computers running a multitasking operating system whi...

Страница 86: ...provided that the command index is larger than zero E9Screen writes the command index and eventually any error and requested answer to the response file Next the user switches back to the user program...

Страница 87: ...n unknown error an unidentified error occurred Implemented Commands and Messages Messages send by E9Screen They do not contain a response string Started E9Screen started communication link Terminated...

Страница 88: ...lean 0 or 1 SetCFast2 real capacitance in Farad SetCFastTau real tau in seconds SetCSlow real capacitance in Farad SetCSlowRange integer 0 1 2 3 off 30 100 1000pF range SetCurrentGain set gain in Ohms...

Страница 89: ...d reserve the AD board string ADReserved otherwise string ADReleased DAWrite syntax DAWrite AD channel Volts DA channel integer 0 to 3 Volts real 10 24 volt no response ExecMacro syntax ExecMacro Macr...

Страница 90: ...eypad 13 6 on numeric keypad 14 7 on numeric keypad 15 8 on numeric keypad 16 9 on numeric keypad 17 Enter 18 minus on numeric keypad 19 plus on numeric keypad 20 period on numeric keypad 21 clear on...

Страница 91: ...irst response E9Screen writes to the message file is Started Thereafter the user can proceed to send commands to E9Screen and read back the response It is advisable to write to the command file in an...

Страница 92: ...n 92 6 E9Screen writes responses to the message files in the following situations On starting batch processing it messages Started Responding to a Get Query or Terminate instruction When an error occu...

Страница 93: ...gnature INTEGER PROCEDURE WriteError Text ARRAY OF CHAR VAR Work ARRAY 0 79 OF CHAR BEGIN IOFiles Message IOFiles GetError Work TermIO WriteString Text TermIO WriteString failed TermIO WriteLine Work...

Страница 94: ...n the first line with a plus i e thus changing the negative signature to positive See above for the explanation IF Signature 0 THEN Work 0 Count 1 IF NOT IOBytes SetPosition CommandFile IOFiles FromSt...

Страница 95: ...NOT CommandFile IsOpen THEN RETURN END IF IF NOT MessageFile IsOpen AND NOT OpenMessageFile THEN RETURN END IF Read the first text line If reading fails or converting to a number fails or the resultin...

Страница 96: ...IF LastSignature NewSignature IF NewSignature Signature OR NewSignature 0 THEN Signature NewSignature 1 Dummy WriteToCommandFile acknowledged END IF END PollForCommands PROCEDURE Startup BOOLEAN VAR...

Страница 97: ...file RETURN FALSE END IF Signature 1 RETURN TRUE END Startup PROCEDURE InitModule BEGIN CommandFile IsOpen FALSE MessageFile IsOpen FALSE Signature 1 LastSignature 1 END InitModule PROCEDURE Shutdown...

Страница 98: ...cted 4 OUT 15 5 IN 2 6 IN 10 7 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 CLIPPING 19 GND 20 GND 21 GND 22 GND 23 Not connected 24 STROBE out 25 OUT 0 26...

Страница 99: ...4 TRIGGER 3 5 TRIGGER 4 6 TRIGGER 5 7 TRIGGER 6 8 TRIGGER 7 9 10 11 12 13 14 15 16 17 Ground IN 18 Power IN 19 Ground IN 20 Power IN 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 The TRI...

Страница 100: ...er supply voltage should not exceed 24V Arrangement for current driven devices Arrangement to generate TTL signals Valve OPTO TRIGGER OUT Power IN EPC 9 Double External 5 24V Power supply Ground IN Gr...

Страница 101: ...ing 18 CSlow 24 CSlow Range 24 Current Clamp Recording 26 Delay 25 E9Screen 16 EPC9 Testing 19 Fast CC Mode 26 Full Test 31 Hotline 9 I mon display 21 Macros ON CELL 24 SET UP 21 WHOLE CELL 25 Make CF...

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