Home

EPC 800 Manual - HEKA Elektronik Dr. Schulze GmbH

1. Call P RE 328GQ e wow o2pa ter Pulse mV p Holaing tmv 0 2 T Averaging Figure 7 6 Automatic or manual C Fast compensation The left panel shows the capacitive spikes resulting from the test pulse and the right side illustrates the effects of an automatic C Fast compensation The amplifier Gain was set to 20 mV pA Note Practical Tips e When the pipette is pushed against a cell the current http www heka com 60 Using the EPC 800 Patch Clamp Amplifier with pCLAMP pulses will become slightly smaller to reflect the increas ing seal resistance when the positive pressure is released the resistance usually increases further Some cell types require more push from the pipette than others but an increase in resistance of 1 5 i e a reduction in the cur rent pulses by this factor is typical e Application of gentle suction should increase the re sistance further and result sometimes gradually over maybe 30 s sometimes suddenly in the formation of a gigaseal which is characterized by the current trace be coming essentially flat again hyperpolarizing the pipette to 40 to 60 mV often helps to speed the seal formation To verify gigaseal formation increase the GAIN to per haps 50 mV pA the trace should still appear essentially flat except for capacitive spikes at the start and end of the voltage pulse e Transien
2. http www heka com 12 5 Technical Data CC Bridge Mode Holding current 1000 pA Low Gain Range Available when switching from VC mode in low gain range 0 005 mV pA 0 2 mV pA IMAX 100nA CC Stim Scaling 10 pA mV InoLrp 50 nA in Local Mode InoLp 100 nA in Remote Mode Medium Gain Available when switching from VC mode in medium gain Range range 0 5 mV pA 20 mV pA Imax 1nA CC Stim Scaling 0 1 pA mV InoLrp 500 pA in Local Mode InoLp 1 nA in Remote Mode Low Frequency Automatic current tracking readjusts the membrane po Voltage Clamp tential to compensate for any slow voltage drift while in LFVC CC mode Range 200 mV 7 of 1 3 10 30 or 100 ps Power Requirements Power Supply Power requirements are 125 Watts Power supply automatically switches the voltage range Operational range is from 90 130 V or 210 250 V at line frequencies of 50 or 60 Hz Ground Lines Signal ground GND is isolated from the chassis by a 10 Q resistor to avoid ground loops It is accessible via a Banana plug on the front panel and also via a connector on the headstage A chassis ground CHAS is accessible via a Banana plug on the front panel and is connected to the ground line of the power cord Dimensions Head Stage DxWxH 90x17x14 5 mm 3 54x0 67x0 57 in Controller DxWxH_ 31 1x48 3x14 5 cm 12 3x19x5 7 in
3. 136 LIST OF FIGURES 7 4 Model circuit schematic 0 56 7 5 Before and after VPorrser in Clampex 58 7 6 Before and after C Fast compensation in Clampex 59 7 7 Before and after C Slow compensation in Clampex 63 7 8 Example voltage clamp protocol in Clampex 66 7 9 Waveform preview of voltage clamp protocol in Clampex 67 7 10 Acquired voltage and current traces of voltage clamp pro tocol in Clampex 2 s s ane See ee Pe oe EO 68 7 11 Example current clamp protocol in Clampex 69 7 12 Waveform preview of current clamp protocol in Clampex 70 7 13 Acquired voltage and current traces of current clamp pro tocol in Clampex 0 2 00002 eee 71 7 14 Configuring telegraph signals in Clampex 73 7 15 Display of telegraph values in Clampex 75 7 16 EPCMaster soft panel in combination with Clampex 77 8 1 Hardware default settings within PatchMaster 81 8 2 Hardware configuration settings within PatchMaster 82 8 3 PatchMaster amplifier window for remote mode of operation 85 8 4 Amplifier gain settings within PatchMaster 86 8 5 I mon V mon and R membrane values within PatchMaster 87 8 6 Loading the EPC800 pro file within PatchMaster 88 8 7 The protocol editor window of PatchMaster showing prede fined protocols as part of the EPC800 pro file 89 8 8 SET UP SEAL and WHOLE CELL protocols 89 8 9 Sett
4. We will explicitly mention the particular operating system versions whenever it is required Apple The EPC 800 Patch Clamp Amplifier is supported by Apple computers running Mac OS X 10 4 or newer An available USB 2 0 port is required for Remote Mode only Throughout the manual we will address all the above Mac OS X versions as Mac OS We will explicitly mention the particular operating system versions whenever it is required 2 5 Support Hotline If you have any questions suggestions or improvements please con tact HEKA s support team The best way is to send an e mail to sup port heka com specifying as much information as possible e Your contact information e The program name e g PATCHMASTER Software e The program version number e g v2 42 e Your operating system and its version e g Mac OS 10 4 Windows XP Pro 32 bit http www heka com 10 Introduction e Your type of computer e g Mac G5 Core i5 2 67 GHz with 3 GB of RAM e Your acquisition hardware if applicable e g EPC 800 Patch Clamp Amplifier ITC 18 Interface e The serial number and version of your EPC 800 Patch Clamp Am plifier e The questions problems or suggestions you have e Under which conditions and how often the problem occurs Please contact the office located closest to you from the listing below We will address the problem as soon as possible Europe HEKA Elektronik GmbH Wiesenstrasse 71 D 67466
5. Table 12 4 Cslow telegraphing values deviation 0 3V http www heka com 128 Appendix Filter in Hz Telegraphing Output in V 100 300 500 700 1K 3K 5K 7K 10K 30K 100K OS S CO NI OD CY BY Co DO FE Table 12 5 Filter1 telegraphing values deviation 0 3V Mode Telegraphing Output in V VClamp CClamp LFVC 100 LFVC 30 LFVC 10 LFVC 3 LFVC 1 NI OD OU eA SN e Table 12 6 Mode telegraphing values deviation 0 3V http www heka com 12 5 Technical Data 129 12 5 Technical Data Head Stage Current measuring resistors High range 50 GQ Medium range 500 MQ Low range 5 MQ Largest measurable currents 200 pA 50 GQ range 20 nA 500 MQ range 2 pA 5 MQ range Input connector Standard BNC Other connections Ground sense input Noise measured with open input 8 pole DC to 1 kHz lt 0 03 pA RMS Bessel filter high gain range DC to 3 kHz lt 0 08 pA RMS DC to 10 kHz lt 0 225 pA RMS Current Monitor Signal Gain 0 005 2000mV pA Bandwidth 100 kHz med and low range 60 kHz high range Filters Filter 1 is a 5 pole 10 to 100 kHz Bessel pre filter Filter 2 is a 4 pole tunable 20 kHz Bessel filter Filter range is controlled by software or from the front panel switch Current Monitor signals are the sum of Filter 1
6. 30 o 70 Delta level mV 0 a i First duration ms 125 200 1000 Delta duration ms 0 0 Digital bit pattem 3 0 Digital bit pattern 7 4 Train rate Hz 3j Pulse width ms i Number of sweeps 500 Allocated time kz 1987 5 of 12000 ms Summary Channel 0 Channel 1 Channel 2 Channei 3 7 Alternate Waveforms Acquisition made Cancel Help Episodic stimulation Unde Frevieit Figure 7 8 Example of how to write a typical whole cell voltage clamp protocol This example is of a voltage ramp from 30 mV to 70 mV http www heka com 7 1 Local Mode 67 i Waveform Preview v T 1 Time s Sweep 1 Visible 1 of 1 gt gt Clipboard Help Figure 7 9 Waveform Preview of the voltage clamp protocol designed in the previous figure In the oscilloscope window figure below the current and voltage traces are displayed These two display signals are selected within the Inputs tab of the Edit Protocol dialog and have already been configured within the Lab Bench dialog where the I monitor was assigned to Analog IN 0 and the V monitor was assigned to Analog IN 1 The output signal was also pre configured in Lab Bench with the Analog Out 0 Digitizer channel being assigned to the V command signal The V command also has to be selected in the Outputs tab of the proto
7. 500 pA 7 1 There will invariably be a small offset potential between the pipette and the bath electrodes Pipette offsets up to 200mV can be compensated manually by turning the VPorrser potentiometer or automatically by pushing the black Auto VP button The values will be displayed on the amplifier LCD display and the effects are observed in the Clampex oscil loscope window see right side of figure below http www heka com 58 Using the EPC 800 Patch Clamp Amplifier with pCLAMP 4 Sr Ee AAAA Ra AAA Figure 7 5 Automatic or manual VPorrger The left panel shows the test pulse before the offset correction and the right side illustrates the effects of an automatic VPorrser correction Note Practical Tips e Before the pipette is inserted into the bath the current trace should be flat except for very small capacitive pulses due to the stray capacitance of the pipette and holder e If there should be no change in the trace upon entering the bath check for an open circuit for example 1 a bubble in the pipette 2 faulty connection to the probe input 3 bath electrode not connected e The surface of the solution is relatively dirty even if as we strongly recommend you aspirate some solution from the surface to suck off dust and contaminants For this reason it is important to apply a small amount of positive pressure to the pipette before you move its t
8. to be manually set in other acquisition programs ex within Lab bench of pCLAMP Current Monitor A BNC cable should be used from this connection to an assigned Analog Input channel of your AD DA interface The EPC 800 USB has one current monitor output with the current signal filtered according to two internal filters Filter 1 is a 5 pole 10 to 100 kHz Bessel pre filter and Filter 2 is a 4 pole 20 kHz tunable Bessel filter Additional information on the relationship between the internal filters and the setting of the filter knob on the front panel can be found in 4 2 2 The current monitor output signal can be viewed on the PATCHMASTER software os cilloscope screen or within other acquisition programs for monitoring the progress of the experiment Positive voltages correspond to currents flow ing out of the pipette The specifics of the control of the filter ranges by the front panel switch will be discussed in the next subsection of this chapter Clipping This LED lights whenever the amplifier saturates in the current monitor pathway The indicator is important in voltage clamp experiments where capacitive artifacts will be subtracted in the host computer the subtraction will work well only as long as no saturation occurs and this indicator serves as a simple monitor of this condition It is particularly useful since it will indicate clipping by internal amplifiers even in cases where because of filtering the output voltage is no
9. 1 pS Figure 7 14 Configuring telegraphs in Clampex The EPC 800 Patch Clamp Amplifier is selected as the Telegraphed Instrument and the Tele graph Connections are chosen to match the physical BNC connections between the amplifier and the Digidata 1440A The third step is to correctly load the appropriate conversion charts for the amplifier gain and frequency and the conversion factor for telegraphed Cm values Instructions on how to write these files can be found in the defaul tuserdefinedinstruments ini file which is located in the Molecular Devices pCLAMP 10 2 folder that was created when the software was installed The correct table for the EPC 800 Patch Clamp Amplifier has already been written and is provided below This table should be copied and saved in the userdefinedinstruments file The appropriate table for the EPC 800 Patch Clamp Amplifier is Once all of the appropriate cable connections have been made and the software is configured properly to accept the amplifier as a telegraphed instrument then the system is ready to be used with the Digidata 1440A and Clampex The amplifier can be used in the same way as previously discussed in the tutorial steps for local mode A very simple test to see if the telegraphing outputs are being read cor rectly is to manually turn the Gain and Filter knobs on the front panel http www heka com 74 Using the EPC 800 Patch Clamp Amplifier with pCLAMP Instrument0 Nam
10. 111 114 Coating 113 Glass Capillaries 111 Polishing 113 Pulling 112 Usage 114 Pipette Holder 109 Pipette Offset 94 Power Switch 31 Practical Tips 58 61 63 65 Probe 17 18 20 107 Adapter Plates 18 Gnd Connector 18 Input Connector 17 R memb 87 R memb R pip 101 Recording Modes 35 39 Current Clamp 36 Low Frequency Voltage Clamp 39 Voltage Clamp 35 References 7 8 Book Chapters 8 Further Reading 7 Original Articles 7 Remote LED 31 Remote Mode 5 83 Safety Guidelines 1 2 Seal Mode 30 Series Resistance Compensation 29 64 98 Show All Controls 100 SIGNAL GND 20 Sound 33 99 101 Static Electricity 15 Stimulus Filter 98 Support Hotline 9 Telegraphing Outputs 32 71 Test pulse 93 Unpacking and Installation 13 15 USB connector 32 Using the EPC 800 Patch Clamp Amplifier with Patch Master 106 Amplifier Window 84 102 Current Clamp Recording 102 Bridge Compensation 104 Voltage Bandwidth 105 Local Mode 83 PatchMaster Protocols 89 Recording Modes 92 Remote Mode 83 Software Configuration 80 Software Installation 79 Using the EPC 800 Patch Clamp Amplifier with pCLAMP 51 77 Configuring Lab Bench 53 Inputs 53 Outputs 53 http www heka com 134 INDEX Hardware Connections 52 Front Panel 52 Local Mode 51 Remote Control 77 Software Installation 52 Telegraphing Configuration 72 Telegraphing Connections 71
11. 19 Setting sound features within PatchMaster SOUND Settings Sensitivity HzMQ and volume in of the sound encoding of R membrane can be specified here To enable the sound op tion press the Sound button The sound function also has to be correctly configured in the PATCHMASTER Hardware Configuration section I Scale and V Scale Scale 1 00 0 00 V Scale Figure 8 20 I Scale and V Scale settings of the test pulse within Patch Master http www heka com Using the EPC 800 USB patch clamp amplifier with 102 PatchMaster I Scale and V Scale can be used to determine the display scaling for the test pulse The value of 1 no display gain corresponds to full scale 10 24V of the built in AD DA converter Thus without display gain one can easily see when the input signal saturates the AD converter If however amplification is needed you should enable the setting Scale Test Pulse in the Misc section of the Configuration window Range Range AD 0 10 v AD 0 v t 10 AD 1 5V AD 2 2 1 Figure 8 21 Hardware scaling of the ITC 18 interface This feature is only present if the EPC 800 Patch Clamp Amplifier is used in conjunction with the InstruTECH HEKA ITC 18 interface It enables the use of the hardware scaling of the ITC 18 For example it is possible to change the measuring range of any of the AD inputs from 10V to 1V 8 4 3 Current Clamp Recording In Cur
12. 9 4 External Shielding 9 5 Pipette Holder and Electrode 9 6 Bath Electrode 10 Patch Pipettes 10 1 Glass Capillaries TOZ PUUE og 0 Pp oe ageing ae aaa Pa L033 Coating i on dae acne BSG alan Hah 10 4 Heat Polishing 0 10 5 Use of Pipettes 0 11 Low Noise Recording 11 1 Measuring the Noise of the Amplifier 11 2 Noise of the Recording Set Up 12 Appendix 12 1 Supported States 12 2 USB Descriptor 12 3 List of EPC 800 USB Commands 12 4 Telegraphing Translation 12 5 Technical Data 0 0 107 111 121 http www heka com CONTENTS v Index 135 List of Figures 137 List of Tables 140 http www heka com 1 Safety Guidelines Please read the instruction manual of the EPC 800 Patch Clamp Ampli fier before putting the amplifier into operation to prevent any possible damage to life and equipment In addition to the instruction manual of the EPC 800 Patch Clamp Amplifier the regulations of prevention of ac cidents applicable to your country VBG 4 in Germany and the relevant rules for safety of the working environment are applicable The instruction manual has been designed such that putting the EPC 800 patch clamp amplifier into operation is comprehensible safe economical and helps to prevent dangerous misuse A safe use of the amplif
13. Mac Please install either the USB dongle Windows or HASP dongle Mac drivers from the downloads gt Dongles section of the HEKA website BEFORE con necting the USB dongle After successfully installing the driver the USB dongle can be connected Note Windows does not allow you to install a driver if you do not have administrative rights Please ensure to login as Administrator before performing any driver installation 8 2 Software Startup and Configuration Upon starting PATCHMASTER will be prompted to set the correct default settings of amplifier type and interface used http www heka com 8 2 Software Startup and Configuration 81 Select Amplifier or AD DA board to use EPC 10 Plus EPC 10 USB OEPC 10 OEPC9 EPC 800 remote mode ITC 16 EPC 800 local mode ITC 18 EPC 8 remote mode LIH 1600 EPC 8 local mode LIH 8 8 EPC 7 and others PCl slot 1 O Activate AD DA board Figure 8 1 Selecting the amplifier as part of the default settings When using PATCHMASTER the amplifier can be run in either local or remote modes Note When first starting PATCHMASTER the mode switch on the front panel of the amplifier has to be set to VC mode If an alternative mode is selected PATCHMASTER will prompt the user to switch to VC mode Once PatchMaster is started the BNC cable connections between the front panel of the amplifier and the AD DA interface must m
14. Tutorial 55 71 Using V Porrsert 51 Gigaseal Formation 59 Whole Cell Configura tion 61 65 Whole Cell Current Clamp 68 71 Whole Cell Voltage Clamp 66 68 the EPC 800 Patch Clamp Amplifier with pCLAMP Remote Control 75 Using the EPC 800 USB patch clamp amplifier with PatchMaster 79 V membrane 86 V mon 87 Voltage Monitor 21 Zap 99 http www heka com List of Figures 2 1 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 4 10 4 11 4 12 5 1 5 2 6 1 7 1 T 2 7 3 EPC 800 USB Patch Clamp Amplifier 3 EPC 800 patch clamp amplifier probe 17 Headstage standard plate ooa a 19 Headstage dovetail plate 2 2 a 19 EPC 800 patch clamp amplifier main unit 19 Bottom Row of Amplifier 20 Gait knope sasa amp dena be Re ee OBB oe Ae x 22 Mode knob stut dird Se ele an ee So a amp Gee AUR ee 23 Filter knob ry 24 gout ee a ed Pe 24 Command potentiometers 2 25 Compensation potentiometers 27 Multi parameter display 220 4 31 Telegraphing outputs sosoo e 32 Voltage clamp mode schematic 0 36 Current clamp mode schematic 37 Series resistance compensation schematic 46 Configuring Lab Bench inputs 53 Configuring Lab Bench outputs for voltage clamp 54 Configuring Lab Bench outputs for current clamp 55
15. and the resulting change in membrane potential caused by the applied current is measured The measured membrane potential will be shown on the LCD panel as Vmon and the signal is available at the Voltage monitor output at the front panel of the EPC 800 Patch Clamp Amplifier For stimulation a command current can be injected while the pipette potential is measured The command current is determined by the sum of the voltages from the External Input CC and the Iyozp control In Current Clamp mode the input of the headstage acts as an high impedance voltage follower circuit see figure The feedback resistor is used for stimulation in Current Clamp mode This can be done by apply ing a defined voltage to the feedback resistor http www heka com 5 2 Current Clamp Mode 37 O CC Stim In O Voltage Monitor Current Monitor Bath Electrode Figure 5 2 Current Clamp Mode The current clamp mode of the EPC 800 USB is called CC Bridge Bridge compensation in current clamp mode acts in a similar way as Rs compen sation does in voltage clamp mode It can be thought of as an enhanced current clamp mode that compensates the voltage drop via the series ac cess resistance of the electrode Rs In this mode the stimulus artifact that is typically generated when injecting current is fully eliminated The current clamp circuitry of the EPC 800 Patch Clamp Amplifier acts as a voltage follower thereby increasing
16. below As we mentioned in chapter 9 General Patch Clamp Setup Practices for low noise the pipette holder must be made from a low loss hydrophobic plastic polycarbonate is one of the best and plexiglas one of the worst materials For our purposes low loss materials are those that show lit tle dielectric relaxation in the frequency range of a few kHz Dielectric relaxation involves the reorientation of dipoles within the material since any dipoles will be in thermal motion thermal reorientation in this fre quency range will result in current fluctuations coupled capacitively into the pipette It is very important that the 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 low noise work is to connect a valve to the pipette suction line and arrange for dry air or nitrogen to flow into the suction line during the time while you change pipettes This will dry out any such aqueous films and keep the noise level low Films of aqueous solutions and dielectric relaxation are also serious prob lems with pipette glass Coating with Sylgard helps because it is hydropho bic and because it has good dielectric properties Also its thickness helps to reduce the capacitance between the pipette interior and the bath This is mainly important because it reduces the coupling of the glass s dielectric
17. com 138 LIST OF FIGURES http www heka com List of Tables 4 1 4 2 5 1 6 1 6 2 7 1 7 2 7 4 8 1 10 1 10 2 12 2 12 3 Gain ranges of the EPC 800 Patch Clamp Amplifier 22 Filter settings of the current monitor 25 Features of current clamp gain ranges of the EPC 800 Patch Clamp Amplifier 2 220004 39 Typical LJ values for different solutions 42 Relationship between R comp settings and membrane bIMe CONSTANTS w nare ie ee Ee 8 a we ee a eee 49 Front panel BNC connections between the EPC 800 Patch Clamp Amplifier and a Digidata interface 52 Telegraphing BNC connections between the EPC 800 Patch Clamp Amplifier and a Digidata 1440A 72 Conversion chart for EPC 800 Patch Clamp Amplifier gain frequency and telegraphed Cm values 74 Front panel BNC connections between the EPC 800 Patch Clamp Amplifier and a HEKA InstruTECH interface 82 Soft glass pipette sources 000 4 111 Hard glass pipette sources o oo 112 Noise sources and relative contributions 117 USB Command handling 00 126 Gain telegraphing values deviation 0 1V 127 140 LIST OF TABLES 12 4 Cslow telegraphing values deviation 0 3V 127 12 5 Filterl telegraphing values deviation t0 3V 128 12 6 Mode telegraphing values deviation 0 3V
18. equipped on the rear panel with telegraphing outputs for Gain Filter Bandwidth Amplifier Mode and C Slow To take advantage of these telegraphing output capabilities the amplifier has to be used with one of the Axon s Digidata series of inter faces that are equipped with telegraphing inputs The Digidata 1440A for example can receive telegraphing inputs for variable gain lowpass fil ter and whole cell capacitance compensation C Slow The telegraphing Mode output of the amplifier will not be used in this example due to a limitation of the software When operated in this mode the amplifier remains a manually controlled instrument with active front panel knobs switches and potentiometers The only difference between this mode and the local mode is that Clampex http www heka com 72 Using the EPC 800 Patch Clamp Amplifier with pCLAMP Telegraphing Output of Telegraphing Input of the EPC 800 USB Digidata 1440A Gain 0 Bandwidth 1 C Slow 2 Table 7 2 BNC connections from the telegraphing outputs of the EPC 800 Patch Clamp Amplifier to the telegraphing inputs of a Digidata 1440A will be able to receive telegraphed values and there are additional BNC cable connections to be made between the amplifier and Digidata and additional configurations to be made within Clampex The following BNC connections should be made from the telegraphing outputs on the rear panel of the EPC 800 Patch Cla
19. example it is a good idea to check the voltage trace on the oscilloscope screen to ensure that the com manded potentials match the recorded potentials If there is a discrepancy than in all likelihood there is a command voltage input inadvertently set http www heka com 7 3 Remote Control through Soft Panel TT GB Membrane Test EEJ FE EpcMaster DEK EE EPC800 Remote b n a pieu V membrane l membrane 2e E Cm I G LFVC V memb pm Wb Ra dW Tau Wt E Hold j z Holding mV o al Averaging nE R series 5 6MOhm Relative Cm f i eee y Rscomp __ot JC o gt Lab Book sic The positive and negative responses are unequal Consider changing the holding level stim Fiter Skz Figure 7 16 Remote control of the EPC 800 Patch Clamp Amplifier with EPCMaster in combination with Clampex In this example EPCMaster was used to set the holding potential Gain Filter and perform Auto V Porrsegr Auto C Fast and Auto C Slow compensations http www heka com 78 Using the EPC 800 Patch Clamp Amplifier with pCLAMP http www heka com 8 Using the EPC 800 USB patch clamp amplifier with PatchMaster The EPC 800 Patch Clamp Amplifier was designed primarily as a versatile stand alone amplifier that can easily be used with any AD DA interface and compatible acquisition software This manual has alread
20. form a smooth tip on the pipette to allow seals to be formed without damaging the cell membrane and to coat the pipette with a suitable insulating coating to reduce the background noise Pipettes can be made from many different types of glass Our impression is that different types of glass work better on different cell types Glass capillaries are available from soft soda glass flint glass or hard glasses borosilicate aluminosilicate Some sources of glass pipettes Soft Glass Supplier OD Non heparinized hematocrit tubing 1 3 mm any scientific supplier Drummond Microcaps 1 4 mm Drummond Scientific Bloomall PA U S A Table 10 1 Soft glass pipette sources 112 Patch Pipettes Hard Glass Supplier OD Kimax 51 1 7 mm Kimble Products Vineland NJ U S A Boralec 100 1 7 mm Rochester Scientific Rochester NY U S A Corning Sealing Glass 7052 7040 1 6 mm Dow Corning Midland MI U S A GCASS 150 4 aluminum glass 1 5 mm A M Systems Everett WA U S A Table 10 2 Hard glass pipette sources Soft glass pipettes have a lower melting point 800 C vs 1200 C are easily polished and can be pulled to have a resistance of 1 2 MQ They are often used for whole cell recording where series resistance rather than noise is the limiting criterion The large dielectric relaxation in soft glass sometimes results in additional capacitive transient components that inte
21. general when a voltage step of size AV is commanded the pipette potential actually receives an initial transient of size AV 1 a due to the compensation effect The technical prob lem comes from the fact that the maximum pipette potential excursion in the EPC 800 USB is about 1 4 V implying that 90 compensation can be used for steps only up to about 120 mV in amplitude Overload of amplifiers obvious in practical use due to the loss of proper transient cancelation will occur if larger pulses are applied unless the COMP setting is reduced http www heka com 48 Theory of Compensation Procedures The degree of R compensation is also limited by stability considerations Stable R compensation requires that the C Fast control is properly set to cancel the fast capacitance transients when the series resistance is high say above 10 MQ misadjusting of C Fast can easily cause oscillation In cases where R is this size or larger it is often advisable to use the slower settings of the R switch which in slowing down the speed of the compen sation feedback makes it less susceptible to high frequency oscillations In cases where R is relatively small on the other hand it is sometimes not possible to use full 90 compensation because of the limited speed of the compensation feedback even in the fastest 2 us setting of the switch This problem arises when the time constant 7 is smaller than about 100 ps and comes from the f
22. http www heka com
23. its original amplitude However if the cell has an unfavorable shape for example a long cylindrical cell or one with long processes the cell capac itance transient will not be a single exponential and the cancelation will not be as complete e If you are a novice to patch clamping it is useful to per form the C Fast and C Slow compensation at least a cou ple of times manually before getting used too much to the convenience of the automatic routines Doing so you will get a better feeling for the quality of a recording and how it is affected by the various parameters especially the input resistance R series Series Resistance Compensation Series resistance R compensation is important when the membrane ca pacitance is large or when the ionic currents are large enough to introduce voltage errors To use R compensation you first adjust the transient cancelation controls including C Fast and 7 Fast if necessary to provide the best cancelation Then you turn on R COMP by selecting the desired speed and turning up the COMP control to provide the desired degree of compensation expressed as a Note The R series control determines along with the http www heka com 7 1 Local Mode 65 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 EPC 800 Patch Clamp Amplifier will think that R i
24. of the EPC 800 Patch Clamp Amplifier The asterisk symbol in the LCD dis play is indicative of the results being obtained through an auto matic procedure The Auto C Fast in this situation can NOT be disabled by pressing and holding the Auto C Fast button on the EPC 800 Patch Clamp Amplifier It has to be turned off through PATCHMASTER by changing either the C Fast or T Fast values C Slow This is used to cancel slow capacitive currents that charge the cell membrane in the whole cell configuration 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 is also limited by the program in order to make the time constant R series C Slow greater than 5 us to prevent oscillations In Local mode C slow compensation is activated by selecting the appro priate range on the front panel knob Compensation can be done manually by turning the C Slow and R Series potentiometers or by pressing the Auto button on the front panel of the amplifier A complete description of the C Slow ranges and gain limitations can be found in chapter 4 Description of the Hardware In Remote mode compensation is activated by selecting the range from http www heka com 8 4 The Amplifier control window of PatchMaster 97 the Range field Compensation can be done by changing the C Slow and the R Series values by clicking and dragg
25. other instrument The EPC 800 USB case was not designed to do this and damage to the front panel will result To minimize noise it is advisable to mount the EPC 800 USB away from devices that emit high frequency signals i e monitors power supplies etc 14 Unpacking and Installation 3 To operate the amplifier in Remote mode and utilize the USB com mands the USB cable should be connected between the USB connec tor on the rear panel of the EPC 800 USB labeled USB to an avail able USB 2 0 port on the computer As soon as the EPC 800 USB is detected by the host operating system the appropriate system files will be initialized and the EPC 800 USB will be ready for use This provides ease of installation and flexibility for moving the EPC 800 Patch Clamp Amplifier from one computer system to another 4 Appropriate BNC cable connections have to be made from the front panel Current and Voltage Monitors to their respective A D Input channels on the AD DA interface The External Input CC and VC should be connected to a chosen D A Output channel on the interface via a T BNC connection Whatever acquisition software is being used it will have to be config ured properly to correspond with the external hardware BNC con nections between the amplifier and the interface Examples of re quired BNC connections are provided in other sections of this man ual see chapters 7 Using the EPC 800 Patch Clamp Amplifier with pCLAMP and 8 Us
26. panel of the Digidata through a T BNC connec tor Voltage Clamp Experiments For the purpose of executing voltage clamp experiments the Analog OUT 0 is assigned the V command signal The units are mV and the scale factor is 100 mV V ai Lab Bench Input Signals Output Signals Digitizer Channels Signals Add Delete Y command Signal units movi iv Default Scale factor m V 100 Sellen es Assistant Figure 7 2 Example of configuring the output signals for voltage clamp experiments Analog Out 0 is assigned to the V command signal Current Clamp Experiments For the purpose of executing current clamp experiments the Analog Out 0 is assigned the I cmd signal The units are nA and the scale factor is 0 1 nA V http www heka com 7 1 Local Mode 55 a Lab Bench Input Signals Output Signals Digitizer Channels Signals Rename lemd Signal units n vil Default Scale factor n v 01 Scale Factor DE Assistant Figure 7 3 Example of configuring the output signals for current clamp experiments Analog Out 0 is assigned to the Icmd signal 7 1 4 Membrane Test with Model Circuit The following tutorial will guide you through most of the basic and some of the unique and more sophisticated features of the EPC 800 Patch Clamp Amplifier It will enable the user to explore the use of the front panel controls while at the same time it allows you to check wh
27. rack mountable 11 4 kg 24 8 lbs http www heka com 131 Index Appendix 121 131 EPC 800 USB Commands 121 Supported States 121 Technical Specifications 129 131 USB Descriptor 121 Auto C Fast 27 59 96 Auto C Slow 28 62 97 Bath Electrode 110 Bridge Compensation 49 104 Capacitance Compensation Con trols 26 29 T Fast 28 C Fast 27 C Slow 28 C Slow Range 28 R Series 29 Chassis GND 32 Clipping Indicator 21 86 Command Signals 24 Command signals 26 Iyoxp 24 VHOLD 24 VPoFFSET 25 Compensation Theory 41 49 Bridge Compensation 49 Capacitance Compensation 44 Offset Compensation 41 44 Series Resistance Compen sation 45 49 Current Monitor 21 Description of the Hardware 17 33 Display Selector 30 Dongle Driver 80 EPC DLL 5 EPCMaster 6 75 External Input CC 20 External Input VC 20 External Shielding 108 Filter 24 Firmware Version 6 Front Panel 52 Gain 21 85 Grounding 108 I mon 87 I Scale and V Scale 101 Input ADC 92 Introduction 3 11 Knob Sensitivity 31 Liquid Junction Potential 42 94 Local Telegraphing Mode 4 71 Local Mode 4 51 83 Low Frequency Voltage Clamp LFVC 39 100 Low Noise Recording 115 119 Amplifier 115 Set Up 115 INDEX 133 Main Controls 85 MODE switch 23 Model Circuit 55 Naming Conventions 9 Noise 30 93 115 Overlay 101 Patch Clamp Setup 107 110 Patch Pipettes
28. saved named and edited and called upon whenever needed they are es sential tools towards automation The protocol file for the EPC 800 Patch Clamp Amplifier is called Epc800 pro and this file will be located within the PATCHMASTER folder when the software is loaded This file should be properly configured in the PATCHMASTER file configuration window as illustrated http www heka com Using the EPC 800 USB patch clamp amplifier with 88 PatchMaster Figure 8 6 File Configuration The EPC800 pro file contains information about the preset and user defined protocols Protocols themselves are created named saved and edited from within the protocol editor window of PATCHMASTER When PATCHMASTER is first loaded and configured for use with the EPC 800 Patch Clamp Amplifier there are already some predefined protocols that have been created as part of the overall Epc800 pro file http www heka com 8 4 The Amplifier control window of PatchMaster 89 Sf Protocol Editor Setup_Epc800 o x 1Examplt_ 20 3 _ Link 4 __ Buffer 5 _SETUP_ 6C SEAL 2 JoJo van igge REPEAT sweeps 0 000s 3 STEP CoE Srov 4 Amplifier C slow 5 IF R series gt 8 0000M Record Macros _ Relative Value _ blocking 6 BREAK protocol 7 END_IF mas Events 1 of 9 Insert Before l Insert After 8 _ Sweep IV 3 END REPEAT Duplicate Delete _ Move _ Annotation Ann
29. separate tabs for input and output signals and virtually any type of signal can be configured for any of these channels In our example Analog IN 0 is physically connected to the Current Monitor of the amplifier and Analog IN 1 is physically connected to the Voltage Monitor of the amplifier In the Lab Bench panel Analog IN 0 is assigned the I monitor signal The units should be pA and the scale factor is 0 001 V pA Analog IN 1 is assigned the V monitor signal The units are mV and the scale factor is 0 01 V mV a Lab Bench a Lab Bench Input Signals Output Signals Input Signals Output Signals Digitizer Channels Signals Digitizer Channels Signals Add Analog IN 2 Delete Analog IN 3 E Add Delete monitor V monitor Scaling Scaling Signal units p z Default Signal units m xii Default Scale factor V pA 0 001 eons tear Scale factor V mV 0 01 Seale Factor Offset pA 0V 0 a Assistant Offset mV 0V 0 Assistant Figure 7 1 Configuring the input signals in Lab Bench for the Voltage and Current Monitor outputs of the EPC 800 Patch Clamp Amplifier 7 1 3 2 Output Signals Both the External Input CC and the External Input VC on the front panel of the EPC 800 Patch Clamp Amplifier are connected to Analog http www heka com 54 Using the EPC 800 Patch Clamp Amplifier with pCLAMP Output 0 on the front
30. shielded 116 Low Noise Recording and grounded his setup sufficiently well to take care of any synchronous noise due to line frequency pickup computer power supplies TV cameras etc Synchronous noise can be readily identified as stationary features on an oscilloscope trace when the oscilloscope is triggered by the appropri ate signal source for example line frequency triggering Grounding and shielding is discussed in chapter 9 General Patch Clamp Setup Practices 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 position near the recording cham ber and power on every piece of equipment that introduces noise lamps oscilloscope camera In a well grounded setup all these components should introduce no more than about 100 fA of additional noise Starting from an intrinsic noise reading of 80 100 fA one observes incre ments in the noise level when the holder and pipette are installed and when an actual recording is made By analyzing these increments you can see where there is the most room for improvement in 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 and 160 fA when the pipette tip is in the bath sealed on a cell These are rms current values which means
31. system sampling and storage in pulse ramp and continuous modes a fully programmable pulse generator a digital oscilloscope and all other features needed for patch clamp electrophysiology and many other applications The complete PATCHMASTER acquisition system can also be http www heka com 6 Introduction batch controlled from another application The user can write their own application with a custom user interface while still benefitting from the advanced features of the HEKA system HEKA provides a free software program called EPCMASTER which can be used to control the functionality of the amplifier in Remote mode It is not an acquisition or analysis program but a software front panel control interface that provides a further level of integration with third party hardware and software In addition EPCMASTER Software is a useful tool to test amplifier functionality and the USB message stream The versatility of the EPC 800 Patch Clamp Amplifier is reflected by the variety of experiments that it can be applied to Besides being used for whole cell voltage clamp experiments and recordings from artificial mem branes or loose patches it also excels in high resolution recordings of single channels Furthermore the amplifier has true current clamp capabilities to enable fast action potential recordings Technically the EPC 800 Patch Clamp Amplifier retains the three noteworthy special features that are common to all HEKA
32. the command name and the current state If a command is send but the argu ment is out of range the FW will choose the closest value of the parameter that is possible If the differences of the value specified by the user and the two valid values are identical the smaller valid value will be used A get command will always be answered by the command and the value Table 12 2 illustrate some examples Command FW response Remarks hello_world unknown_command hello world command is unknown set_mode cv set_mode vc command is known argu ment not valid return cur rent value set_cslowvalue 10 set_cslowvalue 0 command is known argu ment out of range return closest value get_mode get_mode VC command is known current value returned Table 12 2 USB Command handling http www heka com 12 4 Telegraphing Translation 127 12 4 Telegraphing Translation Gain in mV pA Telegraphing Output in V 0 005 0 0 01 0 5 0 02 1 0 0 05 1 5 0 1 2 0 0 2 2 5 0 5 3 0 1 3 5 2 4 0 5 4 5 10 5 0 20 5 5 50 6 0 100 6 5 200 7 0 500 7 5 1000 8 0 2000 8 5 Table 12 3 Gain telegraphing values deviation 0 1V Cslow in pF Telegraphing Output in V Dial Range for Cslow in pF off 0 0 30 0 3 0 10 0 30 100 0 10 0 10 0 100 1000 0 10 0 10 0 1000
33. the audio monitor feature HEKA also provides a PSA 12 sound generator If a HEKA LIH 8 8 interface is used in conjunction with the EPC 800 Patch Clamp Amplifier the built in sound capabilities of this interface can be used In all cases the sound source has to be correctly configured within the PATCHMASTER Hard http www heka com Using the EPC 800 USB patch clamp amplifier with 100 PatchMaster ware Configuration section Reset Selecting this button will reset the EPC 800 Patch Clamp Ampli fier to its initial default configuration It is only applicable for use of the amplifier in Remote mode 8 4 2 Show All Controls When the Show Al tab of the amplifier window is selected there are additional features present on the right hand side of the panel v Low Freq VC Off Low Freq VC 100 LFVC V memb Low Freq VC 30 Pv O Low Freq VC 10 Low Freq C Off Low Freq VC 3 Low Freq VC 1 Figure 8 17 Low frequency voltage clamp LFVC settings within Patch Master Low Frequency Voltage Clamp LOW FREQ VC The low frequency voltage clamp mode is a modified current clamp mode which allows for the measurement of potential deflections such as action potentials or synaptic potentials while the average potential is kept constant at a value chosen by the user LF VC V memb The circuit thus works like a current clamp for fast signals and like a voltage clamp for low frequency signals To achieve this t
34. the dovetail match the headstage housing http www heka com 4 2 Main Unit 19 90 mm Figure 4 2 Standard plate igs are A A A R lt gt 90 mm Figure 4 3 Dovetail plate 4 2 Main Unit Figure 4 4 EPC 800 patch clamp amplifier main unit http www heka com 20 Description of the Hardware The main unit of the EPC 800 Patch Clamp Amplifier contains the power supply the signal processing electronics and all of the controls The bottom level of the front panel consists of the probe connector BNC connections grounding plug and clipping indicator The potentiometers knobs and buttons on the main unit front panel can be divided into six basic functional groups i Gain Mode and Filter ii command signal processing iii capacitance compensation iv series resistance compen sation v display and vi power 4 2 1 Bottom Row of the EPC 800 USB Front Panel SIGNAL EXTERNAL EXTERNAL VOLTAGE GND INPUT CC INPUT VC MONITOR Figure 4 5 Bottom Row of Amplifier PROBE This input accepts the multi pin connector of the head stage SIGNAL GND This banana jack is a high quality signal ground con nection that can be used to ground other parts of the experimental setup as necessary see chapter 9 General Patch Clamp Setup Practices External Input CC Signals from an external stimulus source are applied here they can be summed with the internal stimul
35. this shield is grounded at the microscope 9 4 External Shielding Especially when an unshielded pipette holder is used some electrostatic shielding of the experimental setup is necessary to avoid line frequency pickup from lights and power lines in the room Most experimenters use a table top Faraday cage with a closable front and lead all of the cables e g from the microscope lamp probe cooling system ground lines through a hole in the cage to an equipment rack mounted outside If the pipette http www heka com 9 5 Pipette Holder and Electrode 109 holder is somewhat exposed or if the Faraday cage has an open front a small grounded screen placed near the pipette holder may help 9 5 Pipette Holder and Electrode The pipette holder that was shipped with the amplifier is made of ex tremely low noise polycarbonate having low dielectric loss It is equipped with a BNC connector to fit the headstage of the EPC 800 Patch Clamp Amplifier The design of the pipette holder is such that it virtually elim inates pipette movement and air leakage by virtue of elongation of the screw cap and the addition of a third O ring The choice of materials used in the design of any pipette holder are very important The insulating parts of the holder 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
36. which allows to correct for liquid junction potentials and other offsets It works in conjunction with the Vo operation An online correction requires an Auto Vp operation to be performed before seal formation and LJ to be set to an appropriate value No correction is performed if LJ 0 LJ can be adjusted within 200 mV by dragging the mouse or typing after a double click Note LJ is not changed by the RESET function and can not be set by protocols This restriction is imposed to avoid unintentional 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 asymmet rical solutions are used or the bath solution is changed during an experi ment For the standard liquid junction potential correction the polarity of the entered value should be such that it represents the potential of the bath with respect to the pipette solution For example if the pipette so lution contains glutamate or aspartate with chloride in the bath then the polarity of LJ should be positive 10 mV After an Auto Vo oper ation V membrane 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 Figure 8 11 Liquid Junction and pipette offset features of PatchMaster Vo Pipette Offset Vo displays the offset voltage a voltage which is added to V m
37. 0 0 0 0 Digital bit pattem 3 0 0 0000 0000 0000 D000 0006 Digital bit pattern 7 4 of 0 0000 0000 0000 0000 Number of sweeps 500 Stimulus File Summary Figure 7 11 Example of how to write a typical whole cell current clamp protocol This example is of a 1 nA current injection for 50 ms http www heka com 70 Using the EPC 800 Patch Clamp Amplifier with pCLAMP Waveform Preview Wave End 400 800 Time ms Sweep 1 Visible 1 of 1 gt Clipboard Help Figure 7 12 Waveform Preview of the current clamp protocol designed in the previous figure In the oscilloscope window figure below the current and voltage traces are displayed These two display signals are selected within the Inputs tab of the Edit Protocol dialog and have already been configured within the Lab Bench dialog where the I monitor was assigned to Analog IN 0 and the V monitor was assigned to Analog IN 1 The output signal was also pre configured in Lab Bench with Analog Out 0 Digitizer channel being assigned to the Icmd signal http www heka com 7 2 Local Telegraphing Mode 71 Scope Episodic DER Figure 7 13 Execution of the current clamp protocol displayed in Fig 7 11 with both the current and voltage traces displayed on the scope 7 2 Local Telegraphing Mode 7 2 1 Telegraphing Outputs The EPC 800 Patch Clamp Amplifier is
38. 1 Static Electricity 0 2 2 2 0008 Description of the Hardware AA PrOD ire tet e R E Bla a a APO Bae a a 4 1 1 Probe Adapters o onoo a 4 2 Main Unit s ahai miio SADA a ed BOR aE ad A 4 2 1 Bottom Row of the EPC 800 USB Front Panel 4 2 2 Gain Mode and Filter Knobs 4 2 3 Command Signal Processing 4 2 4 Capacitance Compensation 4 2 5 Series Resistance Compensation ii CONTENTS 4 2 6 Seal Modes nareread Aid a 0 8 eee eee 30 4 2 7 Display Noise and Remote 30 4 2 8 Knob Sensitivity aoaaa 31 4 2 9 Power Switch and Chassis Ground 31 4 2 10 Rear Panel Connectors s sosoo 32 5 Recording Modes of the EPC 800 Patch Clamp Amplifier 35 5 1 Voltage Clamp Mode oaa 35 5 2 Current Clamp Mode aoaaa 36 5 3 Low Frequency Voltage Clamp Mode 39 6 Theory of Compensation Procedures 41 6 1 Offset Compensation 0004 41 6 2 Capacitance Compensation sosoo e 44 6 3 Series Resistance Compensation 45 6 4 Bridge Compensation oaoa 49 7 Using the EPC 800 Patch Clamp Amplifier with pCLAMP 51 TA Local Mode sisi fe seica Ph a i s ete Set 51 7 1 1 Software Installation o ooa a 52 7 1 2 Hardware Connections ooo a 52 Toly Front Panele es mte eaa eei ea WSs 52 7 1 3 Configuring Clampex Lab Bench 53 7 1 3 1 Input Signals oaa 53 7 1 3 2 Output S
39. 44 146 Barry P H amp Lynch J W 1991 Liquid junction potentials and small cell effects in patch clamp analysis J Memb Biol 121 101 117 Peters F Gennerich A Czesnik D amp Schild D 2000 Low frequency voltage clamp recording of voltage transients at constant average com mand voltage J Neuroscience Meth 99 129 135 Book Chapters B Sakmann amp E Neher eds 1995 Single Channel Recording Plenum Press New York e Chapter 1 Penner R A practical guide to patch clamping e Chapter 2 Marty A amp Neher E Tight seal whole cell recording e Chapter 3 Heinemann S H Guide to data acquisition and analy sis e Chapter 4 Sigworth F J Electronic design of the patch clamp e Chapter 6 Neher E Voltage offsets in patch clamp experiments e Chapter 19 Colquhoun D amp Sigworth F J Fitting and statistical analysis of single channel records Neher E 1992 Correction for liquid junction potentials in patch clamp experiments In Methods in Enzymology 207 123 131 Academic Press New York http www heka com 2 4 Naming Conventions 9 2 4 Naming Conventions Windows versions The EPC 800 Patch Clamp Amplifier is supported by 64 and 32 bit ver sions of Windows 8 Windows 7 Windows Vista Windows XP Windows 2000 An available USB 2 0 port is required for Remote Mode only Throughout the manual we will address all the above Windows versions as Windows
40. 8 4 The Amplifier control window of Patch Master This section focuses on the EPC 800 control window of PATCHMASTER The various buttons and commands within this window are discussed and related to the equivalent front panel knobs switches and potentiometers that are located on the front panel of the amplifier The description of all of the front panel controls on the amplifier itself have already been discussed in chapter 4 Description of the Hardware starting on page 17 The acquisition software PATCHMASTER provides the controls and the graphical representation of the EPC 800 Patch Clamp Amplifier by a vir tual panel with buttons In Remote mode PATCHMASTER can con trol all amplifier functions and in Local mode the front panel knobs and switches of the EPC 800 Patch Clamp Amplifier are active and the values are displayed in the software amplifier window In the Notebook window of PATCHMASTER the exchange of communication commands are listed both being sent and received This scrolling can be stopped to be read by pressing on the HELP menu heading of PATCHMASTER A list of the EPC 800 patch clamp amplifier commands used to signal communication between the amplifier and PATCHMASTER are provided in the Appendix Note Alternatively to using the mouse most of the controls in PATCHMASTER can also be changed directly by the keyboard You can see the actual keyboard assignments when you select Show Keys from the Help menu
41. A get_imon both A get_vmon both V get_clipping both string get_serialno both string get_revision remote string Firmware revision is returned in the following format X Y Z get_change both no argument reports the value of the last parameter changed as it applies to cfast cslowrange cslowvalue extstim filter gain lfvc mode rscomp rseries rsrange tfast ihold vhold vp clipping is reported if none of these parameters have changed since the last inquiry The USB string unit busy is returned when a command is received while and auto function is performing http www heka com 12 3 List of EPC 800 USB Commands 125 iter an nitor Auto Csiow 30 ve manuala uto VPottset manuavAuto Chast filter fe lt 10 KHZ manusia uto VPottset manuala uto Ctast filter fe lt 30 KHZ manuala uto VPottset manual Auto Ctust iter ali titer Auto all ran manual A uto VPottset manual A uto Ctast 0 5 20 mvp Miter te lt 10KHz manual VPottset manual Ctast rscomp lt 120 Miter fe lt 10KHZ inoid 50 nA manual VPoftset manual Ctast LFVC tau 1 100 Miter fe lt 10KHz 1 Csiow off manual VPottset manual Ctast Miter te lt 10KHz Inold 50 n manual VPottset manual Cast Figure 12 1 States http www heka com 126 Appendix If a command is send but the argument is illegal the FW will return
42. ASTER by pressing the yellow Auto button in the amplifier window The C Slow and R Series values will be displayed in the PATCHMASTER http www heka com Using the EPC 800 USB patch clamp amplifier with 98 PatchMaster amplifier window as well as on the LCD display of the EPC 800 Patch Clamp Amplifier The asterisk symbol in the LCD dis play is indicative of the results being obtained through an auto matic procedure The Auto C Slow in this situation can NOT be disabled by pressing and holding the Auto C Slow button on the EPC 800 Patch Clamp Amplifier It has to be turned off through PATCHMASTER by changing either the C Slow value or range Rs Comp The series resistance compensation corrects for membrane voltage errors under conditions of high access resistance between pipette and cell interior see Chapter 6 Compensation Procedures The amount of compensation can be changed by dragging the mouse or typing range 0 95 The compensation is based on the value of R series and will be effective only when R comp is not Off i e set to a speed value A de scription of the various settings determining the feedback of compensation can be found in chapter 4 Description of the Hardware Off 100 ps ye Reems ae 2us Figure 8 14 Setting the Rs compensation speed within Patchmaster Stimulus Filter The stimulus can be filtered 2 pole Bessel to reduce the amplitude of fast capacitance transients when the speed of pot
43. Ag AgCl wire Nevertheless a liquid junction potential will develop at the bath salt bridge interface unless a bleeding KCl bridge is used see Neher 1992 Similarly a liquid junction potential will develop during local microperfusion Thus the correction during the episode in low chloride medium will be the sum of this liquid junction po tential and the correction of Example 2 3 mV Taking the value for a low CIT solution e g sulfate Ringer see table we arrive at a value of LJ 3 6 3 mV which should be set during that part of the experiment Note The sulfate Ringer in this case is 6 mV the inverse of http www heka com 44 Theory of Compensation Procedures the value in the table because this potential appears during the measurement with inverted polarity to the convention of Barry amp Lynch Example 4 An outside out or whole cell measurement with Cs citrate based internal solution In this case LJ should be set to 12 mV see table above Example 5 A cell attached measurement with sulfate Ringer in the pipette Two corrections apply 1 the correction for the liquid junc tion potential during the reference measurement 6 mV see table above and 2 the resting potential of the cell We assume the latter to be 60 mV and therefore set LJ to 54 mV In the cell attached mode polarities of the amplifier readout are inverted thus the amplifier will display the physi ological p
44. Lambrecht Pfalz Germany phone 49 0 6325 9553 0 fax 49 0 6325 9553 50 e mail support heka com web http www heka com Canada HEKA Electronics Incorporated 643 Highway 14 R R 2 Chester NS BOJ 1J0 Canada phone 1 902 624 0606 fax 1 902 624 0310 e mail support heka com http www heka com United States HEKA Instruments Inc 2128 Bellmore Avenue Bellmore NY 11710 5606 USA http www heka com 2 5 Support Hotline 11 phone 1 516 882 1155 fax 1 516 467 3125 e mail support heka com web http www heka com http www heka com 12 Introduction http www heka com 3 Unpacking and Installation This chapter provides instructions for unpacking and setting up the am plifier for use 3 1 Unpacking and Connecting the EPC 800 Patch Clamp Amplifier Please follow these steps after receiving the EPC 800 Patch Clamp Am plifier to get to the point where the amplifier is connected and ready to be used 1 When you receive the EPC 800 USB please check the packing list to verify that you have all of required parts e The EPC 800 USB amplifier e The headstage e The model circuit in the box with the headstage e The USB 2 0 cable e The pipette holder e Spare fuses and gold pin 2 The EPC 800 USB can be installed into a standard nineteen inch instrument rack or used as a desktop unit If installing on a rack please do not use the EPC 800 USB as a shelf to support any
45. Manual 1 1 6 gt EPC 800 USB Patch Clamp Amplifier HEA HEKA Elektronik Dr Schulze GmbH Wiesenstrasse 71 D 67466 Lambrecht Pfalz Germany HEKA Electronics Inc 643 Highway 14 R R 2 Chester NS BOJ 1J0 Canada HEKA Instruments Inc 2128 Bellmore Avenue Bellmore New York 11710 5606 USA Phone Fax Web Site Email Phone Fax Web Site Email Phone Fax Web Site Email 49 0 6325 95 53 0 49 0 6325 95 53 50 www heka com sales heka com support heka com 1 902 624 0606 1 902 624 0310 www heka com nasales heka com support heka com 1 516 882 1155 1 516 467 3125 www heka com ussales heka com support heka com Front cover is a current clamp recording made with the EPC 800 USB of action potentials from an isolated guinea pig ventricular cardiomyocyte Data was provided courtesy of Dr Pavel Zhabyeyev and Dr Terence F McDonald Dalhousie University Nova Scotia 2004 2014 HEKA Elektronik Dr Schulze GmbH COME80 4 Contents Safety Guidelines Introduction 2 1 Introducing the EPC 800 Patch Clamp Amplifier 2 2 Firmware Version 0 2 0002 eee 23 References i de eo DAMM ake Ogg a wa ede 2 4 Naming Conventions 000 2 5 Support Hotlines 4 04 ois pee els Ee Cee Ras Unpacking and Installation 3 1 Unpacking and Connecting the EPC 800 Patch Clamp Am POOR He sar ap maana n ony Maesteg Ok Se ne Saree ees 3 1
46. Note Users that may be unfamiliar with some of the controls within PATCHMASTER may find the control descriptions to be helpful The description for any given control is displayed when http www heka com 8 4 The Amplifier control window of PatchMaster 85 the mouse is placed over the item and you have selected Show Tooltips from the Help menu 8 4 1 Main Controls The EPC 800 window provides the amplifier control functions such as gain and filter settings and it enables access to the automatic compensation rou tines of the EPC 800 Patch Clamp Amplifier The virtual amplifier window is essentially the same in both Local and Remote modes of operation with one exception In Local mode C Fast and C Slow automatic compensa tions can NOT be performed through PATCHMASTER and therefore the yellow Auto buttons in the amplifier window in Local mode are grey and can t be executed fzdercoooRemote o Monitor Tuning Show All V membrane 20 mvp A0 0 mv LFVC V memb ae 89 5 pA 43 mV 496 MO l mon V mon R memb Input ADC Recording Mode Current Whole Cell O off Test Pulse Amplitude Length O noise 6 12 pF Cta EE ato range 100 pF C slow ate Rs Comp StimFilt Filter 3 kHz D Reset Z Amplitude _Duration Low Freq YC Off Cne Pulse scale 0 00 vscale Sound Volume 100 Range AD 0 10 Figure 8 3 PatchMaste
47. act that compensated membrane time constant Tc cannot be made smaller than a value that depends on the speed of the R compensation feedback If you turn up the COMP control to try to obtain a smaller Tc you will observe overshoot or ringing in the cur rent monitor signal due to an overshoot in the membrane potential The minimum value for Tc is given approximately by Te min Tu Tf where Ty the effective time constant of the feedback loop is about 2 us for the fast setting and 6 us for the slow setting The corresponding maximum a values are given by Lf Tu Amaxz I Table 6 2 gives maximum a values i e comp settings and the resulting Tc values in the 2 us setting for some values of the uncompensated time constant Tu At the 10 ps setting full 90 compensation may be used without overshoot for time constants 7 greater than about 1 ms the 100 us setting is appropriate for Ty values on the order of 10 ms or longer In practice you can estimate ty from the ratio of the settings of C Slow and R Series For example if C Slow is 10 pF and R is 10 MQ the time constant is 10 pF x 10 MQ 100 ps 6 1 http www heka com 6 4 Bridge Compensation 49 Talus a Telus 90 0 85 13 50 0 80 10 30 0 75 8 22 0 70 7 13 0 60 5 8 0 50 4 Table 6 2 Relationship between R comp settings and membrane time constants The use of the Rs compensation circuitry can be summarized
48. adjust the Bridge Compensation by changing the R setting in the C Slow section This way you have a direct measure of the absolute value of the electrode resistance in Current Clamp mode Note The effect of Bridge Compensation becomes much more prominent when using high resistance electrodes for recording voltage changes 8 4 3 2 Voltage Bandwidth in Current Clamp Recordings The bandwidth of the voltage signal in a current clamp recording is limited by the time constant Rs C Fast With the MC 10 model circuit this time constant calculates to about 30 ps http www heka com Using the EPC 800 USB patch clamp amplifier with 106 PatchMaster 12 0f L Ta SPEER Power Spectrum 29 5 a a e i a log f 15 0 4 3 00 4 00 5 00 Figure 8 24 Power Spectra of voltage recordings from MC 10 in Current Clamp mode No C Fast compensation lower trace 4 pF C Fast com pensation upper trace When increasing the C Fast compensation to e g 4 pF you can already observe in the power spectrum of the voltage trace the increase in recording bandwidth Important note The setting of C Fast is very critical with respect to oscillation Please be careful and do not overcom pensate C Fast in Current Clamp mode C Fast must be set correctly for proper Current Clamp mode operation http www heka com 9 General Patch Clamp Setup Practices 9 1 Mounting the Probe For low noise recording the pipette holder mu
49. ample the low gain range 0 005 to 0 2 mV pA is selected in voltage clamp then upon switching to current clamp Bridge mode the current clamp stim scaling will be set to 10 pA mV corresponding to a maximum command current of 100 nA The Iyo p potentiometer on the front panel can manually be set to 50 nA This is a medium current clamp gain range used in situations where medium current must be injected including e g loose seals It should be mentioned that the medium current clamp gain range has the side effect that it cannot set zero current very precisely This is a limitation when recording while not injecting current For example a jitter of 1 mV of the DA output in the medium current clamp gain range would cause a jitter of 10 pA and injecting 10 pA is not the same as injecting zero current Alternatively if one switches to the current clamp bridge mode from either the medium 0 5 to 20 mV pA or high 50 to 2000 mV pA gain ranges in voltage clamp then the current clamp stim scaling will be set to 0 1 pA mV corresponding to a maximum command current of 1 nA The Inoxp potentiometer on the front panel can manually be set to 500 pA This low current clamp gain range is used in situation where smaller currents need to be injected e g for smaller cells Note Once in current clamp bridge mode the current clamp gain range cannot be changed The Gain settings are internally restricted t
50. ance is 5 1 MQ the membrane resistance is 500 MQ and the membrane capacitance is 22 pF This mode allows testing the C Slow compensation and the current clamp mode Furthermore it is useful to check stimula tion patterns you design within the acquisition software Note This model cell has a long membrane time constant about 10 ms http www heka com 7 1 Local Mode 57 7 1 4 2 Open Pipette and V Porrser With the model circuit in the top position the 10 M setting simulates an open pipette with a resistance of 10 MQ This is useful for applying a test pulse and correcting for offset potentials The Membrane Test dialog of Clampex should be opened with the Bath tab selected The correct pipette resistance should be calculated and displayed in the R field where you should read a value close to 10 MQ For observation of the current pulses it is convenient to set the front panel Gain switch to a setting such that the current through the open pipette is reasonably sized perhaps set the Gain to 5 mV pA Other rec ommended front panel settings are Filter set to 3 kHz RS COMP is off and C Slow range is off The VHOLD THOLD LFVCyotp and V PoFFSET potentiometers should all be set to read 0 on the LCD display With a gain setting of 5 mV pA you should see a rectangular current of about 500 pA in response to a 5 mV test pulse This represents the ohmic resistor you are recording from
51. and 2 Filter 2 is bypassed when the filter knob is set to either 30 or 100 kHz Capacitance Compensation C Fast 0 15 pF 0 to 8us time constant C Slow 30 pF range 1 0 30 pF 100 pF range 1 0 100 pF 1000 pF range 1 0 1000 pF Injection Capaci C Fast compensation signal is injected via 1 pF tors capacitor C Slow compensation signals are injected via a 10 pF capacitor in medium and low gain and via a 1 pF capacitor in high gain range R Series 0 1 MQ 200 MQ 1000 pF range 1 1 MQ 4 200 MQ 100 pF range 3 5 MQ 200 MQ 30 pF range http www heka com 130 Appendix Rs Comp Adjustment Manual range is dependent on cell capacitance Equivalent off 2 us 10 us 100 us Time Constants Range 04 95 In CC Mode Rg Comp serves as Bridge Compensation with a range of 0 120 Pipette Potential Control output Holding potential 500 mV Pipette offset 200 mV Potential monitor 10x Input and Output Specification Minimum Load at Telegraphing 500 MQ Output Impedance at Telegraphing 50 Q Output Range at Telegraphing 10 24 V Minimum Load at Vmon 500 MQ Minimum Load at Imon 500 MQ Input Impedance at VC input 1MQ Input Impedance at CC input 1MQ Output Impedance at Vmon input 20 KQ Output Impedance at Imon input 20 KQ Output Range at VC 10 24 V Output Range at CC 10 24 V
52. as follows When you set the capacitance transient cancelation C Slow R Series C Fast r Fast to minimize the size of the transients when voltage pulses are applied you have also properly set them for series resistance compensation Then you enable R Series and turn up the COMP control to the desired value Any misadjusting of the transient cancelation will be apparent and can be compensated 6 4 Bridge Compensation Bridge compensation in current clamp mode acts in a very similar way to the Rs compensation in voltage clamp mode It basically compensates the voltage drop via the series access resistance of the electrode Rs Fur ther information with illustrations demonstrating the effects of the bridge compensation of the EPC 800 in current clamp mode are provided later in this manual in Chapter 8 Using the EPC 800 USB with PatchMaster http www heka com 50 Theory of Compensation Procedures http www heka com 7 Using the EPC 800 Patch Clamp Amplifier with pCLAMP This chapter will concentrate on the various ways in which the EPC 800 Patch Clamp Amplifier can be used in combination with Axon s pCLAMP software and Digidata interfaces Three modes of oper ation are discussed local mode local telegraphing mode and finally remote mode via control through HEKA s virtual amplifier soft panel Topics covered will be the required hardware connections and software configuration steps This is followe
53. atch potential 6 2 Capacitance Compensation The EPC 800 Patch Clamp Amplifier offers users an automatic procedures for both fast and slow capacitance subtraction The use and behavior of these automatic compensation routines as they pertain to Remote and Local modes of operation are discussed elsewhere in this manual in several places see chapters 4 Description of the Hardware 7 Using the EPC 800 Patch Clamp Amplifier with pCLAMP and 8 Using the EPC 800 Patch Clamp Amplifier with PatchMaster When executing a C Fast or C Slow automatic compensation the ongoing pulse protocols are suspended and short trains of square wave pulses are applied during which time the green Auto LEDs on the EPC 800 Patch Clamp Amplifier front panel will be blinking and the pulses can be seen on an oscilloscope screen The resulting capacitive transients are averaged and then used to calculate the required corrections The algorithm iteratively tries to minimize the RMS amplitude of the current transient elicited by the application of the small square voltage pulse In the case of C Fast compensation for example the search is done over the whole range of C Fast 0 15 pF and r Fast 0 8us values The values of C Fast and 7 Fast that correspond to the RMS minimum amplitude are then stored as the http www heka com 6 3 Series Resistance Compensation 45 new Auto C Fast and Auto 7 Fast values As the algorithm runs external
54. atch the settings in the Configuration Hardware tab of PATCHMASTER http www heka com Using the EPC 800 USB patch clamp amplifier with 82 PatchMaster Figure 8 2 Configuring hardware and connections Once saved this in formation will become part of the PatchMaster set file Based on the software configuration shown above the following cable con nections should be made between the amplifier and interface Front Panel of EPC HEKA Interface 800 USB Voltage Monitor A D Input 0 Current Monitor A D Input 1 External Input VC T connection to D A External Input CC Output 2 Table 8 1 Front panel BNC connections between the EPC 800 Patch Clamp Amplifier and a HEKA InstruTECH interface http www heka com 8 3 Software Operation 83 8 3 Software Operation 8 3 1 Local Mode When operating the EPC 800 Patch Clamp Amplifier in local mode all of the front panel controls of the amplifier are active and PATCHMASTER is constantly reading and interpreting the amplifier parameters The Gain Mode and Filter settings for example are all controlled by the front panel controls and the values are displayed in the amplifier window of PATCH MASTER If you try to set these values directly from the software in this mode it will not work Important note It is recommended that the VgoLD InoLpD and VPorrser potentiometers all be set to read 0 If you try to use these front panel controls you will b
55. back of the pipette e g using a 5 ml syringe Thereafter the pipette is back filled the pipette should only be partially filled just far enough to make reasonable contact with the electrode wire the pipette holder is not filled with solution but is left dry Overfilling the pipette has disastrous consequences for background noise because the solution can spill into the holder wetting its internal surfaces with films that introduce thermal noise Bubbles left in the pipette from filling can be removed by tapping the side of the pipette For low noise recording the electrode holder should be cleaned before each experiment with a methanol flush followed by drying with a nitrogen jet Before you insert a pipette into the holder it is a good idea to touch a hand to a metal surface of the setup to discharge any static electricity that you may have picked up Be sure to tighten the holder firmly enough that the pipette does not move on a scale of 1 um when you give suction Then when you change pipettes during an experiment 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 dried thoroughly http www heka com 11 Low Noise Recording 11 1 Measuring the Noise of the Amplifier The EPC 800 Patch Clamp Amplifier has a particularly low background noise level The noise level is in fact low enou
56. ble examples are highlighted by the three modes of operation of the EPC 800 Patch Clamp Amplifier The EPC 800 Patch Clamp Amplifier can be operated in Local Local Telegraphing and Remote modes The decision of which mode to use depends upon user preference of whether or not to have functionality to operate knobs and switches and upon what data acquisition software and AD DA interface the amplifier is used with The EPC 800 Patch Clamp Amplifier is the most flexible patch clamp amplifier ever produced in that it is a stand alone amplifier which can be combined with any existing AD DA interface and its compatible acquisition software The functionality of the amplifier of course differs slightly depending upon what combination of hardware and software the amplifier is used with In Local mode the amplifier is a manually controlled patch clamp amplifier with all of the front panel knobs and switches active Unlike other manual amplifiers however users do have the option of performing a Vp Offset C Fast and C Slow compensations automatically with the push of a button In this respect the amplifier offers features that previously were reserved for users of the computer controlled EPC 9 or EPC 10 family of amplifiers To operate in Local mode the amplifier can be used with any AD DA interface board Compatible platforms comprise the complete HEKA InstruTECH digitizer family as well as Axon interfaces including older models such as the Di
57. col editor window http www heka com 68 Using the EPC 800 Patch Clamp Amplifier with pCLAMP E Scope Episodic Figure 7 10 Execution of the voltage clamp protocol displayed in Fig 7 8 with both the voltage and current traces displayed on the scope 7 1 4 6 Whole Cell Current Clamp In this mode the resting potential or spontaneous action potentials can be measured in a whole cell recording A constant or time varying current is applied and the resulting change in membrane potential caused by the applied current is measured The voltage is monitored at the Voltage Monitor output of the EPC 800 Patch Clamp Amplifier and the value is displayed on the LCD display when in the I Vmon position As previously discussed with executing voltage clamp protocols the de sired current clamp protocol has to be written in the Protocol dialog of Clampex The figures below illustrate a very simple example of some of the steps for designing and executing a current clamp protocol consisting of a 1nA current injection for 50 ms http www heka com 7 1 Local Mode 69 Edit Protocol cc_smallc pro Mode Rate Inputs Outputs Trigger Statistics Comments Math Waveform Stimulus Waveform Analog OUT Iemd WV Analog Waveform rT Digital Outputs Epochs Stimulus file Intersweep holding level Use holding hd Sample rate Fast Fast 0 o fo fo a o fo 0 0 o 0 0 0 0
58. croscope 108 General Patch Clamp Setup Practices 9 2 Ground Wires It is a good idea to run a separate ground wire from the Signal Ground jack on the main unit to ground large objects such as the isolation table 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 Besides 50 or 60 Hz magnetic pickup there may be some 35 kHz pickup from the magnetic deflection of the computer monitor This pickup becomes visible only when the EPC 800 USB filter is set to high frequencies it can usually be nulled by changing the orientation or spacing of the ground wire from the probe cable 9 3 Grounding the Microscope In most cases the patch clamp is used in conjunction with a microscope it and its stage typically constitute the conducting surfaces nearest the pipette and holder In a well grounded setup the microscope can provide most of the shielding Make sure there is electrical continuity between the various parts of the microscope especially between the microscope frame and the stage and condenser which are usually the large parts nearest the pipette Electrically floating surfaces can act as antennas picking up line frequency signals and coupling them to the pipette Make sure the lamp housing is also grounded It is usually not necessary to supply DC power to the lamp provided that the cable to the lamp is shielded and that
59. d by an example tutorial for using the system with a model circuit to simulate typical experimental conditions of a pipette entering the bath solution canceling potential offsets forming a seal compensation of C Fast breaking the membrane to go to the whole cell configuration compensation of C Slow and finally executing whole cell voltage clamp and current clamp protocols Detailed information related to the installation and general programming of the Clampex software is not covered It is assumed that users of the EPC 800 Patch Clamp Amplifier in combination with Axon hardware and software already possess a general familiarity with these products The scope of this discussion is strictly related to getting the amplifier ready to use and cover some basic functioning of the amplifier in combination with third party equipment 7 1 Local Mode When operated in a local mode the EPC 800 Patch Clamp Amplifier is a completely manually controlled amplifier All of the amplifier settings are controlled directly by the user through the front panel knobs switches and 52 Using the EPC 800 Patch Clamp Amplifier with pCLAMP potentiometers The amplifier can be used with any of Axon s Digidata interfaces and compatible version of Clampex This is possible even with older Digidata models such as the 1200 7 1 1 Software Installation The software being used in the examples given in this chapter is Clampex 10 2 It is assumed a
60. de between the rear panel of the amplifier and the host computer When operated in this mode the front panel knobs and switches of the amplifier are inactive The EPC 800 amplifier commands are public see chapter 12 3 and users are free to write their own interfacing to the instrument without the need of HEKA software or interface boards HEKA also offers a dynamic link library DLL which gives direct access to the EPC 800 Patch Clamp Am plifier and HEKA data acquisition interfaces The DLL can be used with most programming languages such as C Pascal Delphi and Visual Ba sic The DLL provides functions for controlling amplifier settings and for stimulation and data acquisition The EPC DLL package is delivered in cluding a sample program and corresponding code written in Delphi and a C header file as documentation It is supported by Windows System 7 and all older operating systems In addition HEKA supports Mac OS X For Mac platforms HEKA supplies the EPC framework which is equivalent to a DLL on Windows systems The amplifier can be used in remote mode in combination with any of the HEKA InstruTECH series of acquisition interfaces and PATCHMASTER software Within the PATCHMASTER program there is a virtual front panel of the amplifier with a convenient graphics display and mouse and or keyboard operations provide versatility and ease of use In addition to the controls for the amplifier PATCHMASTER contains a powerful data acquisition
61. e The main requirements for a bath electrode are that it have a stable elec trode potential and that it does not disturb the composition of the bathing solution A bare chlorinated silver wire makes a good bath electrode how ever Ag ions are tolerated only by some types of cells such as muscle cells A good alternative is an electrode incorporating an agar salt bridge as illustrated below AgAgCl wire agar m to probe GND Figure 9 1 Example of agar salt bridge reference electrode 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 10 Patch Pipettes 10 1 Glass Capillaries Procedures for fabricating pipettes are presented in detail in the paper Improved patch clamp techniques for high resolution current recording from cells and cell free membrane patches O P Hamill et al Pfliigers Arch 391 85 100 This chapter is a basic summary of some helpful tips The main steps in pipette fabrication are to
62. e HEKA EPC 800 USB Attributes CMPos 10 CMNeg 100 SettingsO_Name gain SettingsO_0 0 0 0 005 SettingsO_1 0 5 0 01 Settings0_2 1 0 0 02 Settings0O_38 1 5 0 05 SettingsO_4 2 0 0 1 SettingsO_5 2 5 0 2 SettingsO_6 3 0 0 5 SettingsO_7 3 5 1 Settings0_8 4 0 2 Settings0_9 4 5 5 Settings0_10 5 0 10 Settings0_11 5 5 20 Settings0_12 6 0 50 Settings0_13 6 5 100 Settings0_14 7 0 200 Settings0_15 7 5 500 SettingsO_16 8 0 1000 Settings0_17 8 5 2000 Settings1_Name frequency Settings1_0 0 0 100 Settings1_1 1 0 300 Settings1_2 2 0 500 Settings1_3 3 0 700 Settings1_4 4 0 1000 Settings1_5 5 0 3000 Settings1_6 6 0 5000 Settings1_7 7 0 7000 Settings1_8 8 0 10000 Settings1_9 9 0 30000 Settings1_10 10 0 100000 Table 7 4 Conversion chart for EPC 800 Patch Clamp Amplifier gain frequency and telegraphed Cm values http www heka com 7 3 Remote Control through Soft Panel 75 of the amplifier and check to see if the same values are being displayed in the Telegraphs section in Clampex Likewise you can turn the C Slow potentiometer on the front panel the same number on the LCD display should be displayed in Telegraphs under the heading Cm Lowpass kHz fo Highpass Hz M Telegraphs Cm 808 11 fF Rm 574 7 MQ Ra Figure 7 15 Display of telegraph values in Clampex 7 3 Remote Control through Soft Panel The EPC 800 Patch Clamp Amplifier can also be used with Clampex software in such a way that the ba
63. e of 200 mV For users of the EPC 800 Patch Clamp Amplifier with HEKA s PATCHMASTER software additional information related to the setting of the liquid junction poten tial within PATCHMASTER can be found in chapter 8 Using the EPC 800 Patch Clamp Amplifier with PatchMaster or within the PATCHMASTER user manual The table below lists the LJ values for some typical solutions Solution LJ 145 K glutamate 8 NaCl 1 MgCl 0 5 ATP 10 NaOH 10 mV HEPES 145 KCl 8 NaCl 1 MgCl 0 5 ATP 10 NuaOH HEPES 3mV 60 Cs citrate 10 CsCl 8 NaCl 1 MgCl 0 5 ATP 20 12 mV CsOH HEPES 32 NaCl 108 Tris Cl 2 8 KCl 2 MgCl 1 CaCl 10 3 mV NaOH HEPES 70 NagSOxq 70 sorbitol 2 8 KCl 2 MgClo 1 CaCl 10 6 mV NaOH HEPES Table 6 1 Typical LJ values for different solutions In each case a liquid junction potential between the given solution and physiological saline 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 amp Lynch Note When applying the above rules for calculating the cor rection LJ two sign inversions of the liquid junction potential are effective for the standard liquid junction potential correc tion First the liquid junction potential that was present during http www heka com 6 1 Offset Compensation 43 the reference measurement disappears during the experiment after seal f
64. e of 7 Fast may be adjusted manually by turning the knob or automatically or semi automatically by performing an Auto C Fast operation C Slow This is used to cancel slow capacitive currents that charge the cell membrane in the whole cell configuration 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 C Slow can be compensated by first selecting the appropriate range see below and either manually adjusting the C Slow potentiometer or automatically or semi automatically by pressing the black Auto C Slow button Pressing the Auto button performs an automatic compensation of both C Slow and R Series These settings are used by the Rg compensation circuitry as the measure of series resistance The automated setting follows the same rules as for V POFFSET and Auto C Fast Note that starting Auto C Slow compensation performes the compensation only once the value is not dynamically adjusted over time During the algorithm optimises C Fast and tau the external stimulus input is deactivated Nevertheless holding potential regardless of its constituents whether applied by external stimulus input of by using the Vhold knob is measured and applied to the cell C Slow Range Selects the range for slow capacitance compensation e Off Turns cancelation off e 30 pF Small cells e 100 pF Small and medium sized cells e 1000 pF Large cells
65. e prompted through the software not to use them We suggest to use PATCHMAS TER itself to set the holding potentials and the offset potential With this approach you will ensure that all of the correct values will be stored with the PATCHMASTER data In the case of VPorrgert it is also suggested to use the Auto Vp from within PATCHMASTER Although the EPC 800 USB has its own Auto V Porfset it is not taken into account by PATCHMASTER 8 3 2 Remote Mode The difference between this mode and the local mode is that in Remote mode PATCHMASTER besides constant reading of EPC 800 parameters additionally allows commands to set parameters at the EPC 800 Patch Clamp Amplifier The commands that are sent have no immediate effects on PATCHMASTER itself until the command is acted upon and the amplifier sends back the resulting status which is then handled the same way as commands received in Local mode In Remote mode the front panel controls on the amplifier are inactive and amplifier settings are controlled through the software http www heka com Using the EPC 800 USB patch clamp amplifier with 84 PatchMaster PATCHMASTER is configured the same way as it is for Local mode except that EPC 800 Remote mode is now selected when choosing the amplifier for establishing the default configuration The external cable connections are the same When configured properly the red REMOTE LED on the front panel of the amplifier will be lit
66. eam In the example given below the EPC 800 Patch Clamp Amplifier is being used with a Digidata 1440A and Clampex software with the EPCMaster control window open In this configuration the red REMOTE LED on the front panel of the amplifier should be on For the most part the knobs switches and potentiometers on the amplifier front panel are inactive The exception to this is the LCD display switch and the Vyorp InoLp and LFVCyotp potentiometers The fact that these potentiometers are ac tive means that users have to be very careful when setting holding values for their experiments In the case of Vyozrp for example there are now three input sources where the holding potential can be set i the front panel Vyorp potentiometer ii the V membrane dialog of the ampli fier panel in EPCMaster and iii the holding setting from within the Clampex program It is suggested that when using EPCMaster in com bination with Clampex to control the amplifier in remote mode that the front panel Vyorp potentiometer be set to 0 mV and not touched Note The parameter values displayed on the front LCD panel of the amplifier will correspond to the values set from within the amplifier window of EPCMaster Even if the Vyorp poten tiometer were inadvertently turned or a holding setting was set from within Clampez these would not show up on the display Users should be very cautious of this When a voltage clamp protocol is executed for
67. eaves V MEMBRANE at that value Note Vo is not changed by the Reset function C fast _ Auto Figure 8 12 C Fast compensation within Patchmaster C Fast This is used to cancel fast capacitive currents that charge the pipette and other stray capacitances range 0 15 pF With nothing con nected to the probe input cancelation is typically obtained at a setting of 1 1 5 pF due to the residual input capacitance of the current measuring amplifier The compensation can be performed manually by dragging the mouse or typing http www heka com Using the EPC 800 USB patch clamp amplifier with 96 PatchMaster In the upper box the total C Fast value is displayed 7 Fast determines the time constant of C Fast up to 8 us The value of 7 Fast may be adjusted by dragging the mouse or typing or automatically by selecting the Auto function Auto C Fast Selection of this button in Remote mode performs an automatic compensation of C Fast and T Fast The procedure uses a rou tine that applies a number of small pulses 5 mV averages the resulting currents and fits an exponential to deduce the capacitance compensation values required to cancel the current Note In Remote mode ONLY C Fast compensation can be performed automatically through PATCHMASTER by pressing the yellow Auto button in the amplifier window The C Fast and T Fast values will be displayed in the PATCHMASTER am plifier window as well as on the LCD display
68. ed current supply to the heater coil is best A mechanical stop to set the length of the first pull is also important for reproducibility 10 3 Coating The capacitance between the pipette interior and the bath and also the noise from dielectric loss in the glass can be reduced by coating the pipette with an insulating agent such as Sylgard Dow Corning Corp Midland MI U S A Sylgard is pre cured by mixing the resin and catalyst oil and allowing it to sit at room temperature for several hours or in an oven at 50 C for 20 min until it begins to thicken It can then be stored at 18 C for many weeks until use The Sylgard is applied around the lower few mm of the electrode to within 10 20 um of the tip and then rapidly cured by a hot air jet or coil Coating should be done before the final heat polishing of the pipette so that the heat can evaporate or burn off any residue left from the coating process 10 4 Heat Polishing Heat polishing is used to smooth the edges of the pipette tip and remove any contaminants left on the tip from coating It is done in a microforge or similar setup in which the pipette tip can be observed at a magnification of 400 800x The heat source is typically a platinum or platinum iridium wire to avoid metal evaporation onto the pipette the filament is coated with glass at the point where the pipette will approach it To produce a steep temperature gradient near the filament which helps make the pipette t
69. embrane to obtain the pipette command voltage It should http www heka com 8 4 The Amplifier control window of PatchMaster 95 be set either by the Auto Vo operation or by manually dragging the mouse after clicking into the item Furthermore Vo is changed automatically by the controlling program whenever the user changes the variable LJ This is necessary for LJ and the Auto Vo operation to interact properly Note It is not recommended that the user change Vo manu ally by turning the V Pos fset potentiometer because this inter feres with the software features for automatic offset correction V Po ffset should be set to read 0 on the front panel display Auto Vo The Auto Vg button calls a procedure for automatic zeroing of the pipette current Thereby an offset voltage Vo to the pipette poten tial is systematically varied until pipette current is zero Range of Vo is 200mV Auto Vo is typically performed before seal formation It works properly only when a pipette is inserted into the bath The Auto Vo pro cedure interacts with the variable LJ to provide for online correction of liquid junction potentials and other offsets see Chapter 6 Compensation Procedures This requires that V Membrane is set to the value of LJ for On Cell and In Out Recording Modes or to the opposite polarity of LJ for Whole Cell and Out Out Recording Modes before the actual zero ing operation is performed Auto Vp does this automatically and l
70. en dial these manually and adjust slightly from this point Note Practical Tips e After a gigaseal is formed the patch membrane can be broken by additional suction or in some cells by high voltage pulses 600 800 mV so called Zap pulse Elec trical access to the cell s interior is indicated by a sudden increase in the capacitive transients from the test pulse and depending on the cell s input resistance a shift in the current level Additional suction sometimes lowers the access resistance causing the capacitive transients to http www heka com 64 Using the EPC 800 Patch Clamp Amplifier with pCLAMP become larger in amplitude but shorter Low values of the access series resistance are desirable and when Rs compensation is in use it is important that the resistance be stable as well A high level of Ca buffering capac ity 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 e Select the appropriate C Slow RANGE and start with C Slow set at a nonzero value If the transient is not too rapid you will be able to see the initial value of the tran stent change as you adjust R Series bring it to zero leav ing no initial step in the transient and then adjust C Slow to reduce the overall size of the transient After an iter ation or two it should be possible to reduce the transient to only a few percent of
71. ential changes is not critical Two settings are available e 2 ps e 20 us Usually a setting of 20 us is sufficient unless very fast currents such as Nat currents are studied The filter range of the EPC 800 Patch Clamp Amplifier is from 100 Hz to 100 kHz In Local mode the filter setting should be controlled through http www heka com 8 4 The Amplifier control window of PatchMaster 99 100 Hz 300 Hz 500 Hz 700 Hz a StimFilt Filter _3kHz 1 kHz vV 20us J 3kHe 5 kHz 7 kHz 10 kHz 30 kHz 100 kHz Figure 8 15 Setting the external stimulus filter within PatchMaster the front panel current filter switch In Remote mode this switch is not active and filter settings should be set through this button in the amplifier window A description of the EPC 800 filters can be found in 4 Description of the Hardware Figure 8 16 Zap sound and reset buttons within PatchMaster Zap A high voltage pulse is applied to the pipette in order to rupture the patch membrane The parameters of the ZAP pulse duration and amplitude can also be specified in the amplifier window In the configu ration window of PATCHMASTER it can be specified whether Zap is always enabled or whether it is restricted to the On Cell recording mode see Zap On Cell only Sound If this control is On a sound is played with its frequency coding for R membrane On Windows computers a sound board with MIDI ca pabilities is required to be able to use
72. entiometer is dependent upon the current clamp range In low current clamp range the http www heka com 4 2 Main Unit 25 Front Panel Filter Knob Filter 1 Filter 2 0 1 kHz 10 kHz 0 1 KHz 0 3 KHz 10 kHz 0 3 KHz 0 5 KHz 10 kHz 0 5 KHz 0 7 KHz 10 kHz 0 7 KHz 1 KHz 10 kHz 1 KHz 3 KHz 10 kHz 3 KHz 5 KHz 10 KHz 5 KHz 7 KHz 10 KHz 7 KHz 10 KHz 30 KHz 10 KHz 30 KHz 30 KHz bypassed 100 KHz 100 KHz bypassed Table 4 2 Filter settings of the current monitor VHOLD HOLD VPorrseT LFVCHOLD Figure 4 9 Command potentiometers potentiometer limit is 50 nA and in the high range is 500 pA in local mode While the instrument is remote controlled by PATCHMAS TER the maximum range for Iyorp is 100 nA and in the high range is 1000 pA The value will be displayed on the LCD panel if Vgorp is selected VPorrser The 10 turn VPorrser potentiometer is used to set an offset voltage that is added to compensate for electrode offset potentials It is typically used for zeroing of the pipette current after the pipette is inserted into the bath The range is 200 mV which is adequate for most stable electrodes The value will be displayed on the LCD panel if http www heka com 26 Description of the Hardware Vp LFVC is selected The offset can be performed manually automatically or semi automatically by pressing the black button next to the VPorrser
73. ether the am plifier is functioning properly In this example the model circuit that was shipped with the amplifier is being used as a substitute for a real patch clamp recording Some of the specifics related to the amplifier s usage with Clampex are highlighted and the screenshots should provide Clam pex users with a good frame of reference 7 1 4 1 The Model Circuit The model circuit should be connected to the probe input via a BNC adapter and the plug goes to the black GND connector on the probe http www heka com 56 Using the EPC 800 Patch Clamp Amplifier with pCLAMP 6pF 10MQ BNC to probe input Plug to GND connector The model cell Figure 7 4 Model Circuit The model circuit provides a switch with three positions simulating the following conditions typically observed during an electrophysiological ex periment e In the top position an open pipette with a resistance of 10 MQ is simulated This mode is useful for applying a test pulse and for correcting offset potentials e The middle position simulates a pipette attached to the cell mem brane after the Giga Ohm seal formation In this setting only a capacitance of 6 pF is left over corresponding to the fast capac itance of a pipette sealed to the cell membrane This mode allows you to test the C Fast compensation e In the bottom position a model cell in the whole cell patch clamp configuration is simulated The input resist
74. gh that in most experimental situations it can be neglected in view of other background noise sources that make larger contributions to the total The intrinsic noise of the amplifier can easily be checked First remove anything from the probe and shield its input with the metallic cap Sec ond the display knob on the front panel of the amplifier should be in the NOISE position The LCD display will show the rms noise current present in the current monitor signal Select the highest feedback resis tor of the preamplifier which has the lowest intrinsic noise by switching into a gain of 50 mV pA or greater The action of the internal filters on the background noise level and the temporal response can be observed by changing the filter setting a filter setting of 3 kHz is suggested In this configuration with a gain of 100 mV pA you should read a noise value below 100 fA Note Because of poor dielectric properties in the internal switch the model circuit introduces excess random noise above the level that can be obtained with a gigaseal There shouldn t be anything attached to the probe other than the shielding cap 11 2 Noise of the Recording Set Up As we consider sources of noise other than the amplifier itself it should be made clear that in this section we are concerned with random noise which is fundamentally due to the thermal motion of electrons and ions we as sume that any user who is interested in low noise recording has
75. gidata 1200 series The Local Telegraphing mode of the EPC 800 Patch Clamp Amplifier is possible by virtue of telegraphing outputs on the rear panel of the amplifier for Gain Filter Bandwidth Amplifier Mode and C Slow values As a result these amplifier features can be fully utilized by use with any AD DA interface having telegraphing inputs For example when used with the Axon Digidata 1440A Clampex software can easily be configured to receive the EPC 800 Patch Clamp Amplifier telegraphs and report the amplifier settings for gain filter and whole cell capacitance compensation http www heka com 2 1 Introducing the EPC 800 Patch Clamp Amplifier 5 The amplifier itself remains under manual control when operated in this mode and the ability to perform automatic adjustments of Vp Offset C Fast and C Slow compensations is still possible The ability to operate the amplifier in this mode exemplifies the versatility of the EPC 800 Patch Clamp Amplifier Not only do users now have the choice to operate the amplifier with non HEKA acquisition software but the amplifier can also be incorporated into experimental set ups with third party digital I O boards having telegraphing inputs as long as the interface is compatible with the chosen software The EPC 800 Patch Clamp Amplifier can be operated in Remote mode in which commands are sent and received to and from the amplifier through USB communication A USB 2 0 connection is ma
76. he following is the USB descriptor information enabling communication with the EPC 800 USB amplifier via USB HEKA s officially registered USB device constants e HEKA_VID 0x16B2 vendor ID e EPC800_PID 0x1003 product ID e EPC800_VER 0x0000 version number 12 3 List of EPC 800 USB Commands The following table shows the complete lines of USB commands to control and set the EPC 800 USB functions Command Mode Arguments or Return Value set_remote both on off set_mode remote ve cc lfvel lfvc3 lfvc10 lfvce30 lfvc100 set filter remote 100 300 500 700 1000 3000 5000 7000 10000 30000 100000 in hertz set_gain remote 0 005 to 2000 in mV pA set_rsrange remote 0 2e 6 10e 6 100e 6 in seconds 122 Appendix set_rscomp remote 0 to 120 of selected rseries value set_cslowrange remote 0 30e 12 100e 12 1000e 12 in farads set_cslowvalue remote 1 to 1000e 12 in farads set_cfast remote 0 to 10e 12 in farads set_rseries remote le5 to 999e6 in ohms set_tfast remote 0 to 8e 6 in seconds set_ihold remote le 9 to le 9 in amperes set_vp remote 2 0e 1 to 2 0e 1 in volts set_vhold remote 5 0e 1 to 5 0e 1 in volts set_lfve remote 2 0e 1 to 2 0e 1 in volts set_exstim remote off 2e 6 20e 6 in seconds auto_offset remote on off on start off deactivate function auto_cslow remote o
77. he measured membrane potential is low pass filtered and compared to the LFVC V memb potential Then a current is injected into the cell to keep the membrane potential at the chosen LF VC potential Since the cell does not distinguish currents entering through the pipette from currents crossing the membrane the low frequency voltage clamp circuit can be considered an additional membrane conductance The time constants and speeds of regulation are described in Chapter 5 Recording modes of the EPC 800 Patch Clamp Amplifier Note When operating in Local mode LF VC is turned on and http www heka com 8 4 The Amplifier control window of PatchMaster 101 the speed of regulation is selected by the front panel Mode switch Although the LFVCyotp potentiometer controls the LFVC potential it is suggested to set this value through the LFVC V memb within PATCHMASTER C memb Rp Overlay One Puise Figure 8 18 Setting sound features within PatchMaster R memb R pip This feature enables the value of R memb to be copied into the R pip This is used to store the pipette resistance in the data file before forming a seal Overlay and One Pulse When the Overlay button is selected the test pulse traces will be overlaid in the oscilloscope window The One Pulse button exceutes one test pulse This is useful when test pulses are off and simply one test pulse is to be outputted Sound Sound Volume 100 Figure 8
78. he patch then all of the additional capacitance transient will be due to the cell capacitance Canceling this transient using the C Slow and R Series controls will then give estimates of the membrane capacitance http www heka com 62 Using the EPC 800 Patch Clamp Amplifier with pCLAMP and the series resistance For adjusting these controls it is a good idea to observe the transients at high time resolution perhaps with 10 kHz filtering This will allow you to observe the effect of the R Series control which sets the initial amplitude of the transient as opposed to the C Slow control which sets the total area After compensating C Fast well the model circuit can be switched to the 0 5 GQ position This will simulate a model cell cell 22 pF membrane capacitance 500 MQ membrane resistance and 5 1 MQ input resis tance in the whole cell configuration This position can be used to verify the C Slow controls and the action of the series resistance compensation with C Slow enabled The figure below is of screenshots of the Membrane Test dialog before and after C Slow and R Series compensation On the right is the capacitance transient due to the cell capacitance The Cm and Rm values are indica tive of the model circuit values On the left is the compensated signal The correct procedure for performing a C Slow compensation is to first select the appropriate range 30 100 or 1000 pF The compensation itself ca
79. hing a grounded metal surface Gnd Connector The black pin jack carries a high quality ground sig nal which is useful for grounding the bath electrode and nearby shields without potential errors that could arise from ground loops This ground is connected directly to the signal ground on the controller through the probe s cable More details on grounding practices will be provided in chapter 9 General Patch Clamp Setup Practices which begins on page 107 Note Since the headstage case is not grounded it needs to be isolated from the micromanipulator otherwise excessive noise will be introduced Note Calibration parameters are unique to each amplifier and head stage combination Thus if you exchange the head stage be sure a new hardware calibration is performed by HEKA 4 1 1 Probe Adapters The headstage of the EPC 800 USB patch clamp amplifier is shipped with two different mounting plates for mechanical connection of the pre amplifier to various micromanipulator systems Standard Mounting Plate The headstage is mounted on a 38 mm x 90 mm x 4 mm plate which has 4 holes with 3 mm diameter Since the plate is wider than the headstage there is room for custom mounting holes on both sides of the headstage Dovetail Mounting Plate The dovetail adapter can be used to connect the headstage to a variety of micromanipulators that require a dovetail connection e g to Sutter MP 285 or HEKA MIM 4 micromanipulator The dimensions of
80. ich does not fall within the intended purpose of the amplifier is not in accordance with the liability regula tions HEKA does not accept liability for any damage caused by misuse of the EPC 800 Patch Clamp Amplifier Manipulations of the instrument are not permissible and lead to loss of liability by the manufacturer If you are uncertain regarding operating interactions safety rules or the instruction manual in general please contact HEKA before putting the EPC 800 Patch Clamp Amplifier into operation The EPC 800 Patch Clamp Amplifier instruction manual does not provide instructions for repair Any necessary repair of the amplifier has to be performed by certified HEKA specialists http www heka com 2 Introduction 2 1 Introducing the EPC 800 Patch Clamp Amplifier In continuing the tradition of providing manually controlled high qual ity patch clamp instrumentation which was established with the EPC 7 Figure 2 1 EPC 800 USB Patch Clamp Amplifier 4 Introduction EPC 7 Plus and EPC 8 Patch Clamp Amplifiers HEKA is pleased to in troduce the EPC 800 Patch Clamp Amplifier The EPC 800 Patch Clamp Amplifier is truly a unique hybrid patch clamp amplifier with its control logic and feature set primarily based upon its predecessor the EPC 8 In comparison to the EPC 8 however the EPC 800 Patch Clamp Ampli fier offers many improvements and new features that increase its overall versatility Some nota
81. ier min imal service costs and no delay in service can be guaranteed only if the instructions given in the operation manual are being followed The in struction manual should always be in proximity to the amplifier Misuse neglected inspection of the instrument or disregarding operating instruc tions may endanger the user and any third party and may cause damage to technical equipment The EPC 800 Patch Clamp Amplifier is manufactured according to cur rently applicable safety regulations The amplifier is to be operated only if working properly The amplifier should be sent immediately for repair if any technical problem occurs which may endanger the safety of any user The EPC 800 Patch Clamp Amplifier is only to be used for its intended purpose as described in the instruction manual Intended purpose in cludes regular inspection and service of the amplifier It is possible to add technical equipment to the amplifier This equip ment is not defined as an instrument according to European Community EC rules Thus equipment can only be added if it is labeled with the CE certification and has an accompanying statement certifying conformity with EC rules Only technical equipment approved by HEKA can be added to the am plifier Information concerning this matter will be provided on request by our technical support team Any further use of the EPC 800 Patch Clamp 2 Safety Guidelines Amplifier and added equipment wh
82. ignals aaa 53 7 1 4 Membrane Test with Model Circuit aaa 55 http www heka com CONTENTS iii 7 1 4 1 The Model Circuit 0 55 7 1 4 2 Open Pipette and VPorrser 57 7 1 4 3 Forming a Gigseal 0 59 7 1 4 4 Whole Cell Configuration 61 7 1 4 5 Whole Cell Voltage Clamp 66 7 1 4 6 Whole Cell Current Clamp 68 7 2 Local Telegraphing Mode ononon 71 7 2 1 Telegraphing Outputs aoaaa 71 7 2 2 Configuring Telegraphs in Clampex 72 7 3 Remote Control through Soft Panel 0 75 8 Using the EPC 800 USB patch clamp amplifier with Patch Master 79 8 1 Software Installation oo 0 0 000 79 8 1 1 Dongle driver onnu 80 8 2 Software Startup and Configuration 80 8 3 Software Operation ooo 2000000008 83 8 amp 3 TocalMo de riean te Grad a SO a g ALE a 83 8 3 2 Remote Mode osuna aae 83 8 4 The Amplifier control window of PatchMaster 84 8 4 1 Main Controls 0008 85 8 4 2 Show All Controls es soci ere a eraa De s 100 8 4 3 Current Clamp Recording 102 8 4 3 1 Bridge Compensation 104 8 4 3 2 Voltage Bandwidth in Current Clamp R cordings t a 40 a 8 ce ed aA 105 http www heka com CONTENTS 9 General Patch Clamp Setup Practices 9 1 Mounting the Probe 9 2 Ground Wires 0 9 3 Grounding the Microscope
83. ing C Fast and tau can either be performed manu ally by turning the 10 turn potentiometer automatically or semiautomat ically by pressing the black Auto button adjacent to the C Fast poten tiometer Selection of this button performs an automatic compensation of C Fast and r Fast As long as the Auto button is still active as shown by the solid green LED next to the black button changes by moving the C Fast potentiometer or 7 Fast knob will effect in relative scale as imple mented for VPorrsger Again the value shown at the display is marked with showing an automatically obtained value Deactivating Auto C Fast the value of C Fast and tau as determined by the Auto operation is lost and the value of them are determined by the reading of the potentiometer Note that starting Auto C Fast compensa tion performes the compensation only once the value is not dynamically adjusted over time During the algorithm optimises C Fast and tau the external stimulus input is deactivated Nevertheless holding potential regardless of its constituents whether applied by external stimulus input of by using the Vhold knob http www heka com 28 Description of the Hardware is measured and applied to the cell Note If the Auto C Fast button is pushed while the ampli fier is NOT in voltage clamp mode an error message will be displayed on the display 7 Fast control This knob determines the time constant of C Fast up to 8us The valu
84. ing the AD inputs and recording mode within Patchmaster 92 8 10 Setting the test pulse parameters within Patchmaster 94 http www heka com LIST OF FIGURES 137 8 11 8 12 8 13 8 14 8 15 8 16 8 17 8 18 8 19 8 20 8 21 8 22 8 23 8 24 9 1 Liquid Junction and pipette offset features of PatchMaster 94 C Fast compensation within Patchmaster 95 C Slow compensation within Patchmaster 97 Setting the R compensation speed within Patchmaster 98 Setting the external stimulus filter within PatchMaster 99 Zap sound and reset buttons within PatchMaster 99 Low frequency voltage clamp LF VC settings within Patch Mastera sidari ts ale OO Bayes ae pa ae ae lo 100 Setting sound features within PatchMaster 101 Setting sound features within PatchMaster 101 I Scale and V Scale settings of the test pulse within Patch Master soporte sep ee et ae lis Oe es Sa hs eck Mak wy eels 101 Hardware scaling of the ITC 18 interface 102 Current injection to MC 10 model circuit with Bridge Com pensation OFF sort s tae foe ale bb Ob WA A ey 104 Current injection to MC 10 model circuit with Bridge Com Penisation ON vee ce select ee BARR eo ele Ge eI eee 105 Power spectra of voltage recordings in Current Clamp mode 106 Example of agar salt bridge reference electrode 110 CAGES pepe 8S E Sa ee eh ar hated Oe ag 1D AG nce hes ae let 125 http www heka
85. ing the EPC 800 Patch Clamp Amplifier with PatchMaster 5 Connect the power cord to the EPC 800 USB The internal power supply used in the EPC 800 USB is an auto switching multi voltage supply that will operate from 90 Volts to 250 Volts Make sure that the EPC 800 USB power cord is plugged into a properly grounded AC receptacle Improper grounding of the EPC 800 USB could result in an electrical shock hazard It is advisable to plug all equipment into a common outlet strip This will minimize power line induced noise in the system 6 Place the EPC 800 Patch Clamp Amplifier in its final place and connect the cable of the headstage to its Probe connector on the front panel of the EPC 800 Patch Clamp Amplifier main unit It is suggested that the amplifier is switched off before connecting the headstage 7 The EPC 800 Patch Clamp Amplifier does not require the installa tion of any drivers It may be that drivers are required by other http www heka com 3 1 Unpacking and Connecting the EPC 800 Patch Clamp Amplifier 15 hardware but the scope of this manual does not cover installation requirements of second source options 3 1 1 Static Electricity The input circuitry of the probe can be damaged by static electricity To avoid this please observe the following rules 1 Avoid touching the input terminal unnecessarily 2 When it is necessary to touch the input e g while inserting a pipette into the holder g
86. ing the mouse by selecting the Auto button in the C Slow section of the amplifier control panel for an automatic compensation of C Slow and R Series or by executing the predefined Whole Cell protocol Off 30 pF Range 100 pF V 100 pF C slow _21 90 pF_ Auto 1000 pF ee Figure 8 13 C Slow compensation within Patchmaster Note When operating the EPC 800 Patch Clamp Amplifier in Remote mode the C Slow range knob on the front panel does NOT have any effect on activating C Slow The range has to be set through PATCHMASTER R series Adjusts the resistance in series with the slow capacitance range 0 1 MO 10 GO to determine the time constant of the C slow transient and also for R compensation Adjustment is limited by the capacitance values and the range as described above In Remote mode the value can be changed manually by dragging the mouse or typing or automatically by clicking on Auto In Local mode R series is adjusted by the front panel potentiometer or by executing an Auto C Slow compensation from the front panel button Auto C Slow Selecting this function in Remote mode performs an automatic compensation of C slow and R series These settings are used by the R compensation circuitry as the measure of series resistance Auto compensation works best when C Fast is canceled beforehand in the cell attached configuration Note In Remote mode ONLY C Slow compensation can be performed automatically through PATCHM
87. ious time constants for the low frequency voltage clamp can be selected 1 3 10 30 100 us These time constants de scribe the speed of regulation The effective feedback speed for the five possible response settings depends on the gain range In the medium gain range 1 100 is approximately the time in seconds whereas in the high gain range it is a hundred times faster Note that the LFVC value of 100 means slow adjustment or tracking whereas the LFVC value of 1 means fast adjustment or tracking to the given set point http www heka com 40 Recording Modes of the EPC 800 Patch Clamp Amplifier http www heka com 6 Theory of Compensation Procedures 6 1 Offset Compensation In all patch clamp configurations a number of offsets have to be taken into account These include amplifier offsets electrode potentials liquid junction potentials and potentials of membrane s in series with the mem brane under study Some of these offsets are fixed during an experiment such as amplifier and electrode offsets while others are variable It is standard practice to take care of the fixed offsets by performing a reference measurement at the beginning of an experiment Thereby an adjustable amplifier offset is set for zero pipette current Thereafter the command potential of the amplifier will be equal in magnitude to the membrane potential if no changes in offset potentials occur The polarity of the command potential will be that
88. ip into the bath and also to avoid going through the air water inter face more than once before forming a seal When you do move the pipette tip into the bath the current trace may go off scale check clipping in that case reduce the gain or adjust the VPorrser potentiometer until the trace reap pears http www heka com 7 1 Local Mode 59 7 1 4 3 Forming a Gigseal Moving the model circuit switch to the middle position will leave only a capacitance of about 6pF connected This simulates a Gigaseal and the C Fast controls can be used to cancel the capacitive spikes resulting from the stimulus test pulse In order to see the small currents resulting from the high resistance of the model circuit the amplifier gain can be set to 20 or 50 mV pA As illustrated in the figure below two fast capacitive transients are coming from the 6 pF capacitor in the model circuit C Fast and 7 Fast compensations can be done manually by turning the front panel potentiometer and knob or automatically by pressing the black Auto C Fast button If performing manually as you approach a value close to 6 pF you should see the spikes becoming smaller As soon as you are overcompensating you will see the spikes going in the opposite direction This indicates that you should decrease C Fast using the model circuit it is not very critical to misadjust 7 Fast Continue adjusting C Fast and 7 Fast until you see an almost flat line Membrane Test
89. ip sharply convergent an air stream can be directed at the filament The amount of current to pass through the filament must be determined empirically for each type of glass but a good place to start is with sufficient current to get the filament barely glowing The typical practice is to turn http www heka com 114 Patch Pipettes on the filament current and move the filament toward the pipette which being stationary should remain in focus Since the opening in the pipette tip is usually at the limit of resolution of viewing you might not see the change in shape at the tip but instead only a darkening 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 10 5 Use of Pipettes Pipettes should be used within 2 3 hours after fabrication 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 pipettes may even be used the next day It is very important to filter the filling solutions e g using a 0 2 um syringe filter Pipettes can be filled by sucking up a small amount of solution through the tip This can be done by capillary force simply dipping the tip for a few seconds in a beaker containing the pipette solution or by applying negative pressure at the
90. isplay the following signals e AD 0 15 Any of the AD channels The AD channel connections have to be configured in the Configuration Hardware section of PATCHMASTER In the example above the voltage monitor is connected to AD 0 and the current monitor is connected to AD 1 These match the external connections discussed earlier in this chapter Recording Mode Sets the Recording Mode e In Out Sets the Inside Out mode e On Cell Sets the On Cell mode e Out Out Sets the Outside Out mode e Whole Cell Sets the Whole Cell mode e C Clamp Sets the Current Clamp mode http www heka com 8 4 The Amplifier control window of PatchMaster 93 Note For cell attached or inside out patch configuration pos itive pipette voltages correspond to a hyperpolarization of the patch membrane and inward membrane currents appear as pos itive signals at the Current Monitor outputs The PATCHMAS TER program compensates for this by inverting digital stimulus and sampled values in these recording configurations such that the stimulation protocols holding voltages and displays of cur rent records in the oscilloscope all follow the standard electro physiological convention In this convention outward currents are positive and positive voltages are depolarized However the analog current and voltage monitor outputs are not inverted in these recording modes Test pulse Test pulses are added to the holding potential and app
91. layed on the LCD panel if Vp LFVC is selected This value determines the average potential during which potential deflections are measured More details of the LFVC mode are provided in the chapter 5 Recording Modes of the EPC 800 Patch Clamp Amplifier 4 2 4 Capacitance Compensation The capacitance compensation circuitry is used to cancel the large artificial currents that flow when the patch potential is suddenly changed for ex ample as done in experiments on voltage activated channels The C Fast circuitry is used to cancel the rapidly decaying currents that charge the pipette and other stray capacitance while the C Slow circuitry is mainly http www heka com 4 2 Main Unit 27 used in whole cell recordings to cancel the slower transients arising from the charging of the cell capacitance The use of these controls is discussed below and in several other places throughout this manual see chapters 7 Using the EPC 800 Patch Clamp Amplifier with pCLAMP and 8 Using the EPC 800 Patch Clamp Amplifier with PatchMaster C SLOW C FAST Figure 4 10 Compensation potentiometers C Fast control This potentiometer is used to cancel fast capacitive currents that charge the pipette and other stray capacitances range 0 15 pF With nothing connected to the Probe input cancelation is typically obtained at a setting of 1 1 5 pF due to the residual input capacitance of the current measuring amplifier C Fast compensation us
92. lied to the pipette the current responses are sampled and displayed Two built in test pulse types are available single or double pulse Additionally any user defined pulse pattern can be used as a test pulse Test pulses are applied at maximal rates depending on the duration specified Amplitude Length Duration and amplitude of the built in test pulses can be specified in the dialog The minimum pulse duration is 1 ms with 100 points sampled per pulse i e the sampling interval is 1 100 times the pulse duration Noise The Noise button can be used to measure the internal noise of the amplifier with shielded probe input or the noise of the environment with open probe input When the noise mode is selected the rms noise is continuously measured and updated For the determination of the noise level there are no pulse outputs and the current is sampled via the active AD channel using the current filter setting It is sampled in sections of 10 times 256 points with a sample interval of 100 ys i e a total length of 256 ms The noise level depends on the gain range and on the current filter setting Reasonable noise values are given in chapter 11 Low Noise Recording http www heka com Using the EPC 800 USB patch clamp amplifier with 94 PatchMaster O off Test Pulse Amplitude Length O noise Figure 8 10 Setting the test pulse parameters within Patchmaster Liquid junction LJ LJ is a variable to be set by the user
93. low and medium gain range only http www heka com 4 2 Main Unit 29 Slow capacitance cancelation ranges 30 100 1000 pF can be set to any desired value However in the high gain range 50 GQ resistor the 1000 pF range will not operate If the gain is set to a value higher than 20 mV pA while the 1000 pF range is selected the auto C Slow cancelation will not be performed An error message will temporarily be displayed on the display Note C Slow compensation is only available in VC mode R Series Adjusts the resistance in series with the slow capacitance total range capability 0 1 200 MQ to determine the time constant of the C Slow transient and also for Rg compensation The adjustment is limited by the selected C Slow range and the actual value of C Slow a 30 pF C Slow range enables an R Series range of 3 5 1000 MQ 100 pF enables a range of 1 1 1000 MQ and 1000 pF enables 1 1000 MQ The value can be set manually of by using Auto C Slow compensation The minimum values of R Series depend on the actual device and might offer even lower values The setting of this control is used by the Rs compensation circuitry as the measure of the series resistance as well 4 2 5 Series Resistance Compensation Rs Comp The series resistance compensation corrects for membrane voltage errors under conditions of high access resistance between the pipette and cell interior see Chapter 6 Theory of Compensation Proce dures sta
94. mp Amplifier to the telegraphing inputs on the rear panel of the Digidata 1440A Again these are examples that can be changed as long as they are configured correctly from within the software 7 2 2 Configuring Telegraphs in Clampex The hardware connections above now have to be configured from within the Clampex program Clampex telegraphs are configured in the Configure gt Telegraphed Instrument dialog The first step is to select the Telegraphed Instrument When the software is first loaded the EPC 800 Patch Clamp Amplifier will not be included in the list of available telegraphing instru ments so it will have to be added as a user defined telegraphed instrument to the UserDefinedInstruments ini file Once added it will be visible to the user in the application s telegraphed instruments configuration box The second step is to choose the digitizer channels to which the telegraphs are connected These should match the physical connections listed in the previous table http www heka com 7 2 Local Telegraphing Mode 73 Telegraphed Instrument Input Channels Telegraphed Instrument HEKA EPC 800 USB Ms Analog IN 1 Analog IN 2 Analog IN 3 Analog IN 4 Analog IN 5 Analog IN 6 Analog IN 7 Analog IN 8 Analog IN 9 Analog IN I0 Telegraph Connections Choose the digitizer channels to which telegraphs are connected Gain Telegraph InputO T Cm Telegraph Input 2 v Erequency Telegraph Input
95. n off on start off deactivate function auto_cfast remote on off on start off deactivate function http www heka com 12 3 List of EPC 800 USB Commands 123 soft_reset remote any auto functions are aborted and the am plifier is re initialized The status of the EPCs800 after the soft reset is Gain ImV pA Mode VC RS Comp off Current filter 3 KHz EXT STIM 20 ps Comp 0 C stow 1 pF C fast 0 R Series 5 MQ C Slow Range off tau fast 0 VHold 0 IHold 0 VPoffset 0 LFVChold 0 Auto Csiow LED off Auto Cfast LED off Auto V Poffset LED off http www heka com 124 Appendix hard_reset remote equivalent to powering the EPC800 off and on Remote is off and all parameters are set based on the current front panel settings get_remote both string get_mode both ve cc lfvel lfve3 lfve10 lfve30 lfve100 get_filter both number in Hz get_gain both number in mV pA get_rsrange both number in s get_rscomp both number in percentage get_cslowrange both number in F get_cfastvalue both number in F get_cfast both number in F get_rseries both number in 2 get_tfast both number in s get_ihold both number in A get_vp both number in V get_vhold both number in V get_lfve both string get_extstim both string get_noise both
96. n be performed automatically by pressing the black Auto C Slow but ton Alternatively it could be performed manually by turning the C Slow and R Series potentiometers With some practice you will develop a good feeling for these parameters and how they affect the recording With in creasing quality of the compensation you should approach the real values of the model circuit and the transients should disappear The compensated signal on the right is a good example of a well compensated whole cell transient http www heka com 7 1 Local Mode 63 Figure 7 7 Automatic or manual C Slow and R Series compensation The left panel displays the capacitance transient due to the cell capacitance and the right side illustrates the effects of an automatic C Slow compensation Important note If either or all three of the automatic rou tines of the EPC 800 Patch Clamp Amplifier are performed through the front panel Auto buttons of VPorfset C Fast or C Slow it is suggested you keep them on If you were to turn the Auto off by pressing and holding the Auto button the val ues obtained during the Auto procedures would be lost and the values from the settings of the front panel potentiometers would be in effect If you wish to manually fine tune an auto com pensation procedure it is best to make a note of the compen sation values obtained during the auto procedure and th
97. n in Voltage Clamp mode It basically compensates the voltage drop via the series access resistance of the electrode Rs With the standard HEKA model circuit this effect is rather difficult to see since the voltage drop across the 5 1 MQ is small The voltage drop across R is seen as an instant step in the voltage trace when injecting a current step into the cell With the model circuit we expect the size of this voltage step to be R istep With R 5 1 MQ and i step 100 pA this initial voltage drop is just 0 51 mV in amplitude In order to reveal this step on the oscilloscope the length of the test pulse should be decreased to 1 ms C Fast set to 0 pF and the resolution of the voltage scaling increased e g use V mon 100 and V scale 20 Figure 8 22 Current injection to MC 10 model circuit with Bridge Com pensation OFF In order to compensate this initial voltage step please turn the R comp ON called Bridge Compensation in current clamp and set the comp knob to 100 Now the circuitry compensates 100 of the value set with the R control of the C Slow compensation http www heka com 8 4 The Amplifier control window of PatchMaster 105 SuisteseesGsencssccbaseencectsecssueseesse aneasinas Faass MOO ys Y 100 pA 999 i i Figure 8 23 Current injection to MC 10 model circuit with Bridge Com pensation ON In case a little step will reappear during the current clamp experiment you can re
98. nable the amplifier to be used in local telegraphing mode provided that the AD DA interface being used is equipped with telegraphing inputs Third party acquisition software programs such as pCLAMP will be able to read the status of these amplifier settings while recording data The knobs and switches on the front panel remain under manual control A more detailed example of how to use the telegraphing outputs of the EPC 800 Patch Clamp Amplifier are provided in the Local Telegraphing Mode section of chapter 7 Using the EPC 800 Patch Clamp Amplifier with pCLAMP and 12 4 TELEGRAPHING OUTPUT GAIN BANDWIDTH MODE C SLOW Figure 4 12 Telegraphing outputs USB This port is the connection to the USB 2 0 port in the host com puter It allows the computer to communicate with the EPC 800 USB in remote mode http www heka com 4 2 Main Unit 33 Sound The EPC 800 USB has a sound generator built in A 3 5 mm output receptacle with a frequency range of 200 Hz to 4 kHz allows con nection of an active speaker or a headset If in manual mode the knobs are in a setting that is not supported e g high gain and CC a beep is output http www heka com 34 Description of the Hardware http www heka com 5 Recording Modes of the EPC 800 Patch Clamp Amplifier The EPC 800 PATCH CLAMP AMPLIFIER is fundamentally an instrument for measuring small electrical currents It uses a current to voltage I V conver
99. nges of the holding potential created by the EPC 800 or by external stimulus input are taken into account The mode can only be accessed in voltage clamp mode To deactivate the mode hold Auto C Slow and Auto C Fast button for some seconds 4 2 7 Display Noise and Remote Display Selector and LCD Panel An LCD panel can display the following parameter pairs I VMon C FAST 7 FAST C Slow R Series RS Range Comp Vp LFVC I VHold and Noise If the Auto display mode is activated the LCD panel will automatically display for 3 seconds the value of any control as it is modified by the user Noise A detailed description of how to record the intrinsic noise of the amplifier can be found in in chapter 11 Low Noise Recording When the display knob is in the Noise position the LCD display will show the RMS noise current present in the current monitor signal http www heka com 4 2 Main Unit 31 RS RANGE COMP REMOTE C SLOW R SERIES VP LEVC C FAST x FAST iVaouo o LIVMON NOISE f AUTO DISPLAY Figure 4 11 Multi parameter display REMOTE This LED lights when the amplifier is controlled and operated through a series of USB commands A USB 2 0 connection is made between the amplifier rear panel and the host computer In REMOTE mode all of the front panel knobs and switches of the amplifier are inactive with the exception of the LCD multi position switch 4 2 8 Knob Sensitivity The knobs COMP C Slow CFas
100. noise into the pipette interior Clearly making thicker coatings especially in the tip region and coating closer to the tip will reduce the pipette http www heka com 118 Low Noise Recording noise The best glass type we know of is aluminosilicate this glass re quires fairly high temperatures in pulling and does not necessarily give the best gigaseals but its dielectric relaxation appears to be about a order of magnitude smaller than soft glass Some improvement is probably to be gained by taking steps to prevent formation of aqueous films on the back end of the pipette It is a good idea to wipe the outside of the pipette to remove any spilled solution fingerprints etc before inserting it into the holder It might also help to treat the inside of the pipette to prevent the formation of a film for example by shooting some dimethyl dichlorosilane 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 noise currents arising in the pipette glass A significant amount of noise seems to arise in the sealed membrane itself and is probably lower in higher resistance seals This noise is gener ally more than one would calculate from the resistance of the gigaseal The usual goal of low noise recording is better time resolution if the noise level is lower you can use a wider filter bandwid
101. not only the speed but also the stabil ity of the circuit Recording and following rapid events such as fast action potentials AP with patch or intracellular electrodes is possible Additional information related to the bridge compensation in current clamp mode of the EPC 800 patch clamp amplifier can be found in the Bridge Compensation section of chapter 8 Using the EPC 800 Patch Clamp Amplifier with PatchMaster The capacitance of the electrode and to some extent the amplifier can be neutralized by the C Fast setting which acts as a capacitance neutraliza tion adjustment in the Current Clamp mode However like capacitance neutralization settings on conventional microelectrode amplifiers exces sive capacitance neutralization can result in oscillation 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 C Fast is then automatically adjusted to neutralize all but the http www heka com 38 Recording Modes of the EPC 800 Patch Clamp Amplifier amplifier input capacitance when you switch to current clamp mode The EPC 800 Patch Clamp Amplifier has two possible current clamp OUT PUT gain ranges When switching from voltage clamp to current clamp bridge mode which of the two current clamp OUTPUT gain ranges being used is dependent upon the voltage clamp gain range setting before switch ing If for ex
102. now act as a bridge balance For the user these changes may be of little consequence and are mainly designed to make current clamp record ing simple and reliable The voltage monitor Vmon should automatically be selected to become your active channel displayed in the oscilloscope Note that the unit of the test pulse amplitude changes from mV to pA as soon as you switch from Voltage Vlamp VC into Current Clamp CC mode PATCHMAs TER uses two 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 will charge the membrane of the model cell at a time constant T Rm X_ Cm 500 MQ 22 pF 10 ms to a final value of Vinae Rm SI 500 MQ 100 pA 50 mV Due to the slower time constant compared with 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 Note In contrast to voltage clamp conditions were T is pro portional to the access or series resistance Rs of the pipette in current clamp experiments T depends on the membrane re sistance Rm http www heka com Using the EPC 800 USB patch clamp amplifier with 104 PatchMaster 8 4 3 1 Bridge Compensation Bridge Compensation in Current Clamp mode acts similar to the R com pensatio
103. ntial or spontaneous action potentials in a whole cell recording The measured membrane potential will be shown on the V mon display while http www heka com 24 Description of the Hardware the current is held at a commanded value I Hold The low frequency voltage clamp LF VC mode is a modified current clamp mode that allows for the measurement of potential deflections such as action potentials or synaptic potentials while the average potential is kept constant at a value chosen by the user LF VC V HOLD FILTER Figure 4 8 Filter knob The EPC 800 Patch Clamp Amplifier has a filter knob on the front panel that ranges from 0 1 to 100 kHz This is an integrated filter comprised of two individual internal filters filter 1 and filter 2 Filter 2 is a 4 pole tunable lowpass Bessel filter which can range depending on the actual instrument up to 20 kHz Filter 1 is a 5 pole 10 to 100kHz lowpass Bessel pre filter Table 4 2 summarizes the various filter combinations of the current monitor 4 2 3 Command Signal Processing These controls consist of the Vyorp JInotp VPorrser and LFVCyo tp potentiometers Vuioxtp The 10 turn Vgorp potentiometer is used to set the holding potential in Voltage Clamp mode The range is 500 mV The value will be displayed on the LCD panel if I Vioxp is selected Iyorp The 10 turn IHorp potentiometer is used to set the holding cur rent in Current Clamp Bridge mode The range of the pot
104. o the selected range i e with a CC scal ing of 10 pA mV only the low gain range 0 005 0 2 mV pA can be used in current clamp mode The table below summarizes the main features of the two current clamp gain ranges http www heka com 5 3 Low Frequency Voltage Clamp Mode 39 Starting IMAX CC Stim IHoLD VC Gain Scaling Knob Ad Range justment mV pA Range Low Gain 0 005 0 2 100 nA 10 pA mV 50 nA Medium 0 5 4 20 or 1nA 0 1 pA mV 500 pA Gain 50 2000 Table 5 1 Features of current clamp gain ranges of the EPC 800 Patch Clamp Amplifier 5 3 Low Frequency Voltage Clamp Mode The low frequency voltage clamp mode is a modified current clamp mode that allows for the measurement of potential deflections such as action po tentials or synaptic potentials while the average potential is kept constant at a value chosen by the user with the LDFVCyoz p potentiometer The circuit thus works like a current clamp for fast signals and like a voltage clamp for low frequency signals To achieve this the measured membrane potential is low pass filtered and compared to the LDFVCyorp potential Then a current is injected into the cell to keep the membrane potential at the chosen LFVC potential Since the cell does not distinguish currents entering through the pipette from currents crossing the membrane the low frequency voltage clamp circuit can be considered an additional mem brane conductance Var
105. of the membrane for whole cell and outside out configurations but will be inverted in the cell attached and inside out configurations In cell attached configuration an additional offset is present due to the resting potential of the cell under study Liquid junction potentials may appear or disappear during the measurement when solution changes are performed or in the case that the pipette solution is different from the bath solution Barry amp Lynch 1991 Neher 1992 Neher 1995 These problems are handled by applying the appropriate corrections and sign inversions during off line analysis An analysis of the underlying offset problem and justification for the procedures can be found in Neher 1995 The rule for calculating the Offset Sum LJ is to form the sum of all changes in offsets which occur between the reference measurement and the test measurement The polarity of a given offset voltage should be taken as viewed from the amplifier input positive if positive side of the voltage 42 Theory of Compensation Procedures source is closer to the input A sign inversion has to be applied if the offset under consideration disappears A procedure how to measure liquid junction potentials is described in Neher 1992 Ion mobilities for calculation of liquid junction potentials can be found in Barry amp Lynch 1991 The EPC 800 Patch Clamp Amplifier enables automatic or manual adjust ment of the offset potential in the rang
106. ormation Second according to Barry amp Lynch the potentials are defined with opposite polarity as those for patch clamp experiments bath vs electrode instead of elec trode vs bath Thus values in the table can be taken as they are and entered as such in the LJ control If however a liquid junction potential appears during a measurement e g during solution changes then only one sign inversion applies In that case the sign of the value in the table must be inverted before adding it to the Correction Sum In the following some specific examples together with explanations will be given In all these cases it is assumed that the reference measurement is performed in standard saline Example 1 An outside out or whole cell measurement with normal saline in the pipette In this case LJ should be set to zero This is one of the few measurements which do not require any correction It is quite unphysiological however Example 2 An outside out or whole cell measurement with KCl based internal solution in the pipette LJ should be set to 3 mV see table in order to correct for the disappearance of a liquid junction potential between the KCl containing pipette and the NaCl based bath solution Example 3 An episode with low chloride bath solution during the ex periment of example 2 It is assumed that the reference electrode in the bath includes a salt bridge such that the change in Cl concentration is not seen by the
107. otation This protocol preceeds each series with AutoC Slow Repeat Status IF etc Result p Figure 8 7 The protocol editor window of PatchMaster showing predefined protocols as part of the EPC800 pro file As shown in the top row numbered 1 through 6 of the figure above there are predefined protocols called Examplel Example2 Link Buffer SETUP and SEAL In this picture the commands compris ing the Example2 protocol are displayed it is a protocol that will execute an automatic C Slow compensation prior to a series and will abort if the value of R Series exceeds 8 MQ Additionally there is a protocol called WHOLE CELL in position 7 These protocols can freely be edited and new protocols can be created all from within the protocol editor window For a complete description of all of the protocol editor features within PATCHMASTER users are encouraged to consult the PATCHMASTER users manual A link to the predefined protocols called SET UP SEAL and WHOLE CELL are also present from within the amplifier window of PATCHMASTER Figure 8 8 SET UP SEAL and WHOLE CELL protocols Set Up This protocol when executed sets the default recording mode to whole cell sets the gain of the amplifier to 5 mV pA create a rectangu http www heka com Using the EPC 800 USB patch clamp amplifier with 90 PatchMaster lar tes
108. own as the prediction pathway in figure 6 1 below and it accelerates the charging of the membrane capacitance by imposing large transient volt ages on the pipette when step changes are commanded this is sometimes called supercharging These voltages would occur due to the action of the correction pathway alone as the large capacitive charging currents elicit pipette voltage changes however when these currents are canceled by the transient cancelation their effect must be predicted by the cancelation circuitry hence the prediction pathway s IV Converter Current Monitor O Cslow Stim In VA Transient Gen Bath Electrode Cv hold Pred Loop Figure 6 1 Series Resistance Compensation Together the two parts of the EPC 800 Patch Clamp Amplifier R compensation circuitry cancel the effects of a fraction a of the series re sistance This means that the charging of the membrane capacitance is accelerated with a time constant under compensation of http www heka com 6 3 Series Resistance Compensation 47 Te 1 a Tu where 7 is the uncompensated time constant Similarly the voltage errors due to membrane currents are also reduced by the factor 1 a The frac tional compensation is determined by the setting of the COMP control on the EPC 800 USB front panel For proper compensation however the circuitry needs to have an estimate of the total series resistance for the co
109. patch clamp amplifiers the range changing capa bility of the headstage the extremely wide bandwidth available from the current monitor circuitry and the integrated transient cancelation auto matically if desired and series resistance compensation functions In Cur rent Clamp mode the EPC 800 Patch Clamp Amplifier acts as a voltage follower similar to classical microelectrode amplifiers which guarantees very fast and accurate membrane potential recordings Magistretti et al 1996 Together these features mean that a single headstage suffices for both single channel and whole cell recordings and that both kinds of recordings can be made with high time resolution and low noise 2 2 Firmware Version After the EPC 800 Patch Clamp Amplifier is started the firmware is shown on the display This manual describes capabilities of EPC800 firmware version 3x0x90 http www heka com 2 3 References 7 2 3 References Further Reading This manual is designed to provide a general guide for setting up and using the EPC 800 Patch Clamp Amplifier Specific examples for the various modes of operation are given and general information about the hardware and basic principles of the EPC 800 Patch Clamp Amplifier functions are provided It is assumed that the reader has some familiarity with patch clamp tech niques Should you be a newcomer to the field perhaps the best place to start would be the paper by Hamill et al 1981 whe
110. potentiometer If an auto V Porrset procedure is executed and the compensation exceeds the 200 mV range an error message will be displayed on the front panel LCD stating Auto Vpoff error Range exceeded One shall reduce the gain and try again After VPorrser is adjusted one shall set the gain back to the intended value and perform auto VPorrser again if required The auto button calls a procedure for automatic zeroing of the pipette current During execution the green LED on the front panel will blink and is completed when the LED stops flashing and remains lit If the display knob is set on VPorrser a will be shown before the digits to indicate an automatic optimization After VPorrser is set by the algorithm one can turn the potentiometer to do fine adjustment Changes on the potentiometer reading will be interpreted on a relative scale The auto feature can be disengaged by pressing and holding the auto button until the green light goes out It is very important to note that once the auto feature is turned off the value obtained during the auto procedure is lost and the value taking effect will be the potentiometer reading Note that starting VPorrser performes the compensation only once the value is not dynamically adjusted over time LFVCyozp When the amplifier is being used in the modified current clamp LFVC mode the LFVC potential is specified by the 10 turn LFVCyoxp potentiometer The range is 200mV and it is disp
111. r fere with good capacitance compensation Hard glass pipettes often have a narrow shank after pulling and consequently a higher resistance Hard glasses tend 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 most glasses is very low soft glasses in particular have a conductivity around 1 kHz that is sufficiently high to become the major source of thermal noise in a patch clamp recording We find that Kimaz glass is a good compromise for whole cell recording Borosilicate and especially aluminosilicate glasses Rae and Levis 1984 have low dielectric loss and are desirable for the lowest noise recordings They do not necessarily form the best seals however this might be due to evaporation of metal onto the glass surface during the high temperature pulling and polishing steps 10 2 Pulling Pipettes are pulled in two stages the first to thin the glass to 200 400 wm at the narrowest point over a 7 10 mm region and the second to pull the two halves apart leaving clean symmetrical breaks Both halves can be http www heka com 10 3 Coating 113 used The length of the first pull and the heat of the second pull are the main determinants of the tip diameter of the final pipette A number of commercial pullers can be used to make pipettes For repro ducibility however a regulat
112. r amplifier window for remote mode of operation Gain Sets the scaling of the current monitor output The range is 0 005 to 2000 mV pA and can be set by dragging the mouse or by pressing the http www heka com Using the EPC 800 USB patch clamp amplifier with 86 PatchMaster up and down keys on the keyboard The gain setting automatically selects one of the three available current measuring feedback resistors in the probe 5 MQ 500 MQ and 5 GQ corresponding to low medium and high gain ranges A full written description and table summarizing the main features and limitations of the gain ranges can be found in Chapter 4 Description of the Hardware 0 005 0 010 0 020 0 050 01 0 2 0 5 1 0 2 0 5 0 Gain V membrane a 10 m jpA 40 0 m 100 200 500 1000 2000 Figure 8 4 Gain and V membrane control Clipping Indicator A blinking box labeled Clip in the Gain title indicates saturation of amplifiers in the current monitor circuitry Like the Clipping LED on the EPC 800 main unit this is a warning that excess artifacts or noise may occur due to the saturation of amplifiers Note This indicator may appear to be more sensitive than the LED on the EPC 800 Patch Clamp Amplifier It is not it just latches the clipping status longer than the LED light V membrane The V membrane control should be used to set a holding http www heka com 8 4 The Amplifier control window of PatchMaster 87 poten
113. re the basic gigaseal techniques are described and the first three chapters of Single Channel Recording B Sakmann amp E Neher eds Plenum Press New York 1995 Certainly it will be worthwhile to read this manual carefully Many users will want to read some of the more advanced and complete discussions of individual topics which can be found in original articles and in the books Single Channel Recording B Sakmann amp E Neher eds Plenum Press New York 1995 and Methods in Enzymology vol 207 Academic Press New York 1992 Original Articles Hamill O P Marty A Neher E Sakmann B amp Sigworth F J 1981 Improved patch clamp techniques for high resolution current recording from cells and cell free membrane patches Pfliigers Arch 391 85 100 Magistretti J Mantegazza M Guatteo E amp Wanke E 1996 Action potentials recorded with patch clamp amplifiers are they genuine TINS 19 530 534 Neher E 1981 Unit conductance studies in biological membranes In Techniques in Cellular Physiology P F Baker ed Elsevier North Hol land Neher E amp Sakmann B 1976 Single channel currents recorded from membrane of denervated frog muscle fibres Nature 260 779 802 http www heka com 8 Introduction Rae J amp Levis R 1984 Patch clamp recordings from the epithelium of the lens obtained using glasses selected for low noise and improved sealing properties Biophys J 45 1
114. rent Clamp mode the cell membrane potential is recorded which can be monitored at the Voltage Monitor output of the EPC 800 Patch Clamp Amplifier and seen on the I Vmon display If C Slow has been compensated up to this point switch from voltage clamp to current clamp recording either by choosing the CC Bridge mode on the front panel Local mode or switch into the Current Clamp mode by selecting C Clamp from the Mode popup in the amplifier window of PATCHMASTER Remote mode In Current Clamp mode you should use membrane in PATCHMASTER http www heka com 8 4 The Amplifier control window of PatchMaster 103 to set a holding current and you can apply stimulus pulses via External Stim INPUT CC The scaling of the External Stim Input CC is auto matically set depending on the selected current clamp OUTPUT gain For a review of the characteristics of the two possible current clamp OUT PUT gain ranges of the EPC 800 Patch Clamp Amplifier see chapter 5 recording Modes of the EPC 800 Patch Clamp Amplifier It should be stressed that this automatic use of appropriate scaling is unique for use of the amplifier with PATCHMASTER and EPCMASTER software With other software programs such as pCLAMP scaling is set manually When you switch to the CC Bridge mode the following things happen in side the EPC 800 main unit C Slow is turned off the maximum Gain is 20 mV pA the maximum Filter setting is 10 kHz and the RS compensation will
115. round yourself first by touching a grounded metal surface http www heka com 16 Unpacking and Installation http www heka com 4 Description of the Hardware The hardware components of the EPC 800 USB patch clamp system con sist of the head stage or probe and the amplifier main unit Specific information about the hardware installation is given elsewhere see chap ter 3 Unpacking and Installation starting on page 13 4 1 Probe EPC 800 S N 800548 ans LS lt A Figure 4 1 EPC 800 patch clamp amplifier probe The probe or headstage of the EPC 800 Patch Clamp Amplifier is con tained in a small enclosure designed to be mounted on a micromanipulator and directly attached to the recording micropipette It contains the sensi tive amplifier that constitutes the current to voltage converter as well as components for injecting test signals into that amplifier On the probe are the following connectors Input Connector This is a Teflon insulated BNC connector The stan dard pipette holder plugs directly into this connector the center pin is the amplifier input and the shield is driven with the command potential Vp Note Avoid touching the probe s input terminal since the 18 Description of the Hardware input circuitry of the probe can be damaged by static electricity When it is necessary to touch the input e g while inserting a pipette into the holder ground yourself first by touc
116. rrection pathway and both the series resistance and membrane capac itance must be known for the capacitance transient cancelation C Slow circuitry In the EPC 800 USB the estimation of series resistance has been combined with the transient cancelation in that the R control has a dual effect Its setting affects both the kinetics of the transient cancelation and the scaling of the correction feedback signal This means that in prac tice the estimation of the series resistance consists of adjusting C Slow and R Series to cancel the transient currents due to the cell membrane capac itance Once this has been done the relative amount of R compensation can then be selected with the COMP control Theoretically it is desirable to compensate as much of the series resistance as possible In practice however a degree of compensation above 90 can involve considerable technical problems and in some recording situations a value below 90 is preferable To illustrate one technical problem con sider the case when a 100 mV potential change is commanded and 90 compensation is in use This degree of compensation means that the cell membrane capacitance will be charged 10 times faster than normally The rapid charging is accomplished in the compensation circuitry by forcing the pipette potential to very transiently reach a potential of 1 V The resulting large current causes the membrane capacitance to charge quickly to its final value of 100 mV In
117. rting on page 41 The amount of compensation can be changed manually by turning the Comp knob The compensation is based on the value of R series and will be effective only when Rs comp is turned ON and set to a particular speed value The following settings determine the speed of feedback compensation e Off Turns compensation off e 100 us Slow compensation e 10 us Fast compensation http www heka com 30 Description of the Hardware e 2 us Very fast compensation The choice of speed depends on the recording time constant and the degree of compensation desired as described in Chapter 6 Theory of Compensa tion Procedures Fast Rs compensation requires more critical adjustment of the controls but provides the maximum voltage clamp speed In Current Clamp mode Rs comp acts as a bridge compensation In this mode only the 100 ws and 10 us speeds are possible 4 2 6 Seal Mode The device provides a seal mode which supports the user performing a seal at a cell Pressing Auto CSlow and CFast simultaneously activates this mode A test pulse is applied on top of the holding potential and the resistance of the cell is gathered The resistance is mapped to a tone which is played if a headphone or speaker is attached to the device The higher the resistance is the higher is the fundamental frequency of the signal that is played During this mode knob settings will be taken into account with a certain delay up to 5 s Cha
118. s larger than it is this can cause oscillation and possible damage to the cell under obser vation Note Practical Tips e How you should set the R compensation controls de pends on the approximate value of the uncompensated membrane charging time constant T which you can cal culate as the product of the C Slow and R Series settings for ecample suppose C Slow is 20 pF and R SERIES is 10 MQ Tu is then 20pF 10MQ 200 us If Tu is smaller than about 500 us you should use the 2 us set ting of the R compensation switch to provide the neces sary rapid compensation however the slower settings will provide compensation that is less prone to high frequency oscillations from misadjusting of the controls How much compensation you can apply is also determined by Ty If Tu is larger than about 100 us you can use any degree up to the maximum of 90 compensation without serious overshoot or ringing in the voltage clamp response For smaller values of Ty the COMP setting should be kept below the point where ringing appears in the current trace e As in the case of patch recording there is rarely need to use the full bandwidth of the Filter in whole cell recording This is because typical membrane charging time constants even after R compensation are considerably longer than 16 us which is the time constant corresponding to a 10 kHz bandwidth Thus the current monitor signal is ex pected to contain no useful information beyond
119. sic amplifier functions are controlled remotely through software For traditional Axon users the equivalent of this would be the MultiClamp commander software for controlling their automatic amplifiers HEKA s EPCMaster remote control software program consists of a virtual front panel of the EPC 800 Patch Clamp Amplifier It is a free program with no requirement for a software protection dongle and is used for con trolling and testing the EPC 800 Patch Clamp Amplifier It can be thought of a soft panel for the EPC 800 Patch Clamp Amplifier and it provides a further level of full integration of the amplifier with pCLAMP software EPCMaster will enable control of the EPC 800 Patch Clamp Amplifier settings but it has no functions for data acquisition or analysis in this case Clampex and Clampfit will be used for these purposes The program http www heka com 76 Using the EPC 800 Patch Clamp Amplifier with pCLAMP however is very useful in that it provides users the option of setting the parameters of the EPC 800 Patch Clamp Amplifier from a software panel instead of manually using the front panel controls Another important point is that the program contains a notebook window allowing the user to see the communication message stream being sent and received for any parameter that changes something In this regard the program is a useful tool for users to test both the functions of the amplifier as well as the message str
120. st be attached directly to the EPC 800 USB probe Although the probe amplifier can tolerate the additional capacitance of a short connecting cable without instability or oscillations we find that the dielectric and electrostrictive properties of coaxial cables introduce excessive noise In typical setups the probe is therefore mounted directly on a 3 axis micromanipulator The EPC 800 USB probe is supplied with a standard plastic mounting plate for mounting on a flat surface see Fig 4 2 Holes can be drilled through the protruding surfaces for attachment to a matching plate or other surface The head stage also comes with a dovetail plate that will fit connections supplied by most leading micromanipulator companies Please remember that the metal case of the probe must remain insulated from ground this is very important Because of the extreme sensitivity of the EPC 800 Patch Clamp Amplifier special care must be taken in grounding all surfaces that will be near the probe input in order to minimize line frequency interference Even one millivolt of AC on a nearby surface 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 s cable directly to the Signal Gnd in the main unit The Gnd terminal on the probe is best used for the bath electrode and perhaps for grounding nearby objects such as the mi
121. stimulus input is deactivated Holding potential applied to external input VC i acquired and then generated in ternally so that the potential for the cell does not change 6 3 Series Resistance Compensation In whole cell voltage clamp recording the membrane potential of the cell is controlled by the potential applied to the pipette electrode This control of potential is not complete but depends on the size of the access resistance between the pipette and the cell interior and on the size of the currents that must flow through this resistance This access resistance is called the series resistance Rs because it constitutes a resistance in series with the pipette electrode Part of the series resistance arises from the pipette itself but normally the major part arises from the residual resistance of the broken patch membrane which provides the electrical access to the cell interior In practice we find that the series resistance usually cannot be reduced below a value about two times the resistance of the pipette alone Series resistance has two detrimental effects in practical recording situa tions First it slows the charging of the cell membrane capacitance because it impedes the flow of the capacitive charging currents when a voltage step is applied to the pipette electrode The time constant of charging is given by Ta Rs x Cm where Cm is the membrane capacitance For typical val ues of Rs 5 MQ and Cm 20 pF the time cons
122. t R Series V Hold I Hold VPOffset and LFVC Hold need to be turned a certain degree before the changes read by the device are interpreted as intended and then change the set tings This prevents unintended changes that would happen using a am plifier with analog controls If the display knob is set to Auto Display the sensitivity is significantly lower Once a knob is turned and the EPC 800 Amplifier interprets the changes as intended the sensitivity is higher After a number of seconds with no changes the sensitivity is decreased again If one wants to make very fine change of e g VHold one should switch the display knob to I VHold 4 2 9 Power Switch and Chassis Ground Power Switch Power ON and OFF http www heka com 32 Description of the Hardware Note Since the calibration settings of the amplifier have been determined for a warmed up amplifier switch on the amplifier at least 15 min before starting an experiment This will ensure that the amplifier has warmed up to regular working tempera ture and calibration parameters are most accurate Chassis GND CHAS The chassis is connected to the ground line of the power cord as is typical of most instruments The Signal Ground Signal GND is separated from the chassis by a 10 Q resistor to avoid ground loops 4 2 10 Rear Panel Connectors Telegraphing Outputs Individual BNC telegraphing outputs for Gain Filter Bandwidth Amplifier Mode and C Slow on the rear panel e
123. t be a mismatch between the front panel controls and the software readings SEAL This protocol will automatically set the default recording mode to whole cell and change the gain to 20 mV pA PROTOCOL SEAL http www heka com 8 4 The Amplifier control window of PatchMaster 91 E Mode 3 whole cell E Gain 12 set gain to 20 0 mV pA Whole Cell This protocol will automatically set the default recording mode to Whole Cell and adjust the gain to 10 mV pA PROTOCOL lt WHOLE CELL E Mode 3 whole cell E Gain 11 set gain to 10 0 mV pA Important note Execution of the SET UP SEAL and Whole Cell predefined protocols only make sense when the EPC 800 Patch Clamp Amplifier is operated with PATCHMAS TER in Remote mode If these protocols are called when oper ated in Local mode it may lead to confusion because there will likely be a mismatch between the actual gain set by the front panel knob of the amplifier as shown in the oscilloscope win dow of PATCHMASTER and what is actually displayed in the gain field within the amplifier window of PATCHMASTER http www heka com Using the EPC 800 USB patch clamp amplifier with 92 PatchMaster Voltage Current AD 2 AD 3 AD 4 AD 5 In Out On Cell AD 6 Input ADC Recording Mode 107 Whole Cell 0 AD 2 Whole Cell no C Clamp Figure 8 9 Setting the AD inputs and recording mode within Patchmaster Input ADC The oscilloscope can d
124. t blinks When it does it means that in ternal amplifiers in the EPC 800 Patch Clamp Amplifier are about to saturate and or that the Current Monitor output voltage is going above 13 V peak on the peaks of the transients and you should readjust the transient can celation controls Otherwise it is likely that the recording will be non linear and subtraction will not work correctly e The fast transient cancelation is not sufficient to cancel all of the capacitive transients in a patch recording This is partly because the pipette capacitance is distributed along the length of the pipette therefore each element of capac itance has a different amount of resistance in series with it so that a single value of T Fast will not provide perfect cancelation The time course of the transients also reflects dielectric relaxation in the plastic of the pipette holder and in the pipette glass These relaxations are not simple ex ponentials but occur on time scales of about 1 ms If you use pipette glass with low dielectric loss e g aluminosil icate 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 these slow relaxations by using the C Slow controls with the C Slow Range set to 30 pF 7 1 4 4 Whole Cell Configuration Breaking the Patch and C Slow Compensation If the fast capacitance cancelation was adjusted as described above before breaking t
125. t cancelation will be essential if you will be giving voltage pulses in your experiment If no voltage jumps are required turn the stimulus off to avoid introducing artifacts If voltage jumps are to be applied switch the GAIN and FILTERS to the values you will be using and adjust C Fast and T Fast to cancel the capacitive spikes mentioned above e Be sure to use Gain settings of 50 mV pA or above for lower noise in single channel recordings Keep the Filter switch set at 10 kHz unless you actually will need the full 100 kHz bandwidth for some reason otherwise you might drive the current monitor output or your recorder s input amplifiers into saturation with the very large amount of high frequency noise Should you use the full bandwidth you should avoid gain settings above 100 mV pA for the same reason e If you are applying voltage pulses to the patch membrane you probably will want to subtract control traces from the traces containing the channels of interest in order to re move the capacitive transients Nevertheless it is im portant to try to cancel the capacitive transients as well http www heka com 7 1 Local Mode 61 as you can in order to avoid saturating any amplifiers the recording medium or the AD converter It is a good idea to set the C Fast and T Fast controls while you ob serve the signal without any filtering beyond the internal 10 kHz filter Then during the recording watch to see if the Clipping ligh
126. t pulse and then performs an automatic compensation of the voltage offsets PROTOCOL SET UP Mode 3 whole cell Gain 10 set gain to 5 0 mV pA medium range PulseAmp 5 0mV set test pulse amplitude PulseDur 5 0ms set test pulse duration compensate voltage offsets Switch on test pulse AutoZero PulseOn TRUE Am wm es Note PATCHMASTER has a built in protocol interpreter that executes command lines of the form Window Controll parameter comment E g the line E Gain 10 would instruct PATCHMASTER to set the gain popup in the EPC 800 window to the 10th value 5 mV pA The predefined protocols are stored in a teat file called Epc800 pro and can be edited with any text editor Please refer to the PATCHMASTER manual for a detailed description on how to record and modify protocols Important note In Local mode it is advised NOT to use the Set Up protocol of PATCHMASTER The reason being is that in this mode the Gain of the amplifier is set through the front panel knob After executing the protocol the Gain displayed in the amplifier window of PATCHMASTER will read 5 mV pA regardless of what the true Gain is according to the front panel knob In addition an Auto V Pos feet will be executed but the front panel Auto LED will not be lit Use of the Set Up protocol makes more sense when the amplifier is operated in Remote mode when the front panel knobs and switches are inactive and there wouldn
127. t saturated 4 2 2 Gain Mode and Filter Knobs GAIN Sets the scaling of the current monitor output The range is 0 005 to 2000 mV pA The gain setting automatically selects one of the http www heka com 22 Description of the Hardware Figure 4 6 Gain knob three available current measuring feedback resistors in the probe 5 MQ 500 MQ and 50 GQ corresponding to low medium and high gain ranges respectively The table below summarizes the main features and limita tions of the gain ranges Low Medium High Feedback Resistor 5 MQ 500 MQ 50 GQ Gain mv pA 0 005 0 002 0 05 20 50 2000 Imax VC 2 uA 20 nA 200 pA Imax out CC 100 nA 1 nA Bandwidth 100 KHz 100 KHz 60 KHz Cslow Ranges 30 100 1000 30 100 1000 30 100 Current Clamp yes yes no RS Compensation yes yes no Table 4 1 Gain ranges of the EPC 800 Patch Clamp Amplifier The lowest gain range may be used for experiments e g bilayers loose patch or large cells in which large currents need to be delivered up to about 2 uA Capacitance compensation of up to 1000 pF is available and Rs compensation can be used for Rg values down to 10 Q in this range In the medium gain range the background noise is larger than in the high gain but the full 100 kHz bandwidth is available and currents of up to about 20 nA can be recorded This range is used mainly for whole cell recordings and for this purpose
128. t this point that this software and the protection dongle driver have been correctly installed on the acquisition computer 7 1 2 Hardware Connections Information about setting up and connecting the EPC 800 Patch Clamp Amplifier have already been covered see Chapter 3 Unpacking and In stallation It is also assumed that the Digidata model of choice is properly powered on connected to the computer and correctly configured for use with the Clampex software These instructions can be found in the pCLAMP 10 user guide 7 1 2 1 Front Panel There are four BNC cable connections that have to be made between the front panel of the EPC 800 patch clamp amplifier and the Digidata interface The connections below are example configurations they can be changed as long as the proper configuration is set from within the software EPC 800 USB Front Panel Digidata Front Panel Current Monitor Analog Input 0 Voltage Monitor Analog Input 1 External Input CC T BNC to Analog Output 0 External Input VC T BNC to Analog Output 0 Table 7 1 Front panel BNC connections between the EPC 800 Patch Clamp Amplifier and a Digidata interface http www heka com 7 1 Local Mode 53 7 1 3 Configuring Clampex Lab Bench Now as the correct hardware connections have been made the input and output signals must be properly configured in the Configure Lab Bench dialog 7 1 3 1 Input Signals Lab Bench has
129. tant is 100 us This time constant is excessively long for studying rapid voltage activated currents such as Nat currents in neurons especially since several time constants are required for the membrane potential to settle at its new 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 Re 5 MQ a current of 2 nA will give rise to a voltage error of 10 mV which is a fairly large error for studying voltage activated currents errors need to be kept to 2 mV at most Electronic compensation for series resistance in voltage clamp systems has been in common use since the days of Hodgkin and Huxley The princi http www heka com 46 Theory of Compensation Procedures ple of the compensation in the case of a patch clamp is that a fraction of the current monitor signal is scaled and added to the command potential correction pathway see Figure 6 1 below When a large current flows in the pipette the pipette potential is altered in a way that compensates for the potential drop in the series resistance This arrangement consti tutes positive feedback and can become unstable when overcompensation occurs The EPC 800 Patch Clamp Amplifier incorporates additional circuitry to allow capacitance transient cancelation to occur while Rs compensation is in use see Sigworth Chapter 4 in Single Channel Recording This is sh
130. ter circuit to convert the currents to an analog voltage which is then made available at the current monitor output for display and recording At the same time that pipette currents are being recorded the potential must be specified and the various operating modes of the EPC 800 PATCH CLAMP AMPLIFIER correspond mainly to different ways of controlling that potential 5 1 Voltage Clamp Mode This is the basic patch clamp mode in which the membrane voltage is con trolled and the transmembrane current required to maintain the clamped voltage is measured The Voltage clamp mode is implemented by the cir cuitry shown in the figure below The pipette potential is derived from the signal applied to External INPUT VC with a variable offset added from the Vyorp control The sum of these two sources is displayed and mon itored as the Vion signal Before being applied to the pipette a further variable offset is added from the Vp OFFSET control to allow the user to cancel electrode offsets 36 Recording Modes of the EPC 800 Patch Clamp Amplifier eo ae ee a a IV Converter Current Monitor Stim In E 5 O ae Vp Ofiset tae Bath Electrode Monitor Oo Figure 5 1 Voltage Clamp Mode 5 2 Current Clamp Mode The Current Clamp mode can be used to measure the resting potential or spontaneous action potentials in a whole cell recording In these ex periments a known constant or time varying current is applied
131. th to observe single channel events of a given amplitude 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 filter bandwidth is the one that makes the rms background noise about 1 10 of the channel amplitude Since one rarely wants to go through the process of choosing the optimum bandwidth during an exper iment the best procedure is to record the data at a wide bandwidth and perform any necessary filtering analog or digital later during analysis of the data In typical voltage clamp whole cell recordings the predominant noise source arises from the combination of the access resistance R and the cell membrane capacitance Cm Above 1 kHz or so the current variance from this source increases with this resistance and capacitance as ao akR Cm so that it is clearly desirable to keep R as small as possible and even more important to select small cells if one is interested in low noise See http www heka com 11 2 Noise of the Recording Set Up 119 the chapter by Marty and Neher 1983 for a more complete description of this and other fine points of whole cell recording http www heka com 120 Low Noise Recording http www heka com 12 Appendix 12 1 Supported States Figure 12 1 shows the states that the device supports 12 2 USB Descriptor T
132. that they are equal to the standard deviation of the fluctuating current Since the noise sources in the patch clamp amplifier pipette holder pipette and patch membrane are statistically independent their contributions to the total noise do not add linearly instead their variances the squares of the standard deviations add This means that the rms reading on the EPC 800 display represents the square root of the sum of the squares of the rms currents from each source Taking this into account one can calculate the relative contributions from the amplifier pipette holder and the combination of pipette immersion and patch noise The table below shows the relative contributions calculated in this way for the optimum situation just described http www heka com 11 2 Noise of the Recording Set Up Noise Source Contribution rms Current Amplifier 35 95 fA Holder 21 73 fA Pipette Patch 44 105 fA Table 11 1 Noise contributions of the EPC 800 USB Patch Clamp Am plifier holder and pipette in an experimental set up The contributions to the variance from the three sources are seen to be comparable in size and improvements in the amplifier noise level will not help very much unless corresponding improvements are made in the other noise sources As it is rms noise values as low as those quoted here are ob tained only with considerable care Some of the important considerations are outlined
133. the special features of the 1000 pF http www heka com 4 2 Main Unit 23 transient cancelation range see C Slow Ranges series resistance compen sation and the current clamp modes are made available The 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 about 60 kHz and the special features mentioned above do not function in this range Slow capacitance cancelation ranges C 30 100 1000 pF can be set to any desired value In voltage clamp mode and high gain range the 1000 pF C Slow range is not supported If inadvertently selected the user will be alerted by an error message on the front panel LCD stating Auto C Slow Error Set lower range The current clamp bridge mode is only possible in the medium and low gain rangese MODE switch The operating modes of the EPC 800 PATCH CLAMP AMPLIFIER are described in detail in chapter 5 Recording Modes of the EPC 800 Patch Clamp Amplifier on page 35 MODE CC Bridge vc 100 Figure 4 7 Mode knob Briefly the VC Voltage Clamp mode is the usual mode for whole cell cell attached single channel loose patch or bilayer recordings in which the pipette current is recorded while the pipette potential is controlled by command signals The CC current clamp Bridge mode can be used to measure the resting pote
134. this band width In whole cell recording the voltage and current monitor signals follow the usual convention with outward currents being positive This is because the pipette has electrical access to the cell interior http www heka com 66 Using the EPC 800 Patch Clamp Amplifier with pCLAMP 7 1 4 5 Whole Cell Voltage Clamp Once C Slow and R Series have been properly compensated you may wish to execute a whole cell voltage clamp recording In this mode the trans membrane current is recorded in response to maintaining the cell to a desired clamped or commanded voltage The current is monitored at the Current Monitor output of the EPC 800 Patch Clamp Amplifier and the value is displayed on the LCD display when in the I Vmon position In Clampex the desired voltage clamp protocol will first have to be written in the Protocol dialog It is not within the scope of this manual to cover the details of how to write such a protocol The figures below simply illustrate a basic example of some of the steps for designing and executing a voltage ramp protocol from 30 mV to 70 mV as Edit Protocol hp 30r30d 70 pro Made Rate Inputs Outputs Trigger Statistics Comments Math Waveform Stimulus Waveform Analog OUT Vemd Info V Analog Waveform Digital Outputs Epochs Stimulus file m Intersweep holding level Use holding Epoch Description A B c Type Step Step Ramp Sample rate First level mV
135. tial in Voltage Clamp mode The V membrane label is converted to I hold in Current Clamp mode and is used for setting the holding current Although the front panel of the EPC 800 Patch Clamp Amplifier has po tentiometers for Vyorp and IHorp when the amplifier is being used with PATCHMASTER these should be manually set to read 0 and the holding potential and current should be set in the amplifier window of PATCH MASTER I mon Displays the actual measured pipette current V mon Displays the actual measured pipette voltage after correcting for liquid junction potentials and offsets provided the zero current potential has been set correctly This may differ temporarily from the holding voltage e g during long stimulation pulses as it indicates the average sum of V membrane and the scaled stimulus voltage R memb The Seal Resistance R membrane is determined from the current sampled during the baseline and the second half of the test pulse R membrane can be encoded into a tone using the Sound feature see below 83 1 pA 39 mv 505 MO I mon V mon R memb Figure 8 5 I mon V mon and R membrane values within PatchMaster One of the more powerful features of PATCHMASTER is the ability to write and record your own protocols previously referred to as Macros as a se quence of commands Virtually all of the buttons and features within the software can be used in the recording of protocols The protocols can be
136. tried The noise level of the pipette holder can be tested by mounting it with the electrode wire installed but dry on the probe input and measuring the noise using the NOISE option on the front panel of the EPC 800 Patch Clamp Amplifier The headstage should be in a shielded enclosure so that no line frequency pickup is visible on an oscilloscope connected to the current monitor output at a bandwidth of 3 kHz or less A good holder increases the rms noise only by about 10 e g from 95 to 105 fA Noise sources are discussed further in Chapter 11 Low Noise Recording The pipette electrode is simply a thin silver wire that is soldered onto the pin that plugs into the probe s BNC connector The chloride coating on the wire gets scratched when exchanging pipettes but we find that this does not degrade the stability very much the wire does need to be re chlorinated occasionally perhaps once per month A wire for the standard electrode holder should be about 4 5 cm long after it is chlorinated an O ring is slipped onto it and the wire is inserted into the holder Chlorinating can be done by passing current e g 1 mA between the wire and another silver or platinum wire in a Cl containing solution e g 100 mM KCl or physiological saline Current is passed in the direction which attracts Cl ions to the electrode wire this produces a gray coating http www heka com 110 General Patch Clamp Setup Practices 9 6 Bath Electrod
137. us if desired The Ext Stim CC is ON or OFF depending on the position of the front panel Ext Stim CC switch External Input VC Signals from an external stimulus source are ap plied here They can be summed with the internal stimulus if desired The combined stimulus signal is passed through a 2 pole filter to round off stepwise changes in voltage This avoids nonlinearities from slew limiting amplifiers in the command processing circuitry and also reduces the am plitude of the current transients from rapid charging of the pipette Two degrees of filtering specified as the rise times time from 10 to 90 of the amplitude of a step change are available in the front panel Ext Stim VC switch on the front panel 2 us which is the minimum required to avoid nonlinearities in the internal circuitry and 20 us which is preferable for http www heka com 4 2 Main Unit 21 all but the fastest measurements to reduce the capacitive transients Voltage Monitor This output signal provides a monitor of the pipette potential A BNC cable should be used from this connection to an assigned Analog Input channel of your AD DA interface It is scaled up by a factor of 10 relative to the potential applied to the pipette The output impedance is 50 Q HEKA s PATCHMASTER software will automatically convert any signal to correct scaling in the MKS system provided that the proper configuration settings are set Appropriate scaling factors will have
138. y covered the example of using the amplifier with a Digidata and pCLAMP soft ware Another option is to use the amplifier with any of the InstruTECH HEKA series of interfaces in conjunction with HEKA s PATCHMASTER software This chapter examines the various modes of operation and par ticulars of the EPC 800 Patch Clamp Amplifier when used with this hard ware and software combination For a complete description and operating instructions for PATCHMASTER users should consult the PATCHMASTER user manual directly 8 1 Software Installation The latest version of PATCHMASTER for Windows and Mac operating sys tems can always be downloaded and installed directly from the downloads sections of our website at www heka com It is suggested using this as the source for the latest software version releases rather than on any CD Rom provided Download and installation of the software should be very straightforward and self explanatory Instructions if needed can be found in the PATCH MASTER users manual and any concerns about software compatibility is sues are addressed in the downloads donelist section of the website or by contacting the HEKA support hotline Using the EPC 800 USB patch clamp amplifier with 80 PatchMaster 8 1 1 Dongle driver To be able to use PATCHMASTER a software protection key or Dongle is required HEKA provides one universal USB port dongle per PATCHMAS TER license that can be used on either Windows or

EPC 800 Manual - HEKA Elektronik Dr. Schulze GmbH

Contents

Download Pdf Manuals

Related Search

Related Contents