current clamp recordings with the epc 10 patch ... - heka

Dr. Christian Heinemann

Current Clamp Recordings with the EPC 10 Patch Clamp Amplifier

Agenda

Part A: Current clamp circuitry

Part B: Sensing membrane potential: Parameters affecting the shape of an action potential

Part C: Stimulation the cell by current injection: How much current can be injected?

CC Circuitry of the EPC 10 amplifier

Current injection path via the feedback resistor of the patch clamp headstage.

I-command (V-Ref)

C-fast comp (V-Ref)

V-mon

500 MOhm

V-Ref

-

+

-

+

1 pF

4,7 pF

-

+

I-mon

Part A 1.2

Voltage follower circuitry, direct and fast measurement of the membrane potential.

Current injection path via the injection capacitor.

Comparison of EPC 9 and EPC 10

The EPC 10 shows no deviations from the command current pulse. Good clamp performance.

Action potential recordings from DRG neurons. All recordings from cells within this presentation are courtesy of the Center for Molecular Biomedicine (CMB), Department of Biophysics, Friedrich-Schiller-University Jena.

Part A 1.3

The EPC 10 with its Voltage Follower circuitry measures the true action potential waveform.

The EPC 9 uses a slower current feedback loop. Clamp performance not perfect.

The EPC 9 gives only a qualitative measurement of the AP waveform.

Equivalent Circuit

Part B 2.1

CellPipette

Cm

Rm

Rs

C-fast

Stimulating in Voltage Clamp

Cm = 22pF

Rm = 500MOhm

Rs = 5.1MOhmV com

Part B 2.1

tauVm=RS∗Cm=112µs

Current charges the cell membrane capacitance, current transient.

During charging the membrane potential time course is dominated by the time constant:

The potential at the membrane drives the current through the Rm. That is, why the current we typically measure also increases with exponential time course.

iRs=iCm+iRm

iRm

V com

Current Recording with C-Slow compensation active.

20mV voltage stimulus drive about 40pA through Rm.

Initially, the constant current mainly charges the membrane capacitance.

Cm = 22pF

Rm = 500MOhm

Rs = 5.1MOhm I com

Stimulating in Current Clamp

Part B 2.2

tau=Rm∗Cm=11ms

iRs=iCm+iRm

With increasing potential the portion of the current that flows through Rm becomes dominating.

This equilibration happens with the time constant given by:

The increase in Vm is typically much slower in CC than VC recordings.

40pA current injection result in a Vm of about 20mV.

If the cell fires an action potential, then the equivalent circuit is reduced to a combination of access resistance and pipette capacitance.

The filtering element is given by:

Sensing an action potential fired by a cell

5,0 pF

Vm

measure

Part B 2.3

tau=RS∗C fast=50µs

BW eff=12∗pi∗tau

=3.18kHz

C-fast

Rs

CC Bandwidth: limited by C-fast?

The time constant Rs * C-fast limits the effective bandwidth of the voltage recording in a current clamp measurement.

C-fast compensation reduces the contributing capacitance and therefore increases the effective bandwidth.

rem. C-fast

theor. Tau meas. Tau eff. BW

5pF 50µs 57µs 2.8kHz




10MOhm

5.0pF measurestimulate

Part B 2.4

tau

AP shape – Affected by C-fast Comp?

Rs = 3.3MOhm and rem. C-fast = 2pF the eff. BW > 20kHz

Part B 2.5

Checking C-fast adjustment in CC mode

Part B 2.8

4.78

pF

5.78 pF

1E-12[V^2/Hz]

1E-13

1E-14

1E-15

1,000 10,000 100,000Hz

Power Spectrum

Voltage recording in CC mode with 100kHz sampling frequency and V-Bessel filter set to 15kHz

CC Bandwidth: limited by Voltage Filter?

The maximum slope is effected significantly by the voltage filter. But in a speed range much faster than the rising phase of an AP.

V-Bessel max. slope

% slope reduction

15kHz 2.156V/s

10kHz 1.781V/s 17%

7kHz 1.531V/s 29%

5kHz 1.187V/s 45%

10MOhm

5.0pF measurestimulate

max. slope

Part B 2.6

AP shape – Affected by Voltage Filter?

The maximum slope of the AP is not significantly affected at V-Filter settings above 1 kHz.

A

B

C

D

Part B 2.7

Stimulation Artifact: Bridge Compensation The initial fast voltage step over Rs can be

compensated using the Bridge Compensation.

100% of the Rs value can be compensated. Adjust Rs or % value for fine tuning.

measure - V

stimulate - ICm = 47.0 pF

Rm = 500 MOhm

Rs = 100 MOhm

Part C 3.1

Stimulation Artifact: Bridge Compensation A fast enough time constant of Bridge

Compensation eliminates the voltage step completely. measure - V

stimulate - ICm = 47.0 pF

Rm = 500 MOhm

Rs = 100 MOhm

Part C 3.3

100µs

10µs

AP Recordings with high access Resistance estimated Rs about 70-80 MOhm, 80 pA

current injection induce about 6 mV voltage drop.

Recordings with Bridge Comp On and Off. Note change in AP amplitude. Slope is not affected.

Part C 3.2

How much current can be injected into a cell?

Voltage Measurement Limit: The maximum voltage that can drive the current through the input resistance is 1V in the standard EPC 10 operating mode and 5V with „Extended Stimulus Range“ turned on.

Compliance Limit: The overall voltage that can drive current through the circuitry is appr. 12V.

Cm = 22.0 pF

Rm = 500 MOhm

Rs = 5.1 MOhm Rf = 500 MOhm

max. compliance voltage = appr. ± 12 V

input resist. = Rm + Rsvoltage measuring limit

Part C 3.4

Example 1: 10nA injection via 500MΩ

i

i

Cm = 22.0 pF

Rm = 500 MOhm

Rs = 5.1 MOhm

i

Rf = 500 MOhm



Part C 3.5

CC-Gain I max

0.1 pA/mVRf = 500 MΩ

1 nA


10 nA

10 pA/mVRf = 5.1 MΩ

100 nA


1 µA

URf= i∗Rf=10nA∗500MOhm=5V

Rinput=1V10nA

=100MOhm

i limit=1V500MOhm

=2nA

Ucompl=1V+5V=6V

extended StimulusRange:URf= i∗Rf=10nA∗500MOhm=5V

Rinput=5V10nA

=500MOhm

Ucompl=5V+5V=10V

Example 2: 1µA injection via 5.1MΩ

i

i

Cm = 22.0 pF

Rm = 500 MOhm

Rs = 5.1 MOhm

i

Rf = 5.1 MOhm



Part C 3.6

CC-Gain I max


1 nA


10 nA


100 nA


1 µA

URf=i∗R f=1µA∗5.1MOhm=5.1V

R input=1V1µA

=1MOhm

i limit=1V500MOhm

=2nA

Ucompl=1V+5.1V=6.1V

extended StimulusRange :URf=i∗R f=1µA∗5.1MOhm=5.1V

R input=5V1µA

=5MOhm

i limit=5V500MOhm

=10nA

Ucompl=5V+5.1V=10.1V

Selection of the CC Gain Range

Part C 3.6

CC-Gain I max


1 nA


10 nA


100 nA


1 µA

high input resistance

low input resistance

Attention: High CC Gain might cause Voltage offsets!

Part C 3.7

CC-Gain I max


1 nA


10 nA


100 nA


1 µA

0.3mV∗100pAmV

=30pA

Uoffset=500MOhm∗30pA=15mV , model cell−WholeCell Mode

Uoffset=10MOhm∗30pA=0.3mV , model cell−PipetteBath Mode

Current stimulus is generated by 16bit DAC with a typical resolution of about 0.3mV/bit.

What happens if we are just off by one bit?

Note:High CC-gains can not be used or tested with high input resistances, e.g.

model cell in Whole Cell Mode!

Ext. Stimulus Range: 5 x stronger injection

CC-Gain I max (R input), standard range

I max (R input), extended range


1 nA (1 GΩ) 1 nA (1 GΩ)


10 nA (0.1 GΩ) 10 nA (0.5 GΩ)


100 nA (10 MΩ) 100 nA (50 MΩ)


1 µA (1 MΩ) 1 µA (5 MΩ)

5x more current at given input resistance

current injection possible at 5x higher input resistance

Part C 3.8

Summary - CC related EPC 10 Features Voltage Follower Circuitry: True

Current Clamp

C-fast Compensation: increase bandwidth of CC recording

small Rs makes C-fast setting less critical

Voltage Filter: analog filter for the voltage signal

filtering with 5kHz or more typically does not affect the AP shape

4 different CC Stim Gains: for injection of large currents

amount of injected current depends on input resistance

with Rs lower than 10MOhm artifact typically less than 1mV

5x more current at given input resistance

given current at 5x higher input resistance

Bridge Compensation: remove current injection artifact

Extended Stimulus Range: 5x stronger current injection

Thank you!

Questions ?

current clamp recordings with the epc 10 patch ... - heka

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