NPI BA-01X Operating Instructions Manual Download Page 30 | Manualshive

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BA-01X User Manual

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version 1.9

page 30

Figure 15: Approaching the cell, forming a gigaseal and establishing the whole cell

configuration

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Summary of Contents for BA-01X

Page 1: ...ESCRIPTION FOR THE BA 01X INTRACELLULAR BRIDGE MODE AMPLIFIER VERSION 1 9 npi 2014 npi electronic GmbH Bauhofring 16 D 71732 Tamm Germany Phone 49 0 7141 9730230 Fax 49 0 7141 9730240 support npielectronic com http www npielectronic com ...

Page 2: ...nt Panel 6 3 3 Description of the Rear Panel 15 4 Headstage 16 4 1 Headstage Elements 16 5 Setting up the BA 01X 17 6 Passive Cell Model 18 6 1 Cell Model Description 18 6 2 Connections and Operation 19 7 Test and Tuning Procedures 21 7 1 Headstage Bias Current Adjustment 21 7 2 Offset Compensation 22 7 3 Capacitance Compensation 22 7 4 Bridge Balance 24 7 5 Electrode Selection 24 8 Sample Experim...

Page 3: ...er 1 carefully It contains general information about the safety regulations and how to handle highly sensitive electronic instruments Signs and conventions In this manual all elements of the front panel are written in capital letters as they appear on the front panel System components that are shipped in the standard configuration are marked with 3 optional components with ê In some chapters the u...

Page 4: ...is to be operated only at 115 230 Volt 60 50 Hz AC Please check for appropriate line voltage before connecting any system to mains Always use a three wire line cord and a mains power plug with a protection contact connected to ground protective earth Before opening the cabinet unplug the instrument Unplug the instrument when replacing the fuse or changing line voltage Replace fuse only with an app...

Page 5: ...ferential input 3 BA 01X System This manual is related to the standard configuration of the BA 01X system consisting a standard headstage and standard calibrations of bridge balance electrode resistance display range etc Other configurations are available e g if the BA 01X system is used only for whole cell patch clamp recordings with suction electrodes the BA 01X system can be delivered with adap...

Page 6: ...0 MΩ dependent on the preset current range With this headstage the system can be used either with high resistance electrodes for intracellular recordings or with low resistance suction electrodes for whole cell patch clamp recordings To cover all the needs of electrophysiological research all systems have a large variety of operation and control elements such as cell penetration mode BUZZ ELECTROD...

Page 7: ..._____________________________________________________________________________ ___________________________________________________________________________ version 1 9 page 7 Figure 1 BA 01X front panel view the numbers are related to those in the text below ...

Page 8: ... of display 2 is mV OSCILLATION SHUT OFF unit The OSCILLATION SHUT OFF unit consists of 5 OSCILLATION SHUT OFF LED 6 DISABLED RESET switch and the 30 THRESHOLD potentiometer 5 OSCILLATION SHUT OFF LED Indicates whether the OSCILLATION SHUT OFF circuit is active LED red or not LED green 6 DISABLED RESET switch Switch to DISABLE the OSCILLATION SHUT OFF unit or RESET the circuit RESET is for resetti...

Page 9: ...ENT RANGE LED and 10 CURRENT RANGE switch 9 CURRENT RANGE LED LED indicating the actual current range of the amplifier LED ON BA 01X is in x10 range LED OFF BA 01X is in x1 range 10 CURRENT RANGE switch Switch for setting the current range of the amplifier x1 max 12 nA into 1 GΩ x10 max 120 nA into 100 MΩ HOLDING CURRENT unit The HOLDING CURRENT unit consists of 12 HOLDING CURRENT nA potentiometer...

Page 10: ... to Imax maximum positive current or Imax maximum negative current The maximum current is dependent on the setting of the current range see also 10 15 BUZZ push button Push button to activate the BUZZ circuit duration set by 19 To facilitate the penetration of the cell membrane the BUZZ circuit is provided which is based on oscillations caused by overcompensating the capacitance compensation syste...

Page 11: ...his may lead to an incorrect reading of the electrode resistance see also 31 especially when electrodes with high resistances are used 18 HEADSTAGE connector The HEADSTAGE is connected via a flexible cable and an 8 pole connector to the mainframe see also chapter 3 3 Caution Please always adhere to the appropriate safety regulations see chapter 1 Please turn power off when connecting or disconnect...

Page 12: ...nal current stimulus is determined by the amplitude of the input voltage signal input voltage signal output current signal Figure 3 input output relation using CURRENT INPUT 25 on off switch Switch to enable on or disable off CURRENT INPUT via 0 1 nA V connector Hint To avoid interferences always switch to off position if the INPUT is not used 26 CURRENT INPUT 0 1 nA V connector Analog input BNC c...

Page 13: ...timulus set by STEP SIZE 7 and gated by GATE TTL STEP SIZE 28 or to select the polarity of the gated stimulus gated stimulus positive 0 gated stimulus disabled gated stimulus negative 28 GATE TTL STEP SIZE connector With this input a current step gated stimulus can be generated set by the digital potentiometer STEP SIZE 7 and the polarity switch 27 This current step is gated by a positive digital ...

Page 14: ...lls since this current may stimulate or damage the cell Important With high resistance electrodes REL 20 MΩ the displayed value is dependent on the setting of the capacitance compensation POTENTIAL OUTPUT unit The OUTPUT unit consists of 32 POTENTIAL OUTPUT FROM HEADSTAGE connector 33 POTENTIAL OUTPUT GAIN switch and 34 POTENTIAL OUTPUT mV connector 32 POTENTIAL OUTPUT FROM HEADSTAGE connector BNC...

Page 15: ...older counter clockwise using a screw driver The appropriate fuse type is listed above the holder 4 CHASSIS This connector is linked to mains ground green yellow wire protective earth 5 GROUND This connector is linked to the internal system ground which has no connection to the 19 cabinet CHASSIS and the mains ground to avoid ground loops MODES of OPERATION connectors 6 REL connector BNC connector...

Page 16: ...hat fits into the BNC connector of the headstage or into an electrode holder adapter optional see also Optional accessories in chapter 2 The electrical connection between the electrolyte and the headstage is established using a carefully chlorinated silver wire Chlorinating of the silver wire is very important since contact of silver to the electrolyte leads to electrochemical potentials causing v...

Page 17: ...trument into a grounded outlet o Connect the headstage to the HEADSTAGE connector 18 Figure 1 at the BA 01X o Connect a cell model see chapter 6 Connect a digital analog timing unit or a stimulation device to CURRENT INPUT or to GATE TTL STEP SIZE if you intend to use the gated stimulus unit o Connect a store oscilloscope or a data recording device i e a computer with data acquisition system to th...

Page 18: ...odel consists only of passive elements i e resistors that simulate the resistance of the cell membrane and the electrodes and capacitances that simulate the capacitance of the cell membrane A switch allows simulation of two different cell types a cell with 50 MΩ and 22 pF CELL 1 represents an astrocyte like cell or a small cell with 200 MΩ membrane resistance and 100 pF membrane capacitance CELL 1...

Page 19: ... is assumed that all connections are built as described in chapter 5 Checking the configuration o Turn POWER switch of the amplifier off a For simulation of an experiment using a suction electrode o Connect the BNC jack labeled 10MΩ of the cell model to the BNC connector PEL of the headstage b For simulation of an experiment using a sharp electrode o Connect the BNC jack labeled 50MΩ of the cell m...

Page 20: ... 5 Figure 7 to SEAL position The 1 GΩ resistor simulates the SEAL resistance when forming a GIGASEAL in patch clamp experiments Simulation of intracellular recording Intracellular recordings can be mimicked with one of two cells with different properties Use the 50 MΩ electrode connector 1 Figure 7 for an experiment with sharp electrodes or the 10 MΩ electrode connector 3 Figure 7 for simulating a...

Page 21: ...hapter 3 2 The tuning procedure must be performed regularly about once a month since the bias current changes over time If very small currents are used in the 10 pA range the procedure must be repeated in shorter intervals The tuning procedure is performed using high value resistors and or a cell model It cannot be performed with an electrode since there are always unknown potentials involved tip ...

Page 22: ...Capacitance Compensation High resistances of electrodes and stray capacitances Cstray form a low pass filter which deteriorates the shape of recorded intracellular signals see also Figure 11 The frequency response bandwidth of the amplifier is improved considerably by using the capacitance compensation function This function is based on positive feedback negative capacitance circuit The tuning of ...

Page 23: ...________________________________ version 1 9 page 23 Important The capacity compensation for the electrode does NOT work if the oscillation shut off circuit is activated This may lead to an incorrect reading of the electrode resistance see also 31 especially when electrodes with high resistances are used Figure 9 Tuning of the capacitance compensation using a 100 MΩ resistor ...

Page 24: ...he POTENTIAL OUTPUT at the oscilloscope and adjust the BRIDGE BALANCE as shown in Figure 10 using the BRIDGE BALANCE potentiometer 22 Figure 1 After adjustment you should see a straight voltage trace without artifacts caused by the potential drop at REL Figure 10 illustrates the BRIDGE BALANCE procedure using a 100 MΩ resistor that represents the electrode The current stimuli were generated using ...

Page 25: ...________________ ___________________________________________________________________________ version 1 9 page 25 Sometimes the performance of electrodes can be improved by breaking the tip or by using the BUZZ or ELECTRODE CLEAR facilities of the amplifier Figure 10 Tuning of the BRIDGE BALANCE using 100 MΩ resistor ...

Page 26: ...to the BNC connector and the Ag AgCl pellet or the agar bridge for grounding the bath with GND at the headstage o Make the basic settings see chapter 0 Again It is of major importance that the BA 01X systems are used only in warmed up condition i e 30 minutes after turning power on o Adjust BIAS CURRENT to zero if necessary see chapter 7 1 o Reconnect the STIMULUS INPUT and or the GATE TTL STEP SI...

Page 27: ...membrane the electrode resistance increases the bridge balance appears undercompensated extracellular action potentials APs are recorded the acoustic monitor signal changes o Set the BUZZ DURATION potentiometer to one fourth and apply a BUZZ to the electrode o If you are lucky the tip of the electrode is now inside the cell o If necessary readjust the BRIDGE BALANCE as shown in Figure 12 o You rea...

Page 28: ...rode The measurements were done using a cell model with 100 MΩ membrane resistance 100 pF membrane capacitance and 100 MΩ electrode resistance A Cstray and VREL not compensated bridge not balanced B Cstray compensated and VREL not compensated C Cstray and VREL compensated bridge balanced Cm membrane capacitance Cstray electrode stray capacitance REL electrode resistance Rm membrane resistance τCm ...

Page 29: ... positive pressure to the pipette to prevent settling of particles at the tip o Apply test pulses to the pipette about 10 pA The resulting voltage signals at the pipette are very small 50 µV with a 5 MΩ electrode o Approach the cell until the voltage signal changes a Figure 15 Often you can observe a slight dent in the cell membrane o Release pressure from the pipette Now forming of the seal is in...

Page 30: ..._______________________________________________________________________ ___________________________________________________________________________ version 1 9 page 30 Figure 15 Approaching the cell forming a gigaseal and establishing the whole cell configuration ...

Page 31: ... in the agar bridge are damaged 3 There is an unwanted GND bridge e g caused by a leaky bath 4 The headstage or the amplifier has an error Solutions 1 Chloride the silver wire again 2 Exchange the pellet or chloride the silver wire in the agar bridge 3 Try to find the GND bridge and disconnect it e g by sealing the bath 4 Contact npi Problem 2 Even if no stimulus is given a current flows through t...

Page 32: ...Herz A V 2002 Energy integration describes sound intensity coding in an insect auditory system J Neurosci 22 10434 10448 o Hutzler M Lambacher A Eversmann B Jenkner M Thewes R Fromherz P 2006 High resolution multi transistor array recording of electrical field potentials in cultured brain slices J Neurophysiol o Kettenmann H Grantyn R eds 1992 Practical Electrophysiological Methods Wiley Liss New ...

Page 33: ...ordings Journal of Neurophysiology 93 3548 3559 o Zeck G and P Fromherz 2001 Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilized on a semiconductor chip PNAS Vol 98 no 18 10457 10462 Juxtasomal Filling Loose Patch Techniques General o Auger C Marty A 2000 Topical Review Quantal currents at singlesite central synapses J Physiol 526 1 3 11 o Barbour B Isope ...

Page 34: ...physiology techniques J Neurosci Methods 126 91 98 o Roberts W M Almers W 1992 Patch Voltage Clamping with Low Resistance Seals Loose Patch Clamp In Rudy B Iversen L E eds Ion Channels Methods in Enzymology 207 Academic Press San Diego o Strickholm A 1961 Impedance of a Small Electrically Isolated Area of the Muscle Cell Surface J Gen Physiol 44 1073 1088 Tracer injection juxtasomal filling and ex...

Page 35: ...ge 150 pA current adjustable with trim potentiometer OFFSET range 200 mV ten turn control CAPACITY COMPENSATION range 0 30 pF ten turn control BUZZ overcompensation of capacitance compensation BUZZ duration 2 5 100 ms slightly dependent on the setting of capacitance compensation timer controlled with linear control accessible with remote switch Electrode clear circuit Application of max DC current...

Page 36: ...XX X nA Inputs Current stimulus input via BNC connectors sensitivity dependent on preset CURRENT RANGE x1 1 nA V or 0 1 nA V x10 10 nA V or 1 nA V Step gate input via BNC connector gated stimulus with digital control of current step size resolution x1 10 pA resolution x10 100 pA polarity selectable with toggle switch holding current range x1 10 nA holding current range x10 100 nA adjustable with t...

Page 37: ...UT unit 12 CURRENT RANGE unit 9 electrical connections 17 electrode 22 artifacts 28 capacity compensation 22 offset compensation 22 selection 24 ELECTRODE RESISTANCE TEST 14 front panel view 7 gated stimulus unit 13 general description 5 GROUND connector 14 headstage 16 elements 16 HEADSTAGE connector 11 HOLDING CURRENT unit 9 literature 32 model circuit sharp electrode 26 model circuit suction pa...

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