PC-505B User`s Manual (Rev. 050816)
Contents
1. eese eese eene eene tnr enenn tenete enne en 21 Auto Zero CONITOL CRE CKOUL cei sas oW RR e Mee ete a eve SA aee reve a eve M GAA SES gus 2l 2V commands checkout RII euh RUNI NI EEE RE 22 Cap Comp heckoul e da het ed ee Ue her Ee EHE cd e deep Eee b i e dE hae gera b ed A 22 PC 505B Manual Rev 050816 Current clamp checkout d asain dani ev heil iii e Mm i ee Rees 23 THEORETICAL CONSIDERATIONS scsssssssssssssesssvssescescssescescossscssossencessssasensessssassessassecssossencesssense 25 Chloriding sIlver WIFe ric oe eese ee treten onore ee erre Sea sa pea esot repose ot Ssss sosoo aep oS ae EE EE Nee eoa ESS oossoo See 25 Chloriding by electrolysis ei diei ee et tte eot Ree dert eb eee HER ERE Sb E epe STAR ad 25 Chloriding chemically 2 iaa iiu e tii iet i e a m i E EC RA E Ha Ed 23 El ctrode holders sicsssccaksscccicisssosssesesstcecavescssessseccessvanssaeedecasaseseacsavastessbesesansesdesensdsesvedsesesoeeaceseseessoesseosses 25 Care atid useof holders a e te ae Ane E 26 Cleaning and storage 2 ide eee etie ee e eee died eiae ea ie edet chats dette uda Den 26 Replacing holder parts ius Aig EAB Ag I n ARE a BOS 26 Reference electrodes isinske 26 APPENDIX eL 28 usse E 28 Accessories and replacement Parts siesvsiersecocsasssesevornessndsseensssnvessverseseccenonesevenscesedde2. The average length of time a gating channel will remain in the closed state mean open time The average length of time a gating channel will remain in the open state mini jack A small plug on the headstage to which the electrodes are attached model cell An electric circuit designed to model the electrical characteristics of a biological membrane open probability The calculated probability of finding a channel open at time t given that the channel is in a closed state at time t 0 oscilloscope A device used to monitor voltages within an electrical circuit output current See Im OUTPUT SYNC A pulsed signal appearing at the OUTPUT SYNC BNC on the instrument rear panel Used to synchronize the PULSE GENERATOR Or CAP TEST signal to an external device such as an oscilloscope periodic That which repeats itself at regular intervals perfusate The solution being perfused perfusion The exchange of one solution with another plasma membrane The surface membrane of a cell Contrast with an intracellular membrane which is a membrane contained entirely within the cell potentiometer A single or multi turn dial used to make a continuously varying selection with a range In its heart this is a variable resistor pulse code modulator A device which converts an analog signal into a form acceptable for storage on VCR tape Also converts data previously stored on VCR tape back into an analog signal
3. Set scope to 50 mV div Set the METER SELECT switch to the Junction Zero setting and adjust meter reading to 10 mV with the JUNCTION ZERO control Verify that scope reads 10 times the panel METER reading 2 div at these settings Using JUNCTION ZERO control adjust METER to 0 mV Set the METER SELECT switch to the 2Vc position Verify that METER reads zero Set VOLTAGE COMMAND toggle to positive Adjust VOLTAGE HOLD control fully CW Verify the METER reads a minimum of 200 mV Set oscilloscope to 1 V div Verify that scope reads 10x the meter reading e g 2 V for a meter reading of 200 mV Switch VOLTAGE HOLD toggle to minus Redo steps 9 and 10 Switch VOLTAGE HOLD toggle to positive Using VOLTAGE HOLD control adjust until meter reads 150 mV Switch METER SELECT to Junction Zero Adjust JUNCTION ZERO until METER reads 100 mV Set METER SELECT to V t h IN Verify that METER reads holding potential of 150 mV Set METER SELECT to 2Vc Verify that meter reads sum of holding potential and junction offset 250 mV Set METER SELECT control to Veth IN Verify that the adjustment of JUNCTION ZERO control does not affect the meter reading Set the VOLTAGE HOLD toggle to off Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 21 20 21 Set the METER SELECTOR switch to Junction Zero and adjust the METER to zero Return amplifier to initial settings External DC Voltage comman
4. signal polarity Defined as the sign applied to a current generated through a membrane in the presence of an applied holding potential The electrophysiological definition is determined by the membrane such that an outward directed current and a depolarizing potential are both positive single channel Refers to a solitary channel protein functioning within a measurement milieu step potential A functionally instantaneous change in potential from one value to another time constant In a system governed by exponential kinetics this is the time required for a value to change to 1 e of its initial value where e 2 71828 is the base of the natural logarithm transient Momentary transmembrane That which spans a membrane or is referred from one side of a membrane to the other Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 39 trim pot An adjustable variable resistor used for making fine adjustments to a circuit TTL Transistor Transistor Logic Voltage ranges used to define an on or off state in binary devices 0 0 8 V defines a logic 0 state and 2 4 5 0 V defines a logic 1 state unitary channel conductance A measure of the ability of a channel to pass an ion from one side of the membrane to the other An intrinsic property of a single channel which depends on the ionic species under consideration Determined by measuring the current through an open channel in the presence of a
5. WHOLE CELL and OUTPUT A final section contains the LCD METER as well as the METER SELECT and POWER switches This section will be referred to as the METER block Front panel COMMANDS block The COMMANDS block contains the VOLTAGE and CURRENT HOLDING controls the JUNCTION ZERO controls and a COMMAND SELECT toggle switch selecting internal or external command inputs This block also contains several controls for adjusting the sensitivity of input commands as well as controls for the ZAP function Voltage and current commands The VOLTAGE and CURRENT HOLD controls provide independent modification of holding potential and holding current settings within the ranges 200 mV and 1 0 nA respectively A MODE toggle switch in the OUTPUT command block is used to select between voltage clamp V or current clamp Ic modes Placing the instrument in current or voltage clamp mode activates either the VOLTAGE Or CURRENT HOLD controls respectively This structure allows switching between voltage and current clamping configurations without the need to readjust settings VOLTAGE and CURRENT HOLD controls are not attenuated by COMMAND SENSITIVITY settings Internal command external command and command sensitivity The COMMAND SELECT toggle switch selects between an internally generated internal command or an externally generated external command command applied to the COMMAND IN BNC located on the instrument rear panel The C
6. COMMAND IN attenuator is modified to allow command inputs up to 2 V SPEED TEST is used to re adjust the rear panel boost trimmers normally only required when replacing a headstage These adjustments are factory set for the headstage supplied with the instrument CMR BOOST and GAIN trimpots are factory set User adjustment may be required if a headstage is replaced Adjustments instructions are provided with the new headstage Grounds Circuit and chassis ground connectors black and green respectively are binding posts supplied with a shorting link For most recording situations the shorting link can remain connected with no detrimental effect in amplifier performance However there are occasions where line noise can be reduced if the two grounds are isolated from each other We recommend that you test both connected and unconnected configurations to determine which is best for your setup Headstage The headstage or probe is a low leakage current solid state current to voltage converter which is switch selectable between two feedback resistors The headstage input terminal is a 1 mm jack connector that accepts a 1 mm pin on the electrode holder Pin jacks on the side of the headstage provide for grounded and or driven shield applications The headstage housing is a metal enclosure which serves as a shield which is driven by the command signal The attached metal rod is used for mounting to a micromanipulator and is insulate
7. Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 23 NO m sf 10 11 12 13 14 15 16 17 18 Set scope to 5 V div Set COMMAND SELECT to internal command Set COMMAND SENSITIVITY toggle to on Scope should now display large amplitude positive and negative pulses Clipping LED will be lit Using FAST CAP COMP controls minimize amplitude and duration of pulses Leading edge transients will remain due to characteristics of model cell Set PROBE RESISTER to low Connect model cell to headstage in whole cell mode Turn C SLOW toggle switch on Adjust C SLOW and SERIES R controls until leading edge of square wave transitions smoothly from one peak to the other Full transition should take approximately 1 ms Remaining leading edge transients can be minimized by small adjustments to the FAST CAP COMP controls Turn CORRECTION toggle switch on Verify that control decreases transition time without increasing amplitude of spike Turn CORRECTION off Turn LEAK SUBTRACTION on Verify that control adjusts amplitude of square wave Remove model cell from headstage Return amplifier to initial settings Current clamp checkout l 2 3 4 5 6 7 8 9 10 11 Attach model cell to headstage in bath mode Set MODE toggle to h Set METER SELECT to Vm Using JUNCTION ZERO control adjust METER to 0 mV Set METER SELECT to h IN Set CURRENT HOLD toggle to positive Ad
8. driving force transmembrane potential at different potentials Measurements made within the Ohmic range of the channel s response will graph as a straight line The slope of this line when plotted as current I vs potential V will yield the conductance or inverse resistance of the channel under these conditions Vm HOLD The transmembrane potential generated by the amplifier and applied to the headstage This driving force appears in addition to any other driving forces which may be present Ve The user selected potential set in the COMMANDS APPLIED TO REFERENCE block of the amplifier Warner Instruments A Harvard Apparatus Company
9. negligible when compared with cell membrane and single channel resistances Correction Correction is used to apply voltage compensation to the amplifier to correct for the voltage drops due to the series resistance This control is adjusted until the output signal just begins to oscillate at the leading edge ringing of the TEST PULSE during experimental setup The control is activated by a toggle switch Leak subtraction Leak subtraction is used to compensate for leakage currents to the bath through the pipet membrane seal resistance RS or through the membrane patch For leaky seals RS 1 GQ it is important to correct for the error caused by the shunt resistance of the leak Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 10 This error can usually be ignored with tight seals RS 10 100 GQ Fully counterclockwise rotation of this control switches it off OUTPUT command block The OUTPUT command block ZU OT contains controls for selecting different I operating modes and headstage resistors The instrument gain and filter controls can also be found here Probe resistor select The headstage resistor is selected with the PROBE RESISTOR select switch The high position selects the 50 GO resistor for both patch and bilayer headstages models LC 201 HC 202 and HB 205 respectively The low position is used for whole cell recording and selects the 500 MO resistor in
10. the LC 201 headstage and the 50 MQ resistor in the HC 202 headstage It is not available in the HB 205 headstage The low resistor is automatically selected when in current clamp mode Clamp mode switch In voltage clamp mode MODE switch in upper position all command controls are active with the exception of the CURRENT HOLD command in COMMANDS block The range of voltage clamp potentials at the headstage input is 200 mV Zero current mode MODE switch in center position is a transition mode between voltage clamp and current clamp It disengages all commands and functions from the headstage with the exception of the ZAP controls The primary uses for zero current mode are 1 to protect the preparation when switching between voltage and current clamp modes and 2 to preset the voltage hold or current hold settings prior to switching to voltage or current clamp modes respectively In current clamp mode MODE switch in lower position all commands are again active The current range at the preparation is limited by the 10 V input maximum and the headstage resistor as follows e 20 nA with the 500 MQ resistor e 200 nA with the 50 MQ resistor Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 11 Gain The GAIN control selects the amplifier gain in units of mV pA The gain scale on this control is modified by the PROBE RESISTOR select toggle xO 1 or x1 0 for the low setting and x10 for the hig
11. 5 pA i e within 1 div of zero on oscilloscope for this setting Set PROBE RESISTER toggle from low to high Set FILTER to 1 kHz Set oscilloscope voltage base to 0 5 V div Move oscilloscope cable from I OUTPUT on instrument front panel to Lj V OUTPUT on instrument rear panel Redo steps 7 11 Return amplifier to initial settings Junction zero checkout l 2 Set the METER SELECTOR switch to the Junction Zero Adjust the JUNCTION ZERO control COMMANDS block until the meter reading is 10 mV Verify that Vm and ZVe on the METER SELECTOR switch give the same meter reading as Junction Zero 10 mV Set METER SELECT to Vcth IN and check that the METER reads zero Place METER SELECT switch in Junction Zero position Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 20 10 11 12 Adjust the JUNCTION ZERO control to its maximum setting fully CW Check that the METER reads gt 120 mV for Junction Zero Vm and XVc METER SELECT settings Adjust the JUNCTION ZERO control to its minimum setting fully CCW Check that METER reads x 120 mV for Junction Zero Vm and XVe METER SELECT settings Set METER SELECT to Junction Zero Adjust JUNCTION ZERO until METER reads zero Return amplifier to initial settings Internal DC voltage command checkout l 2 S o usb Ow Uu cb 10 11 12 13 14 15 16 17 18 19 Connect oscilloscope to V x10 BNC on front panel
12. OMMAND SENSITIVITY controls are comprised of an on off toggle and a sensitivity selector The SENSITIVITY SELECTOR attenuates the internally generated TEST PULSE signal and any externally generated command voltages connected to the COMMAND IN input BNC by factors of x0 1 x0 01 and x0 001 Internal or external commands are activated by moving the COMMAND SENSITIVITY toggle into the on position When switched off these modifiers are disconnected from the COMMAND pathway Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 8 When internal command is selected a 1 V p p line frequency 50 60 Hz square wave TEST PULSE is generated by the instrument The TEST PULSE is attenuated as described above and is available for adjustment of capacitance compensation measurement of pipet resistance or for monitoring the formation of a gigohm seal at the electrode tip When external command is selected the TEST PULSE circuitry is disabled and commands appearing at the COMMAND IN BNC are attenuated and available to be applied to the headstage Junction Zero and Auto Zero These controls set the pipet current to zero after the pipet is placed in the test solution and prior to seal formation This circuitry is used to compensate for electrode potentials liquid junction potentials and other offset voltages and establishes a zero baseline reference potential The 10 turn JUNCTION ZERO control manually adjusts Im between 120 mV
13. PC 505B Manual Rev 050816 Warner Instruments Whole Cell Patch Clamp Amplifier Model PC 505B Warner Instruments 1125 Dixwell Avenue Hamden CT 06514 800 599 4203 203 776 0664 203 776 1278 fax PC 505B Manual Rev 050816 Table of Contents INTRODUCTION ee M 4 NOMENCLATURE Do 5 ext COMVENULIONS 45555 s0isssicesincsssesdsossecsesnsoncdonednoaaesastenadesssedinkeadeassoveoscoassancsdasdescdesdeeccessseeedovednsnaesenssoasense 5 Device panel abbreviations scsssssssssscssssccssscssssscssssccssscssesssssssscssssssssssssessssssessssscssesssssssssssescssseces 5 Signal Polarity CONVENCONS EROR se osto sss 5 Membrane current Li tire dhe itis eel eiie i ieee hed ah edd ae 5 Membrane potential V m osised s anera sese esee esent NEN enne rn nre e earn ns enne nnne sene nnns s nnns nnn 6 CONTROE DESCRIPTION ER 7 OMG ame CE E H 7 COMMANDS DIOCK 332 60 eia aem eee EE Re e e E 7 FAST CAP COMP fast capacitance compensation command block sese 8 WHOLE CELE command DOCK crscrssccarvesscsaneshsccabiesiscnscassccassessacuacassivariessccadcdboccabvessedanedsadcassesscaareadacenedsss 9 OUTPUT Command block et e des de dei sa eee Cp ee ede eden Coste dde 10 METER SECON cse cech e Eee i e e et eiue E te eere eu te ee Ee tb e ben ede te dee 11 Front and rear panel BNC s and connectors esee eese eese een
14. R REAR PANEL Ve COMMAND IN voltage OUTPUT METER REAR PANEL XVc sum of all command and METER compensation voltages Ve h IN V plus HOLDING voltage METER P STAT potentiostat mode OUTPUT REAR PANEL Signal polarity conventions Membrane current lm Im is indicated as positive when cations flow outward from the pipet tip through the cell membrane and into the bath and or when anions flow from the bath into the pipet With outside out patch and whole cell preparations this corresponds to the conventional Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 6 physiological definition of outward transmembrane current With inside out or cell attached patches the physiological transmembrane current equals minus the indicated Im Membrane potential Vm Membrane potential is defined as pipet potential minus bath potential With outside out patch and whole cell preparations this corresponds to the conventional physiological definition of transmembrane potential With inside out or cell attached patches the physiological transmembrane potential equals minus the indicated Vm Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 7 CONTROL DESCRIPTION The instrument front panel is divided into several control blocks Controls within these blocks are dedicated towards a common functional purpose Each control block is outlined in blue and is clearly labeled as COMMANDS FAST CAP COMP
15. TS block of the amplifier gain telegraph A defined voltage dependent on the gain setting appearing at the associated BNC at the rear of the amplifier Used to communicate the gain setting to external devices ground loop A loop formed from multiple connections into the circuit ground plane by the same device The flux of magnetic fields through this loop can induce small currents within the ground plane resulting in increased noise in the circuit Careful consideration of the interconnection between several devices is often required to identify ground loops headstage A low gain amplifier placed as close to the preparation as possible Used to amplify small currents to a range sufficient for the main amplifier to accept lm A measure of the current passed through an open channel in the presence of a driving force Operationally the current appearing at the Im OUTPUT of the amplifier Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 38 intracellular Situated or occurring within a cell junction potential A difference in conductivity between two dissimilar materials will appear as a small voltage when the two materials are brought into contact This voltage is termed the junction potential LED Light Emitting Diode The red green or yellow lighted indicators on the front of many devices LED s are preferred indicator light sources due to their low power consumption mean closed time
16. Therefore we recommend that Ag AgCl electrodes be connected to the bath through agar salt bridges to maintain a constant Cl concentration near the electrode In addition the isolation provided by the agar bridge will prevent Ag ions from contaminating the baths First clean the wire by wiping with a clean tissue wet with alcohol or a standard laboratory detergent then rinse well with distilled water Wiping in this way can help to straighten the wire If using alcohol or similar solvent avoid getting it onto the polycarbonate holder body as this can weaken the structure Chloriding by electrolysis Dip the Ag wire to the required depth in a solution of 0 1m NaCl or KCl optionally made slightly acidic with HCl and arrange to pass positive current from the Ag into the solution For the indifferent electrode in the solution a cleaned carbon rod from a discarded 6 V lantern battery works well Another thicker Ag wire will also work but most other metals are likely to contaminate the AgCl coat Pass current at a density of about 1 mA cm for about 1 minute or until adequately plated For a 2 cm length of 0 01 wire this is about 150 uA When well plated the surface should be uniformly light grey Reversing the current polarity occasionally while plating ending with the Ag positive tends to make a more stable electrode If available a low frequency signal generator at about 0 1 Hz with a slight positive bias is convenient The electrolyte sol
17. adstage to the amplifier and place the headstage into the shielded enclosure Faraday cage Do not make attachments to the headstage inputs at this time Insure that the Faraday cage is grounded as described above 4 Plug the amplifier voltage source and oscilloscope in 5 Power up the voltage source and oscilloscope Initial configuration Set all controls on the PC 505B to the values specified in the following table With the exception of the power switch this initial configuration will be used to return the amplifier to a known condition to begin each sub section during the instrument checkout Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 18 Begin each checkout section by returning the PC 505B to this known configuration Front panel controls Control Control block Initial setting POWER METER off VOLTAGE HOLD COMMANDS fully CCW toggle switch off CURRENT HOLD COMMANDS fully CCW toggle switch off COMMAND SELECT COMMANDS external command COMMAND SENSITIVITY COMMANDS off x0 1 JUNCTION ZERO control COMMANDS zero mV AUTO ZERO toggle COMMANDS off ZAP toggle COMMANDS safe ZAP control COMMANDS 0 1 ms C FAST 1 FAST CAP COMP fully CCW C FAST 2 FAST CAP COMP fully CCW FAST T1 FAST CAP COMP fully CCW FAST T2 FAST CAP COMP fully CCW C SLOW WHOLE CELL fully CCW C SLOW toggle WHOLE CELL o
18. anar INSTRUMEN and the internal holding voltage h Full scale reading is CORPORATION t 200 mV XV This selection reports the sum of all command voltages Mathematically XV Vet h IN JUNCTION ZERO AUTO ZERO SERIES R It does not include LEAK SUBTRACTION Full scale range is 200 mV Vm This selection reports the transmembrane potential when in current clamp mode Full scale range is 200 mV Im This selection reports the transmembrane current when in voltage clamp mode Full scale range is 1999 pA Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 12 RMS noise This selection reports the root mean square RMS value of the noise filtered to a bandwidth of 1 kHz This reading is valid only when the PROBE RESISTOR select is set to high since the amplifier gain changes for other settings The expected reading for the 50 GQ resistor with open input and properly shielded from 60 Hz interference is approximately 0 040 pA Full scale range is 1 999 pA RMS Front and rear panel BNC s and connectors The PC 505B has input and or output BNC s on both front and read panels These include Im and Vm outputs V x10 and Vm x10 outputs GAIN and FILTER TELEGRAPHS Im Vm mode telegraph SYNC OUTPUT and COMMAND INPUT Front and rear panel layouts are described below With the exception of the rear panel COMMAND IN BNC all connector sleeves are connected to circuit ground and are insulated fr
19. anufacturer s Address 1125 Dixwell Avenue Hamden CT 06514 Tel 203 776 0664 Equipment Description Instrument Amplifier Safety requirements for electrical equipment for measurement and laboratory use Equipment Class Class I Model Numbers PC 505B I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Place Hamden Connecticut USA Signature Cah pr Full Name Burton J Warner Position President Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 35 WEEE RoHS Compliance Statement EU Directives WEEE and RoHS To Our Valued Customers Harvard Apparatus is committed to being a good corporate citizen As part of that commitment we strive to maintain an environmentally conscious manufacturing operation The European Union EU has enacted two Directives the first on product recycling Waste Electrical and Electronic Equipment WEEE and the second limiting the use of certain substances Restriction on the use of Hazardous Substances RoHS Over time these Directives will be implemented in the national laws of each EU Member State Once the final national regulations have been put into place recycling will be offered for those Harvard Apparatus products which are within the scope of the WEEE Directive Products falling under the scope of the WEEE Directive available for sale after August 13 2005 will be identified w
20. aph settings 13 Im gain mV pA telegraph out V 0 05 0 5 0 1 1 0 0 2 1 5 0 5 2 0 1 0 2 5 2 0 3 0 5 0 3 5 10 4 0 20 4 5 50 5 0 100 5 5 200 6 0 500 6 5 1000 7 0 FILTER TELEGRAPH Output BNC reporting the filter setting in use Telegraphs range from 0 2 to 2 0 V in 0 2 V steps as shown below Filter telegraph settings Frequency Hz telegraph out V 50 0 2 100 0 4 200 0 6 500 0 8 1k 1 0 2k 1 2 5k 1 4 10k 1 6 20k 1 8 Bypass 2 0 SYNC OUT Sync out provides a TTL pulsed output for synchronizing an oscilloscopes or other equipment with the internally generated 50 60 Hz TEST PULSE or SPEED TEST signal Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 14 Rear panel connectors and controls In addition to input and output BNC s the instrument rear panel also contains the headstage cable connector calibration trimmers and selector function switches for PSTAT and SPEED TEST modes Headstage cable connector The headstage connects to the instrument via an 8 pin DIN connector PSTAT switch The PC 505B is converted to operate as a potentiostat for voltammetric measurements by a clockwise rotation of this switch When switched on the front panel PSTAT indicator LED will light In this mode the maximum electrode voltage hold potential is increased to 1 V and the
21. coded and organized for quick reference and convenient operation Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 5 NOMENCLATURE Text conventions This manual refers to amplifier controls at three functional levels control blocks specific controls within a block and settings of specific controls To minimize the potential for confusion we have employed several text conventions which are specified below Since our goal is to provide clarity rather than complexity we welcome any feedback you may wish to provide Warner Instrument product numbers are presented using bold type References to instrument panel control blocks are specified using UNDERLINED SMALL CAPS References to specific controls within a block are specified using SMALL CAPS References to individual control settings are specified using italic type Special comments and warnings are presented in highlighted text Any other formatting should be apparent from context Vv Vv ON ON WV Device panel abbreviations Many controls on the PC 505B have abbreviations associated with them Several of these abbreviations are listed here for quick reference In addition these and other terms have been collected and are included in a Glossary at the back of this manual Term Meaning Sections CAP COMP capacitance compensation FAST CAP COMP In output current OUTPUT METER REAR PANEL Vin membrane voltage OUTPUT METE
22. d changes in applied membrane potential These effects if uncompensated can drive the headstage amplifier into saturation which can result in the loss of several ms of data while the headstage recovers WHOLE CELL command block The WHOLE CELL command block contains controls for C SLOW SERIES R CORRECTION and LEAK LEAK SUBTRACTION 10pF Turn SUBTRACTION These controls are used to compensate ON for the effects of membrane capacitance and access resistance when the amplifier is used in whole cell mode With the exception of LEAK SUBTRACTION these controls are only available when the PROBE RESISTER in the OUTPUT command block is in low mode C Slow This control is used to compensate for the whole cell membrane capacitance The compensated capacitance can be read directly from the lockable 10 turn control The associated toggle switch disables c sLow allowing comparison with the uncompensated signal This control is not available in patch mode since the FAST CAP COMP circuitry is used to address capacitances in this mode Series R Series resistance compensation is used to compensate for the voltage drop across the electrode and access resistance in the experimental setup This control is used to adjust the time constant of the C SLOW control facilitating measurement of membrane resistance which can be read from the dial This control is not available in single channel recording mode since series resistances are
23. d checkout 1 on fa QU ROO d 10 11 12 13 14 15 Apply a 1 VDC signal to COMMAND IN BNC on instrument rear panel Connect oscilloscope to V x10 output BNC on instrument front panel Set the METER SELECT switch to XVc Set COMMAND SELECT COMMANDS block to external command Set COMMAND SENSITIVITY to xO 1 Turn COMMAND SENSITIVITY toggle to on Set oscilloscope to 0 5 V div Verify that METER reads 100 1 mV and that oscilloscope reads 1 V 2 div at these settings Set COMMAND SENSITIVITY toggle to x0 01 Set oscilloscope to 50 mV div Verify that METER reads 10 mV and that oscilloscope reads 100 mV 2 div at these settings Set COMMAND SENSITIVITY toggle to x0 001 Set oscilloscope to 5 mV div Verify that METER reads 1 mV and that oscilloscope reads 10 mV 2 div at these settings Return amplifier to initial settings Internal AC voltage command checkout 1 2 3 4 5 6 7 Set COMMAND SELECT COMMANDS block to internal command Set COMMAND SENSITIVITY to xO 1 Set COMMAND SENSITIVITY toggle to on Connect V x10 output BNC to oscilloscope Set oscilloscope voltage base to 0 5 V div and time base to 5 ms div Verify that oscilloscope displays appropriate square wave 1 V p p at line local frequency of 50 or 60 Hz Return amplifier and oscilloscope to initial settings Auto Zero control checkout 1 Disconnect voltage source from COMMAND IN BNC Warner Instrument
24. d from the headstage housing A 2 5 m cable with 8 pin DIN connector attaches the headstage to the mating connector on the instrument rear panel Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 15 Headstage types applications and features Headstage Types Application Features LC 201 Patch Whole Cell 50 GQ 500MQ headstage Single channel currents up to 200 pA Whole cell currents up to 20 nA HC 202 Patch Whole Cell 50 GQ 50MQ headstage Single channel currents up to 200 pA Whole cell currents up to 200 nA HB 205 Bilayer studies 50 GQ headstage for bilayer Currents up to 200 pA Bilayer capacitance up to 250 pF Electrode holders Electrode holders connect the glass micropipet electrode to the headstage A fine chlorided silver wire the actual electrode makes electrical contact between the headstage input and the electrolyte solution in the micropipet Holders are machined from polycarbonate to minimize electrical noise and are custom bored to accommodate various diameter pipet electrode glasses The micropipet is secured with a rubber gasket and a polycarbonate screw cap matching the OD of the pipet glass A 1 mm pin makes electrical contact with the silver wire and plugs the holder onto the headstage A 1 16 OD access port is provided for applying suction to seal the pipet tip to the cell membrane ee Model Cell A model cell is included wi
25. e ennt KENNE SAARNAK nnne nennen 12 H adstage 5 8 AR ASTI uu Ae idee ted ae UH Pine aR 14 Electrode holders uice a R cce eccles e tee ex eL deae dede eti ree dede at ede eti dede dR 15 Model Cell i aii iet re heel t Ud eee d HER EL dE TU chants LE ELE Hen 15 SETUP AND INITIAL TES EE G0S Z n nttne 16 JETTA L GE AEE AAEE ERE 16 Instrument grounding eoru ro hoo eo neo ae ea eos a cucuecsenecodevesgsvescseaeecesetssussdedessesseteenedensobessucesests 16 Headstage precautlons i iieeeee se entere en ren ote ee oin oti ea Feo ee to poosi SRo Eee Ea es en ee ena eee be erae Pese eoe en eoo ra Pie Pe oe in 16 neues ucro M 17 Test Procedures 17 Initial Connec ONS i e e ote e e oa TNC bahia wea walle x ea es aah ou ba ae lle o eve aa eo dede ec eae Des 17 Initial conftguration iae eh eise et edis tes ehh e dE RR O RAEE a e d Haan 17 Headstage inputs OpenRMS noise checkout eese eene nennen 18 RMS NOREC REE KOU nae aie e ut TEO Ge Ee dote Ae BO 19 T OULDUL CHECK OUL erisin e TEES eia EEEE EENE dte etre N E 19 Junction zero CheGkOUtu u s e et EAE AEAEE Oa AAN AAE EE ESE EE to e dae eva Ee eaae eara Ded 19 Internal DC voltage command checkout eee esee eene entente enne nennen nein rentre nns 20 External DC Voltage command checkout eee esses esent ener 21 Internal AC voltage command checkout
26. ff SERIES R WHOLE CELL fully CCW LEAK SUBTRACTION WHOLE CELL fully CCW click off CORRECTION WHOLE CELL fully CCW CORRECTION toggle WHOLE CELL off PROBE RESISTOR select OUTPUT high MODE OUTPUT V GAIN OUTPUT 10 mV pA FILTER OUTPUT 10 kHz FILTER toggle OUTPUT active METER select METER In Oscilloscope setup Control Setting Time base 2 ms div Voltage base 1 V div Trigger line triggered Connect Im output on instrument front panel to the oscilloscope Headstage inputs Open Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 19 RMS noise checkout 1 2 Set the METER SELECT switch to the RMS noise position Check for a noise reading on the METER at or below 0 038 pA If the value reported is high then relocate or adjust the headstage and shielding to minimize noise inputs Im Output checkout 1 2 3 4 5 o 10 11 12 13 14 15 16 17 Set scope voltage base to 0 5 V div Set GAIN to 100 mV pA Set FILTER to 1 kHz Check that the METER reads 0 pA Switch PROBE RESISTER toggle from high to low Verify that the METER reading stays within 0 pA Switch PROBE RESISTER toggle from low to high Check that oscilloscope reads 0 0 0 5 pA i e within 1 div of zero on oscilloscope for this setting Set PROBE RESISTER toggle from high to low Set FILTER to 50Hz Set oscilloscope voltage base to 50 mV div Again check that oscilloscope reads 0 0 0
27. gar gel is usually employed for this purpose The AgCl reference electrode goes into one end of the tube and the other end is applied to the bath Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 28 APPENDIX Specifications HEADSTAGES LC 201B Headstage single channel currents to 200 pA 50 GQ 500 MQ whole cell currents to 20 nA HC 202B Headstage single channel currents to 200 pA 50 GQ 50 MQ whole cell currents to 200 nA HB 205B Bilayer Headstage for artificial bilayer capacitances up to 250 pF 50 GO modified currents to 200 pA Noise referred to input Measured with an 8 pole Bessel DCtolkHz 0 038 pA RMS filter input open 50 GO resistor DCto5kHz 0 170 pA RMS Bandwidth 25 kHz VOLTAGE CLAMP COMMANDS Command In t 10 V maximum AC or DC applied to input Voltage Hold 200 mV with 10 turn control Junction Zero 100 mV with 10 turn control Internal Test Pulse 1 Volt 50 60 Hz line frequency square wave attenuated by COMMAND SENSITIVITY Command Sensitivity attenuates at x0 1 x0 01 and x0 001 Zap 1 0 Volt Pulse adjustable duration from 0 1 to 10 ms CURRENT CLAMP COMMANDS Command In 1000 pA maximum with Command Sensitivity x0 1 100 pA maximum with Command Sensitivity x0 01 10 pA maximum with Command Sensitivity x0 001 Current Hold 1 nA with 10 turn control Internal Test Pulse 1 nA 50 60 Hz square wave Command Sensitivity at
28. h setting LED s indicate the selected gain scale modifier The clipping LED lights when the instrument Im output exceeds the amplifier limits 4 Pole Bessel filter The Im output signal can be filtered using the internal low pass 4 pole Bessel filter selectable from 50 Hz to 20 kHz in 9 steps The full amplifier bandwidth of 25 kHz is available by selecting bypass with the ACTIVE BYPASS toggle switch The filtered In signal is present at two BNC outputs labeled Im located at both the front and rear panels Voltammetry The PC 505B can be used as a potentiostat for voltammetric measurements A rear panel POTENTIOSTAT SWITCH increases maximum output of the VOLTAGE HOLD control in the COMMANDS block to 1 V and increases the maximum COMMAND IN signal to 2 V The P STAT LED lights to indicate on status for this switch METER section The six METER switch settings interact with other controls as noted Due to the limiting bandwidth of the Patch Clamp PC 505B meter display any high frequency signals presented will gt EF be reported as its DC time average value d Junction zero This selection reports the voltage METER SELECT POWER supplied by the JUNCTION ZERO control used to Eve Vm Ve h im compensate offset voltages present in the setup Full IN Ce Junction scale reading is 120 mV Zero ROSE V h IN This selection reports the sum of the COMMAND IN voltage Vc after attenuation by command sensitivity w
29. ined potential CMD IN Command Input An external input into the amplifier allowing the application of user defined command voltages to the headstage Connection is usually via BNC command sensitivity Selectable scaling of CMD IN input Attenuation values of CMD IN are xO 1 x0 01 and x0 001 command voltage The voltage applied to the headstage resulting in a desired transmembrane potential in the system under study control blocks Organization of controls on the amplifier into functional groups Blocks are delineated by titled blue boundaries Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 37 current voltage relationship A measure of the way in which the current varies as a function of the applied voltage In an Ohmic device obeys Ohm s law or V R this relationship is linear An understanding of the current voltage relationship of a channel yields information about that channel s function depolarization A biological membrane in which charge separation has resulted in transmembrane voltage is termed polarized Electrically depolarization refers to any action which tends to reduce the degree of polarization Biophysically a polarized membrane has a resting transmembrane potential between 40 and 90 mV relative to the inside of the cell An action which tends to increase the polarization e g increase the transmembrane potential to say 100 mV is termed hyperpolariza
30. insure good contact with the pin connector Reassemble the wire and seal into the holder taking care that the seal sits squarely in the hole and that the wire doesn t jam in the holder on its way through Reference electrodes A reference electrode in the recording chamber maintains the bath at circuit ground potential the reference potential for all measurements It is also the return path for currents Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 27 from the pipet electrode A variety of Ag AgCl reference electrodes are available from Warner Instruments A simple reference electrode can be made from a silver wire Use wire somewhat thicker than the one in the pipet electrode holder The end applied to the bath should be chlorided as described on page 26 such that the Ag AgCl coating can be submerged with no bare Ag exposed to solution The free end is connected to circuit ground Any exposed Ag surface that could potentially contact the bathing solution should be insulated with a waterproof coating of epoxy cement insulating varnish or similar durable waterproof coating This precaution provides a stable baseline as long as the bath solution is not changed When bath solutions are to be changed during an experiment a KCl salt bridge can be used to minimize changes in the junction potential that accompany these solution changes A small glass or plastic U tube filled with saturated or 3 M KCl in warmed 2 a
31. ith a wheelie bin symbol Two Categories of products covered by the WEEE Directive are currently exempt from the RoHS Directive Category 8 medical devices with the exception of implanted or infected products and Category 9 monitoring and control instruments Most of Harvard Apparatus products fall into either Category 8 or 9 and are currently exempt from the RoHS Directive Harvard Apparatus will continue to monitor the application of the RoHS Directive to its products and will comply with any changes as they apply Special Collection Disposal Required Do Not Dispose Product with Municipal Waste Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 36 Glossary A D converter Analog to Digital converter Computers are inherently digital while the voltage or current output from an amplifier is analog Therefore a signal must be first converted to a digitized form before a computer or its software can accept it Desirable features in an A D converter include rapid signal conversion small step resolution and low noise analog Continuous or non discrete Often dynamically varying Compare to digital bandwidth The range of frequencies a device is capable of processing with minimal distortion A bandwidth of 1 Hz indicates that the device can faithfully process a signal occurring once per second 1 Hz The larger the bandwidth the faster the device Bessel filter A device used t
32. just CURRENT HOLD control until METER reads 50 mV Set METER SELECT to Vm Verify that METER reads 0 mV e g CURRENT HOLD command is disabled in I mode Set MODE toggle to Ic Set METER SELECT to Im Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 24 12 13 14 15 Adjust CURRENT HOLD control until METER reads 500 pA Set METER SELECT to Vm Verify that meter reads 5 1 mV corresponding to the holding potential required to pass a 500 pA current through a 10 MQ resister Return amplifier to initial settings This completes the functional checkout of the instrument Please contact our technical support staff if you have any questions or experience any problems Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 25 THEORETICAL CONSIDERATIONS Chloriding silver wire Silver silver chloride electrodes act as signal transducers by converting ionic currents in solution to an electric current within a wire This is achieved by utilizing a reversible oxidation reduction reaction between the electrode and Cl ions in solution The chemical reaction is Cl Ag amp AgCI e The potential developed by one electrode is proportional to the standard electrochemical potential for Ag AgCl plus the Cl concentration at the solution electrode interface Since this potential is dependent on Cl a voltage bias will be introduced by changing the solution Cl concentration
33. lamp mode Sync Out TTL signal for synchronization oscilloscope with test pulse OPERATING CONDITIONS Equipment is intended to be operated in a controlled laboratory environment Temperature 0 40 C Altitude sea level to 2000 m Relative humidity 0 95 PHYSICAL DIMENSIONS Power Requirements 110 130 or 220 250 VAC 50 60 Hz Main Unit 9 x 42 x 30 cm Hx W x D Headstage 1 9x3 5x 5 7 cm Hx Wx L with 1 8 m cable Headstage Mounting Rod 6 3 mm x 6 3 cm Dia x L Shipping Weight 11 4 kg Warranty Three years parts and labor Axon pClamp and Instrutech Pulse Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 Accessories and replacement parts 30 Model Number Order Number Description Headstages When ordering additional or replacement headstages please reference the serial number of your PC 505B LC 201 64 0004 50 GQ 500 MQ feedback resistors HC 202 64 0005 50 GQ 50 MQ feedback resistors HB 205 64 0006 50 GO 500 MO feedback resistors modified for bilayer Standard cable length is 1 8 meters Lengths up to 3 05 meters 10 are available Electrode Holders One electrode holder is supplied with each headstage The standard model is QSW AxxP straight Holder part numbers are completed by replacing the xx with 10x the OD in mm of the pipet glass to be used QSW A10P 64 0821 for 1 0
34. mm OD pipette glass QSW A12P 64 0822 for 1 2 mm OD pipette glass QSW A15P 64 0823 for 1 5 mm OD pipette glass QSW A17P 64 0978 for 1 65 mm OD pipette glass QSW A20P 64 0824 for 2 0 mm OD pipette glass parts Other style holders such as 90 45 and microperfusion are available Contact our Sales Department for complete details on holders capillary tubing and replacement Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 31 Warranty and service We recommend that all questions regarding service be referred to our Support Department Normal business hours are 8 30 AM to 5 30 PM EST Monday through Thursday and 8 30 AM to 5 00 PM on Friday Our offices are located at 1125 Dixwell Avenue Hamden CT 06514 and we can be reached by phone at 800 599 4203 or 203 776 0664 Our fax number is 203 776 1278 In addition we can be reached by e mail at support warneronline com or through our Web page at http www warneronline com Warranty The model PC 505B is covered by our Warranty to be free from defects in materials and workmanship for a period of three years from the date of shipment If a failure occurs within this period we will either repair or replace the faulty component s This warranty does not cover instrument failure or damage caused by physical abuse or electrical stress inputs exceeding specified limits In the event that instrument repairs are necessary shipping charge
35. nessdenseseseasdenecsoasssns 30 Warranty and Service ceo res tcc eonun Fo n Eig nooo oinpe eier Sae Yee ona ro SWR eeN Eor SS Tease o pF ke aas CERE eue PE Te Yo eR VER piesi 31 WSANEY EE 3l Seryice HOlgs ee itr eco ed p e toe Presse de pie e due be Peri cep ERE DA ode rese etd 3 RECOMMEND BXc rm 32 Bowie X 33 UD eC 36 PC 505B Manual Rev 050816 4 The Warner model PC 505B Whole Cell Patch Clamp Amplifier is an extremely low noise resistive feedback patch clamp amplifier designed for whole cell single channel and bilayer applications The unique circuitry and dedicated design of this amplifier allows Warner Instruments to present a superior quality instrument at a cost significantly below that of many of our competitors Principal features of the PC 505B include Y EE LL 10 00M M Low noise levels of 0 038 pA RMS at 1 kHz Built in RMS noise monitor Voltage and current clamp modes with independently selectable Vno and Inoia Bandwidth to 25 kHz Internal 4 pole low pass Bessel filter with rear panel filter telegraph Built in test generator Automatic junction potential compensation Two range fast capacitance compensation Series resistance and C slow capacitance compensation Correction circuitry Adjustable duration zap circuit for whole cell membrane penetration Output gain selection with rear panel gain telegraph Front rear panel controls and connectors are color
36. o attenuate the high frequency components of a signal The cutoff frequency of a filter is normally defined as the frequency at which the amplitude of the signal is attenuated by 3 dB A higher order filter i e 8 pole vs 4 pole will attenuate the high frequency components more rapidly An 8 pole Bessel filter attenuates at 14 dB per octive BNC connector A type of connector used to connect coaxial cables to high frequency electronic equipment CAP COMP See capacity compensation capacitance A capacitor can be represented by a small break in a conducting pathway bounded by two parallel plates The electric field generated across the space between the plates in the presence of an applied voltage maintains a charge density on each plate The numerical measure of a capacitor s ability to maintain charge separation at a given potential is its capacitance Capacitors effectively block DC currents while passing AC currents Has units of Farad F capacity compensation The process wherein the current generated when charging a capacitor is subtracted or compensated from the output signal channel conductance See unitary channel conductance chassis ground A connection used to link the amplifier chassis to an external potential circuit ground The potential to which all other potentials within the circuit are referenced Also a connection used to link the reference potential of the amplifier circuit to an externally def
37. om the chassis Front panel BNC s Ve x10 Output BNC reporting the sum of all voltages applied to the headstage input Vm x10 Output BNC reporting the transmembrane potential active only in current clamp mode I Output BNC reporting membrane current in both voltage and current clamp modes Rear panel BNC s In Vm OUT Output BNC reporting membrane current when instrument is in voltage clamp mode and Vm x10 when instrument is in current clamp mode Im Wm TELEGRAPH Provides TTL logic to indicate voltage clamp or current clamp mode O I clamp 1 V clamp COMMAND IN Differential input BNC for voltage command from external sources e g signal generator or computer Since the center pin and sleeve are the and inputs respectively the sleeve is not grounded The applied voltage is attenuated by the COMMAND SENSITIVITY switch and is available in both voltage and current clamp modes The COMMAND IN input BNC is disengaged when TEST PULSE is active internal command when the COMMAND SENSITIVITY toggle switch is off or when the amplifier is in zero current mode MODE selector switch set to Io GAIN TELEGRAPH Output BNC reporting Im gain as a combination of the GAIN switch selection and the gain multiplier x0 1 x1 0 or x10 dependent on the headstage in use Telegraphs range from 0 5 to 7 0 Vin 0 5 V steps as shown below Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 Gain telegr
38. s A Harvard Apparatus Company PC 505B Manual Rev 050816 22 o won anu fF 9p M ee C Op M PU Ee MD ee ge Se ge 11 12 13 14 15 Connect model cell to headstage in patch mode Set METER SELECT to Im Set PROBE RESISTER to high Set COMMAND SENSITIVITY toggle to off Set COMMAND SELECT COMMANDS block to external command Use JUNCTION ZERO control to set Im output on METER to 10 pA Switch AUTO ZERO on and press AUTO ZERO BUTTON until meter reads zero Switch AUTO ZERO off Re zero Im output using manual JUNCTION ZERO control Return amplifier to initial settings commands checkout Connect model cell to headstage in patch mode Connect Im output BNC to oscilloscope Set oscilloscope to 1 V div Set METER to Im Verify PROBE RESISTER is set to high Set COMMAND SELECT to external command Set COMMAND SENSITIVITY toggle to off Set VOLTAGE HOLD to positive Use VOLTAGE HOLD to set Im output to 10 pA on METER positive hold adjustment should give positive current output Set GAIN to 10 mV pA Verify that oscilloscope reads 1 V 1 div at these settings Set VOLTAGE HOLD to negative Verify that METER reads 10 pA Verify that oscilloscope reads 1 V 1 div at these settings Return amplifier to initial settings Cap Comp checkout 1 2 3 Connect model cell to headstage in patch mode Set PROBE RESISTOR to high Set VOLTAGE HOLD control to off Warner
39. s to the factory are the customer s responsibility Return charges will be paid by Warner Instruments Inc This warranty is not extended to electrode holders since these items are considered disposable Service notes A If the instrument POWER light fails to light check the fuse at the rear panel If the fuse is found to be defective replace it with a 3AG 1 2 amp normal blow fuse 1 4 amp for facilities using 220 240 V line voltages If the replacement fuse also fails please call Warner Instruments for assistance B Occasionally a knob on the front panel will loosen after long use These are collet style knobs and are tightened with a screw located under the knob cap To gain access to the adjustment screw pry the cap off with a thin bladed screwdriver or similar tool C Should service be required please contact the factory The problem may often be corrected by our shipping a replacement part Factory service if required will be expedited to minimize the customer inconvenience D Instruments are inspected immediately upon receipt and the customer is notified if the repair is not covered by the warranty Repairs can often be completed in 1 2 days from our receipt of the instrument If factory service is required please observe the following instructions 1 2 3 Package the instrument with at least 3 inches of cushioning on all sides Use the original shipping carton if it is available Insure the shipment for its f
40. t filling solutions onto exposed bare silver above the AgCl coating in the pipet within the body of the holder into the suction port or especially onto the pin jack which could damage the headstage Should this happen disassemble the holder rinse thoroughly with distilled water dry thoroughly and reassemble Cleaning and storage After use rinse any deposits from the holder body the Ag wire pin jack and suction port and allow to dry To protect the silver wire from getting bent in storage the holder can be capped with a short piece of 3 8 ID plastic tubing or a 50 x 10 mm ID plastic vial with a small nylon set screw Replacing holder parts The rubber seal at the pipet end of the holder can be replaced if it becomes damaged with repeated pipet insertions A spare is supplied with the holder and additional gaskets can be ordered as required The gasket is easily removed with a small pair of blunt forceps The silver wire is also replaceable Replacement is necessary when the wire becomes hopelessly kinked or badly jammed as by carelessly inserting into pipets To replace the wire unscrew the pin connector and remove the rubber seal Pull the wire from the small hole in the center of the seal and replace it with a new piece about 6 cm long Insert the wire through the seal from the pipet side so that 5 mm extends beyond the pin connector side Bend this end by 90 at the seal and fold the end of the wire back across the seal to
41. tential It can be used to drive any additional guard shielding such as a foil covering or conductive paint applied to the pipet electrode Insure that the guard shielding never touches ground Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 17 Headstage preparation Using the proper handling precautions described above connect the headstage cable connector into the rear panel probe receptacle and connect the Im output BNC to an oscilloscope The headstage probe normally requires a grounded enclosure Faraday cage to shield it from 50 60 Hz line interference The shield should be grounded to the black circuit ground jack on the rear of the PC 505B chassis Place the headstage into the shield enclosure and run the grounding cable to the amplifier in the same bundle as the headstage cable ems Test Procedures To perform the following tests of the instrument you will need these tools and components e The PC 505B amplifier e The headstage mounted into a shielded enclosure e An oscilloscope with BNC cables e An adjustable DC voltage source e The model cell shipped with the instrument Initial connections Place the amplifier on a counter and make the following connections between the instrument and oscilloscope 1 Connect the Im output BNC on the instrument front panel to the oscilloscope 2 Connect the DC voltage source to the COMMAND IN BNC on the instrument rear panel 3 Connect the he
42. tenuates at x0 1 x0 01 and x0 001 CAPACITANCE COMPENSATION C FAST 1 0 1 to 1 75 us Voltage Mode O to 5 pF C FAST 2 0 33 to 8 5 us Voltage Mode O to 15 pF C SLOW O to 100 pF with 10 turn control Series R O to 10 MO with 10 turn control Correction O to 9096 of series R Leak Subtraction 50 GQ Headstage Resistor o to 50 GO 500 MO Headstage Resistor o to 500 MO Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 29 50 MQ Headstage Resistor o to 50 MO FRONT PANEL OUTPUTS I m membrane current gains of 0 05 to 10 mV pA with 50 MO headstage resistor gains of 0 5 to 100 mV pA with 500 MQ headstage resistor gains of 5 to 1000 mV pA with 50 GQ headstage resistor Ve x10 summation of all commands amplified by 10 Vm x10 membrane voltage amplified by 10 I m Output Low Pass Filter 4 Pole Bessel 3 dB points 0 05 0 1 0 2 0 5 1 2 5 10 and 20 kHz Bypass allows full bandwidth Panel Meter 3 1 2 digit LCD full scale reading Junction Zero 199 9 mV V h IN sum of all commands and V hold t 199 9 mV XV Sum of all commands t 199 9 mV Vm membrane voltage in current clamp mode t 199 9 mV In membrane current t 1999 pA RMS noise 1 999 pA REAR PANEL OUTPUTS Gain Telegraphs 0 5 to 7 0 V in 0 5 V steps Filter Telegraphs 0 2 to 2 0 V in 0 2 V steps Im Vm telegraph TTL logic O I clamp mode 1 V c
43. th the instrument This model cell has three connectors one to simulate the bath 10MO to ground one each to simulate a membrane patch and one to simulate a whole cell application An additional model cell is included with the HB 205 bilayer headstage to simulate a planar lipid bilayer membrane The model cell is used in the following familiarization instructions and is a valuable tool in troubleshooting problems with actual setups Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 16 SETUP AND INITIAL TEST Line voltage Power line voltage requirements for the PC 505B are specified on the serial number nameplate attached to the chassis rear They are wired for either 100 130 VAC or 220 240 VAC at either 50 or 60 Hz Check to be sure the PC 505B is wired for the line voltage and frequency to be used Instrument grounding The power cord is fitted with a three prong grounding type plug and should be plugged into a properly wired three wire grounded receptacle This internally grounds the PC 505B chassis to the power receptacle ground and insures safe operation of the equipment Headstage precautions The PC 505B headstage is a high impedance static sensitive device as noted on the protective envelope in which it is shipped The device can be seriously damaged by static discharge or inadvertent grounding Therefore to insure proper operation and long life we recommend you follow these precautions 1 Alwa
44. tion while depolarization refers to any action which decreases the transmembrane potential It should be noted that by this definition a transmembrane potential of 100 mV is still depolarized digital Quantized or discrete Normally refers to information manipulated by a computer All processes within a computer are discrete and are composed of 0 s and 1 s The universe we interact with is functionally analog therefore information we wish to manipulate with a computer must be digitized prior to use by the computer DIN connector Deutsche Industrie Norm A German standard for electronic and industrial products DIN connectors can be 3 to 6 pin plugs with the same outer diameter and appearance electrode One terminal of a voltage source which can either supply or collect current electromagnetic From physics An electric current induces a magnetic field and a changing magnetic field induces an electric current Therefore these two entities are related to each other and are combined into electromagnetism electrophysiologist A scientist who combines the disciplines of physics electrical engineering and physiology to the study biological systems Faraday cage A grounded conducting enclosure which shields its interior from external electric fields Named after Michael Faraday who first described it in 1875 gain The numerical value of the amplification of a signal by an amplifier User selectable in the OUTPU
45. ull value Include with the shipment an explanation of the problem experienced Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 32 Recommended reading Sakmann and Neher Single channel Recording Plenum Press 1983 Hamill Marty Neher Sakmann and Sigworth 1981 Pflugers Archiv 391 85 100 Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 33 Certifications Declaration of Conformity CE MARKING EMC Application of Council Directive 89 336 EEC Standards To Which Conformity EN55022 Class A Is Declared EN61000 3 2 EN61000 3 3 EN50082 1 1992 EN61000 4 2 EN61000 4 3 ENV50204 EN610000 4 4 EN610000 4 8 EN610000 4 11 Manufacturer s Name Warner Instruments LLC Manufacturer s Address 1125 Dixwell Avenue Hamden CT 06514 Tel 203 776 0664 Equipment Description Instrument Amplifier Equipment Class ITE Class A Model Numbers PC 505B I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Place Hamden Connecticut USA Cah pr Full Name Burton J Warner Signature Position President Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 34 Declaration of Conformity CE MARKING LVD Application of Council Directive 73 23 EEC Standards To Which Conformity Is EN61010 1 1993 Declared Manufacturer s Name Warner Instruments LLC M
46. ution can be saved and reused indefinitely Chloriding chemically Immerse the clean Ag wire in Clorox solution until the wire is uniformly light gray about 1 10 min Rinse well with tap water then with distilled Electrode holders The standard holder supplied with PC 505B is the QSW AxxP straight body style It uses a 0 010 diameter 99 99 pure silver wire to couple the signal from the micropipet solution to Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 26 the input pin of the headstage amplifier Before use the wire must be plated with silver chloride AgCl to within 2 3 mm of the end cap which secures the micropipet A 2 mm OD port on the side of the holder is used for applying pressure or suction through standard 1 16 ID flexible plastic tubing Each holder is made to accommodate a single specified diameter of pipet electrode glass designated by xx in the part number where xx 10x the OD in mm For example QSW A15P specifies 1 5 mm OD pipets Care and use of holders Both ends of the pipet tubing should be lightly fire polished before pulling micropipets in order to avoid scraping AgCl from the wire surface and to prolong the life of the rubber gasket that holds the pipet in place Fill pipets with only enough electrolyte to cover several mm of the AgCl coating when inserted into the holder This minimizes stray capacitance and the noise level while recording Take care to avoid getting pipe
47. with immediate response With the AUTO ZERO toggle switch in the on position auto zero LED on depressing the associated pushbutton zeros the current automatically The settling time is rapid when used with MQ pipet resistances but can take several seconds if used with GO resistances For complete compensation hold the pushbutton in until Im on the METER reads zero Zap The ZAP function generates an internal 1 5 V pulse FAST CAP COMP TAS which is applied to the headstage The pulse duration may be adjusted from 0 1 to 10 ms as indicated A safe active toggle switch arms the circuitry and the pulse is initiated when the ZAP button is depressed FAST CAP COMP fast capacitance compensation command block The two pairs of controls C FAST 1 FAST t and C FAST 2 FAST 15 are used to adjust the amplitude and time constant tau of the circuitry that compensates for capacitative currents due to the pipet and other stray capacitances This is achieved by applying an appropriate capacitative countercurrent to the headstage lt pesosparcHCLAMP input These controls are disabled in current clamp mode Warner Instruments A Harvard Apparatus Company PC 505B Manual Rev 050816 9 When properly adjusted FAST CAP COMP controls have two important functions 1 to closely align the voltage clamp waveform at the cell membrane to the command signal waveform and 2 to minimize large current transients associated with rapi
48. ys discharge static electricity from your body before handling the headstage Your body has a capacitance of around 100 200 pF to ground and can acquire enough static charge by handling Styrofoam touching the face of a video monitor walking across a dry carpet wearing polyester clothing etc to alter your electric potential by as much as 10 kV relative to ground A discharge of this magnitude through the headstage can render it useless We recommend that you electrically discharge yourself either by firmly contacting a securely grounded part of the setup with a lightly moistening a finger or by wearing a grounded wrist strap available from any electronics store 2 Do not ground or apply a low impedance signal the headstage input connector pin 3 Do not ground the headstage case when the power is on This includes allowing the headstage case to inadvertently contact any grounded components This is necessary since the case is both isolated from ground and is driven at the command potential 4 Two 1 mm pin jacks are provided on the side of the headstage for grounded and or driven shield applications The circuit ground pin jack is insulated from the case and is identified by a black collar It is intended to be used for the bath ground and or shielding around the electrode and holder It is not intended as a general equipment ground The uninsulated pin jack makes direct contact with the headstage case and is driven at the command po
Download Pdf Manuals
Related Search