Molecular Devices pCLAMP 11 Software Suite User Guide

Setting Up Clampex Software for Data Acquisition
Rev. H
January 2021
Welcome to the Guide
Setting Up Clampex Software for Data Acquisition is a step-by-step guide that explains how to integrate Clampex
software with your amplifier and digitizer for data acquisition. It describes typical setup configurations for three
representative amplifiers—the Axopatch™ 200B amplifier, MultiClamp™ 700B amplifier, and Axoclamp™ 900A
amplifier. At the end of the guide, not only will you be ready to acquire data using the explained configurations,
you should understand how to set up new configurations tailored to your needs.
We suggest you open Clampex software—and MultiClamp or Axoclamp Commander software if you are following
the MultiClamp or Axoclamp software sequence—and toggle between the guide and programs as you move
through the guide, using the Alt + Tab key combination.
This guide assumes you are using a Digidata® Data Acquisition System 1550 series (including the 1550, 1550A,
and 1550B) digitizer and that you have connected it to your computer. Similarly, if you have amplifiers other than
those featured, you should be able to extrapolate the provided instructions to your other amplifiers.
Contents
Note: Each
is a hyperlink.
Configure Digitizer
Select a Sequence
Axopatch Sequence
MultiClamp sequence
Digitizer–Amplifier Connections
Digitizer–Amplifier Connections
Configure Telegraphs
Configure Telegraphs
Create Signals
Create Signals
Set Scale Factors
Configure Protocols
Configure Protocols
Configure Sequencing Keys
Axoclamp sequence
Digitizer–Amplifier Connections
Configure Telegraphs
Create Signals
Configure Protocols
Contacting Molecular Devices
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p3
Configure Digitizer
The first step in the setup is to configure Clampex software with a digitizer.
When Clampex software is first installed it is in “Demo” mode. This mode uses simulated data, and
is excellent for exploring the application. Now, however, we want to connect to a digitizer for real
data acquisition.
Configure Digitizer 1
Select Digitizer from the
Configure menu.
The Digitizer dialog opens, showing Clampex software in
Demo mode.
Click Change.
To continue the configuration, select your model of Digidata digitizer.
Digidata 1550 (including 1550A and 1550B)
Digidata 1440A
For research use only. Not for use in diagnostic procedures.
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Configure Digitizer 2
Digidata 1550, 1550A, and 1550B Digitizers
Select Digidata 1550 Series, Digidata 1550B Series, or
Digidata 1550B Series from the list.
If Clampex software reports
Not present, click Scan.
Clampex software should then report your Digidata digitizer properties.
There is no calibration necessary with the Digidata 1550,
1550A, or 1550B digitizers.
This completes the configuration of the Digidata 1550,
1550A, or 1550B digitizer you have purchased, and it is
ready to be used with Clampex software.
If you experience problems, check the suggestions on the
next slide.
For research use only. Not for use in diagnostic procedures.
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Configure Digitizer 3
Digidata 1550, 1550A, and 1550B Digitizers
If clicking Scan does not detect the Digidata 1550, 1550A or 1550B digitizer, check the power and the
USB 2.0 connections to the digitizer.
The green POWER LED on the front of the digitizer should be illuminated when the power is on and the
digitizer is connected to the computer using a USB 2.0 cable. When the digitizer is recognized by
Clampex software, the yellow READY LED will turn on as well.
If problems persist, contact Molecular Devices.
For research use only. Not for use in diagnostic procedures.
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Configure Digitizer 4
Digidata 1440A
Select Digidata 1440A Series from the list.
If Clampex software reports
Not present, click Scan.
Clampex software should then report your Digidata digitizer properties.
There is no calibration necessary with the Digidata 1440A digitizer.
This completes the configuration of the Digidata 1440A digitizer, and it is
ready to be used with Clampex software.
If you experience problems, please check the suggestions on the next slide.
For research use only. Not for use in diagnostic procedures.
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Configure Digitizer 5
Digidata 1440A
If clicking Scan does not appear to detect the Digidata 1440A digitizer, check the power and the
USB 2.0 connections to the digitizer.
The green POWER LED on the front of the digitizer should be illuminated when the power is on and
the digitizer is connected to the computer through a USB cable. When the Digidata 1440A digtizer is
recognized by Clampex software, the yellow READY LED will turn on as well.
If problems persist, contact Molecular Devices.
For research use only. Not for use in diagnostic procedures.
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Select a Sequence
With the digitizer configured, the next step is to connect the amplifier. Choose the sequence you want
to follow according to your amplifier type:
Axopatch Sequence
Follow this sequence if your amplifier telegraphs via cable connections. This includes Axon
Instruments’ Axopatch series amplifiers, and most non-Axon Instruments amplifiers. Follow this
sequence also if your amplifier does not support telegraphs.
MultiClamp Sequence
Follow this sequence for the MultiClamp 700B amplifier. Parts of the sequence also apply to
MultiClamp 700A amplifier and GeneClamp 500B amplifier.
Axoclamp Sequence
Follow this sequence for the Axoclamp 900A amplifier.
For research use only. Not for use in diagnostic procedures.
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Axopatch 200B
Axopatch Sequence
This sequence describes how to set up two distinct data-acquisition “protocols”, that might
be used in whole-cell recording, for an Axopatch 200B amplifier.
With these protocols you will be able to switch between current and voltage clamp and,
without any changes to your physical setup, have only to load the appropriate protocol to be
sure you are receiving the right signals, with the right units and scaling.
Move through the sequence page by page, or skip sections with the links below—but note
that the discussion assumes the setup from earlier sections:
Digitizer–Amplifier Connections
Configure Telegraphs
Create Signals
Set Scale Factors
Configure Protocols
For research use only. Not for use in diagnostic procedures.
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Axopatch 200B
Digitizer – Amplifier Connections
In this section we put in place the cabling between the digitizer and Axopatch 200B amplifier.
Axopatch Connections 1
We want the following signals:
Voltage Clamp
Digitizer Analog Output
Digitizer Analog Inputs
• Membrane current—scaled
• Membrane potential—set gain
• Command potential
Current Clamp
Digitizer Analog Output
Digitizer Analog Inputs
as well as:
• Membrane potential—scaled
• Membrane current—unscaled
• Command current
Telegraphs
• Gain telegraph for current clamp
• Gain telegraph for voltage clamp
For research use only. Not for use in diagnostic procedures.
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Axopatch Connections 2
Clampex software allows for more than one signal to be sent, at different times, on each channel (the
relationship between signals and channels is more fully explained in the Create Signals section).
Because we are never in current clamp and voltage clamp at the same time, signals associated with
these modes can share channels.
Specifically, the following signals can share channels:
• the scaled input signals for current and voltage clamp
• the command signals for current and voltage clamp
• the telegraphs for current and voltage clamp
The eight signals and telegraphs from the previous slide, then, require only five digitizer-to-amplifier
connections. These are described on the next slide.
For research use only. Not for use in diagnostic procedures.
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Axopatch Connections 3
TELEGRAPH OUTPUTS
TELEGRAPH
INPUT 0
(on digitizer rear
panel)
GAIN
FREQUENCY
MODE
CELL
CAPACITANCE
SCALED
OUTPUT
10 Vm
OUTPUT
DATA
NOT VALID
OUTPUT
EXT. COMMAND
INPUT
FRONT SWITCHED
ANALOG IN 1
SPEED
TEST
INPUT
FORCED
RESET
INPUT
EXT. COMMAND
INPUT
REAR SWITCHED
I OUTPUT
(10 kHz)
ANALOG OUT 0
ANALOG IN 2 #
Connect the rear panel BNCs on the Axopatch 200B to the digitizer ports as indicated.
* The SCALED OUTPUT BNCs on the front and rear of the amplifier are equivalent—you may prefer to use the front panel
port.
# Make sure the switch to the right of the 1 OUTPUT (10 kHz) BNC is in the down position: β mV/pA
For research use only. Not for use in diagnostic procedures.
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Axopatch Connections 4
In this setup, the following connections will carry different signals or telegraphs for voltage-clamp and
current-clamp modes:
 SCALED OUTPUT—ANALOG INPUT 0: reads membrane potential in current clamp, and
membrane current in voltage clamp
 EXTERNAL COMMAND INPUT—ANALOG OUT 0: for command current in current clamp and
command potential in voltage clamp
 GAIN—TELEGRAPH INPUT 0: telegraphs gain irrespective of clamp mode
This leaves:
 10 Vm OUTPUT—ANALOG IN 1: reads membrane potential in voltage clamp, and
 I OUTPUT—ANALOG IN 2: reads membrane current in current clamp
Finish
With physical connections set up, we now need to configure telegraphs.
For research use only. Not for use in diagnostic procedures.
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Axopatch
Configure Telegraphs
Telegraphs are analog signals sent from the amplifier to Clampex software registering key amplifier settings.
Clampex software recognizes Axopatch 200B telegraphs for gain, lowpass filter frequency and whole cell
capacitance neutralization. These are reported in the Real Time Controls in Clampex software, and are written into
the header information for data files recorded under those settings.
Most importantly, gain telegraphs enable automatic data scaling in Clampex software. When you change the
Axopatch 200B amplifier gain settings, Clampex software automatically rescales the Y axis in the Scope window,
and similarly sets the Y axis scaling for any data files recorded under the new settings.
We will enable just the gain telegraph in this demonstration.
Axopatch Telegraphs 1
Open Telegraphed Instrument from the
Configure menu.
Select the digitizer input channel that you have the Axopatch
amplifier scaled output connected to. Signals on this channel are
the ones that are affected by changes in gain settings, so we must
associate our telegraphs with this channel.
In our case, we have connected the scaled output to Analog IN #0
(Connections), so select this channel.
For research use only. Not for use in diagnostic procedures.
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Axopatch Telegraphs 2
Select Axopatch 200B from the Telegraphed Instrument
list box.
The Telegraph Connections section of the dialog
is enabled. Here, in the Gain field, select
Telegraph Input 0, the digitizer telegraph input
channel receiving the gain telegraph.
For research use only. Not for use in diagnostic procedures.
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Axopatch Telegraphs 3
This completes the dialog for our purposes.
Click OK to exit.
Clampex software displays the warning shown at
right, alerting us of the need to set scale factors for
all signals using the input channel we have set
telegraphs for, in our case, Analog IN #0.
We proceed to this in the next two sections.
Finish
We have configured Clampex software so that for any data signal received via digitizer channel
Analog IN #0, telegraph information about the amplification of that signal is also received. We now
have to create signals for this channel so that the telegraphed information is put to use.
Users with different cable-telegraphing amplifiers should follow the procedure outlined here, but
select their own machine in the Telegraphed Instrument list box. In the Telegraph Connections
section, they will be offered options for the telegraphs supported by their amplifier.
For research use only. Not for use in diagnostic procedures.
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Axopatch
Create Signals
In this section we create the signals required for the two protocols we are going to define, and assign
these to input and output channels. We also note some additional telegraph options.
Before starting it is important to be clear on what signals and channels are:
 Signal: a set of name, unit, scale factor and offset, by means of which the voltage inputs and
outputs at the digitizer are represented in Clampex software as the parameter being read at,
or delivered to, the preparation.
 Channel: a cable connection to the digitizer, identified by the name of the BNC port where
connection is made, e.g. Analog IN #0, Digital OUT #2.
As already noted, analog channels can be configured for different signals at different times. In this
section we name the signals we will need and give them appropriate units. We set scale factors for
these signals in the next section.
Axopatch Signals 1
Open Lab Bench from the Configure menu —
or use the button:
The Lab Bench opens with the Input Signals tab
on top, and digitizer channel Analog IN #0
selected. We have the amplifier’s scaled output
connected to this channel (Connections), so we
need to create two signals — one each for
current and voltage-clamp in association with it.
For research use only. Not for use in diagnostic procedures.
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Axopatch Signals 2
Click Add in the Signals section, opening the Add Signal dialog.
Type in “Im_scaled”— the name we will give the scaled
membrane current signal for voltage clamp.
Click OK.
With the new signal selected in the
Signals list, the rest of the Lab Bench
shows options and settings for that signal.
First is scaling.
We want to read “Im_scaled” in picoamps
— i.e. have picoamps as the Y axis units
for the Scope window and subsequently
recorded data files.
Select p from the Signal units list box, and type A in the
adjoining field.
For research use only. Not for use in diagnostic procedures.
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Axopatch Signals 3
Now we add a signal — still for channel
Analog IN #0 — for reading membrane
potential in current clamp. Click Add again.
This time call the signal Vm_scaled.
Click OK.
Vm_scaled is to be read in millivolts.
Set the signal units appropriately, as
we did for “Im_scaled”.
For research use only. Not for use in diagnostic procedures.
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Axopatch Signals 4
There are 2 more input signals to create — one each for the 10 Vm OUTPUT and I OUTPUT ports.
We connected the 10 Vm port to Analog IN #1
(Connections). Select this channel and add a new
signal, 10_Vm, as we did for previous signals.
Configure the signal for millivolts.
We connected the I OUTPUT port to Analog IN #2.
Select this channel and add new signal I_Output.
Configure this signal for picoamps.
For research use only. Not for use in diagnostic procedures.
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Axopatch Signals 5
This completes the input signals. Before creating output signals, however, reselect Analog IN #0 as the
digitizer channel, and Im_scaled as the signal.
Notice the options for additional
filtering in the lower half of the tab.
In the Telegraphs section, because we
have set up a gain telegraph for this
channel, the amplifier gain is reported.
This combines headstage and output
gain, so, for example, a headstage gain
β = 0.1 and output gain α = 10 gives a
reported gain of one.
Because we have not set up lowpass filter frequency or whole
cell capacitance neutralization telegraphs, we are given the
option of typing in values for these.
For research use only. Not for use in diagnostic procedures.
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Axopatch Signals 6
Now we create output signals for the command waveform for each of current and voltage clamp.
Still in the Lab Bench, go to the Output
Signals tab. Ensure Analog OUT #0 is
selected as the digitizer channel. This is
the channel we have connected to the
amplifier EXTERNAL COMMAND INPUT
FRONT SWITCHED port (Connections).
Just as for the input signals, add a new
signal, V_clamp, and set the units to
millivolts. This will carry the command
signal for voltage clamp.
Next, still for Analog OUT #0, add I_clamp, and configure
for nanoamps. This is the signal we will use to deliver the
command in current clamp.
For research use only. Not for use in diagnostic procedures.
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Axopatch Signals 7
Finish
We have created four input and two output signals, giving them units and associating
them with particular digitizer channels:
Voltage Clamp
Current Clamp
 Im_scaled
 Vm_scaled
 10_Vm
 I_Output
 V_clamp
 I_clamp
Next, and still in the Lab Bench, we must set scale factors for each of these signals.
For research use only. Not for use in diagnostic procedures.
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Axopatch
Set Scale Factors
Clampex software must be configured so that voltage differences received and produced by the digitizer
represent the actual currents and voltages produced and received by the cell. We have gone some way
towards this by defining appropriate units for our signals, but it remains to set scale factors for these.
Setting scale factors is greatly simplified with the Scale Factor Assistant. Note however, that although
this can be used for all output signals, for input signals it is intended for use with scaled signals only, i.e.
signals on channels connected to the amplifier SCALED OUTPUT port. In this section then, we set some
scale factors using the Assistant, and some manually.
Axopatch Scale Factors 1
We will set the scale factor for
“Im_scaled” first — the signal for
reading membrane current in
voltage clamp.
We will use the Scale Factor
Assistant for this signal.
On the Lab Bench Input Signals
tab, with Im_scaled on digitizer
channel Analog IN #0 selected,
open the Assistant.
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 2
Because we indicated the amplifier type when we
configured the gain telegraph for this channel, the
Assistant automatically opens with the correct
dialog for the Axopatch 200 amplifier.
Most of the work in the Assistant consists of
simply copying amplifier settings into the dialog.
Mode Setting
“Im_scaled” is for use in voltage clamp, so select
V-Clamp for the amplifier mode option.
Config Setting
This section, where you would otherwise indicate the headstage gain that you are using is disabled, as
the combined headstage and amplifier gain is being telegraphed.
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 3
Signal Units
In section 3 choose whether to read cell
current in picoamps or nanoamps. We
have already set this value in the
previous section, so keep the pA setting
we entered there.
Entering “nA” here does not affect the
scale factor, but simply causes it to be
expressed in terms of this unit.
Gain
The amount of gain applied to a signal is important for calculating the scale factor. In our case the total
amplifier gain — i.e. the combined headstage (b) gain and amplifier output (a) gain — is telegraphed to
Clampex software, and this value is reported, just as in the Telegraphs section of the Lab Bench Inputs tab.
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 4
Two scale factor values are reported at the bottom of the Assistant:
 Scale factor at unity alpha × beta gain = 0.001 V/pA
This is the scale factor that applies when the combined headstage and output gain is one.
This value depends on amplifier circuitry and never changes (though it can be expressed in terms of
nanoamps if this is selected as your preferred unit).
Since we have the gain telegraph enabled, this is the value that will be reported in the Lab Bench
scale factor field.
 Scale factor at the current alpha × beta gain = [ ]
This is the scale factor that will be used if data are acquired under the current gain settings in the
Axopatch software. With the gain telegraph enabled, this value does not appear anywhere else in
Clampex software.
This scale factor changes if you change the gain setting, but you need to close and reopen the
Assistant in order to have this reported in the Assistant.
Click OK to close the Scale Factor Assistant.
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 5
The scale factor at unity
α × β gain—0.001 V/pA has been
written into the scale factor field.
When gains telegraphs have been
enabled, as in this case, this value is
displayed no matter what the gain on
the amplifier is set to. The scaling
applied to incoming data is
automatically adjusted for the gain at
the time of acquisition, but the value
reported in this field does not change.
There is generally no need to set offsets for the amplifiers
used in electrophysiology, so this completes setup for
“Im_scaled”. We now move on to the second scaled and
telegraphed input signal, “Vm_scaled”, for which we will again
use the Scale Factor Assistant.
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 6
Still with Analog IN #0 as the digitizer channel, select Vm_scaled and
again open the Assistant.
This time, the signal is for use in current-clamp mode I-Clamp Normal,
so select this in the Mode group.
This is all you need to do. Again, the amplifier’s total gain setting is
reported and used to calculate the second of the scale factors at the
bottom of the Assistant. And again, only the unity-gain scale factor is
reported in the Lab Bench, though incoming data will be scaled for the
gain at the time of acquisition.
The scale factor at unity gain is 0.001 V/mV,
i.e. 1 mV/mV, which is what we expect when there is no amplification.
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 7
Setting Scale Factors Manually
The scale factor for a signal is found by taking the unity-gain scale factor for the amplifier port the signal will use, and
multiplying by the amount of amplification applied.
For the Axopatch 200B amplifier, this procedure is summarized in either the names given to the BNC ports, or in
information provided beneath the ports on the amplifier panel.
For example, the SCALED OUTPUT port has information:
 I: α β mV/pA
 Vm: α mV/mV.
The first of these means that, for current, when combined headstage and output gain is one (α × β = 1) the Axopatch
outputs one millivolt per picoamp input, or 0.001 V/pA.
The second means that for voltage, with output (α) gain of one (headstage gain is not relevant in this case), the Axopatch
outputs one millivolt per millivolt, or 0.001 V/mV.
Note that these are the unity-gain scale factors reported by the Scale Factor Assistant for the previous two signals.
We must now apply this to our two remaining input signals: “10 _Vm” and “I_Output”.
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 8
10_Vm
We created 10_Vm for digitizer channel Analog IN #1.
Select this channel and the signal.
Analog IN #1 is connected to the 10 Vm port on the
amplifier. This port outputs membrane voltage with a set
gain of 10. A unity-gain scale factor of one millivolt per
millivolt (0.001 V/mV) multiplied by the set gain value:
0.001 V/mV × 10 = 0.01 V/mV
This is the scale factor for signals read from this port.
Type 0.01 in the scale factor field.
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 9
I_Output
Select digitizer channel Analog IN #2, and I_Output.
The channel is connected to the
I OUTPUT port on the amplifier, which outputs
membrane current at one of two scaling options
reported on the panel:
100 β mV/pA and β mV/pA.
We have the switch in the down position (β mV/pA),
and the Config switch on the front panel should be
set at Whole Cell β = 1. The scaling factor we set is:
1 mV/pA = 0.001 V/pA
Set this value in the scale factor field.
This completes the scale factors for all the input signals.
Now for the two output signals.
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 10
Command Signals
We will use the Scale Factor Assistant to set the scale
factor for the voltage clamp command signal first.
On the Output Signals tab, select Analog OUT #0 and
the signal V_clamp.
Open the Scale Factor Assistant and select Axopatch 200 series
from the first dialog box.
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 11
The signal “V-clamp” is for use in voltage clamp, so select V-Clamp in the
Mode Setting section.
This leaves just the Ext. Command Input section to complete.
The Axopatch 200B amplifier has two command input ports — the selection here
informs the Assistant of the one we have connected our command cable to. We
used the front-switched port.
Reading off the panel we see that in voltage-clamp mode this port has a set
scaling of 20 mV/V.
The rear-switched port, in contrast, scales command signals at 100 mV/V.
Select 20 mV/V. This is reported at the bottom of the dialog.
Click Finish to close the Assistant and transfer the scale factor to the Lab Bench.
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 12
For “V_clamp“, the choice of amplifier input BNC was the sole determinant of the scale factor, and this
could have been easily read off the panel and entered manually. For current clamp, headstage β gain
becomes relevant.
Select the signal I_clamp in the Lab Bench and open the
Scale Factor Assistant.
Again, choose the Axopatch 200 series and go to the next dialog.
Select I-Clamp Normal as the mode, as “I_clamp” is the command
signal for current clamp.
As we are passing current with this signal, the Config setting,
switched on the front panel of the Axopatch amplifier, is relevant.
It determines the headstage β gain.
We are setting up for Whole Cell recording with β = 1.
Select this option.
The calculated scale factor (2 nA/V) is reported at the bottom of
the Assistant, and in the Lab Bench scale factor field when the
Assistant is closed (next slide).
For research use only. Not for use in diagnostic procedures.
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Axopatch Scale Factors 13
Again, we might have set the scale factor manually.
Under the front-switched EXT. COMMAND BNC on the
amplifier the scaling rate for current clamp is given as:
2 ÷ β nA/V
We have β = 1, hence a scale factor of
2 nA/V.
Finish
We have completed all that we need to do in the Lab
Bench. We have created all the signals we wanted, and set
scale factors for these. Press the OK button to close the
Lab Bench with the new signals intact.
It remains now to assemble the new signals into two
groups, according to their use for current or voltage clamp.
This is done in the protocol editor.
For research use only. Not for use in diagnostic procedures.
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Axopatch
Configure Protocols
Protocols in Clampex software are complete sets of acquisition parameters, including options for
command waveforms and preliminary data analysis. Particular signals, defined in the Lab Bench, are
specified for each protocol.
In this section we create two simple protocols, one each for current and voltage clamp, incorporating the
signals we have just defined.
Axopatch Protocol 1
Open the protocol editor by selecting New Protocol
from the Acquire menu.
Note: If a previously saved protocol is not loaded in
Clampex software, it uses a place-holder protocol,
labeled “(untitled)”. If this is currently loaded you can
open the editor to create a new protocol by selecting
Edit Protocol, or by clicking the button:
The currently loaded protocol is reported in the status
bar at the bottom of the main Clampex software window.
We will begin by setting up the protocol for
voltage clamp.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p44
Axopatch Protocol 2
The front tab of the protocol editor
has controls for, amongst other
things, acquisition mode, sampling
rate, and trial hierarchy.
The default acquisition mode is
episodic stimulation—the only mode
that allows a command waveform to
be generated. We want to generate
a command, so leave this setting. In
fact, all the default settings on this
tab can be left as they are, but take
time to note key parameters such as
the Sampling Rate (10 kHz), the
number of samples per sweep, and
the number of sweeps per run.
The sweep start-to-start interval is set at Minimum, so each
new sweep starts as soon as the previous one is finished.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p45
Axopatch Protocol 3
Next go to the Inputs tab.
Here you select digitizer input channels for the protocol,
and the signals you want to carry on these.
For voltage clamp, we want two input signals—one scaled
signal for membrane current, and a second unscaled
signal to monitor membrane voltage. We created these in
the Lab Bench—“Im_scaled” and “10_Vm”—associating
them with channels 0 and 1. Now we incorporate them
into the protocol.
Channel #0 should be already checked. Open the list box
beside it and select “Im_scaled”.
Then check Channel #1 and select 10_Vm.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p46
Axopatch Protocol 4
Go to the Outputs tab.
We configured the signal “V_clamp” to deliver the voltage
clamp command waveform, on output channel #0.
Select V_clamp from the Channel #0 list box.
Additionally, we will set a holding level for this
output. This is maintained all the time the
protocol is loaded except for when specific
output commands are generated.
Enter –50 in the V_clamp holding level field.
The units are millivolts, from our Lab Bench
configuration of this signal.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p47
Axopatch Protocol 5
Although we will not make any changes for the purposes of our protocol, it is worth taking a quick look at
the trigger settings.
Go to the Trigger tab.
Default settings give “Immediate” trial starts. This means
Clampex software is armed for data acquisition as soon as
you select Record, or View Only, from the Acquire menu —
or click the buttons:
Record
View Only
The default trigger source is “Internal Timer”.
This triggers the command waveform and data acquisition immediately the trial is started, continuing
through to the end of the trial automatically.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p48
Axopatch Protocol 6
Now go to the Waveform tab, Channel #0 tab,
where outputs are defined for ANALOG OUT #0.
A default waveform is already defined—we will
delete this and create our own simple stimulus,
but first familiarize yourself with some key
settings on this tab.
The Analog Waveform check box enables analog
command definition. Selecting Epochs means we
define the waveform using the table in the middle
of the tab. In this, the sweep can be divided into
up to 50 sections (epochs) A–AX, and a
waveform defined for each of these.
The Epoch Description table in the Waveform tab
includes cut and paste functionality.
Note that the confirmation that “V_clamp” is the
signal carrying the output waveform. Click Info to
see the V_clamp vital statistics. Note too that in the
Epoch Description table, First level and Delta level
have the correct units (mV) for “V_clamp”.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p49
Axopatch Protocol 7
Now to the definition of our waveform.
We will configure an output with a simple step, increasing the
amplitude of the step with each sweep.
In column A of the epoch description table, keep Step in the
Type row, but click First level and type –50. This sets the
output level for epoch A in the first sweep of the run. Our
entry of –50 mV maintains the holding level we set on the
Outputs tab.
Click in Delta level and type 0. This keeps the first level setting for subsequent sweeps — i.e. epoch A is
maintained at –50 mV for each of the 10 sweeps in the trial.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p50
Axopatch Protocol 8
Now to set the period for epoch A.
Click First duration and type in 50 for a 50 ms duration.
Click Enter to see this reported below the table.
This completes epoch A. Now we configure the step, in epoch B.
Click Type in column B (currently set to “Off”).
Select Step from the popup menu.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p51
Axopatch Protocol 9
Set the level for the first sweep at –100 mV.
For this epoch, because we want an incrementing step level
from sweep to sweep, we enter a delta level. Click in the Delta
level cell and type 20. This forces the step level up 20 mV with
each successive sweep.
We have 10 sweeps starting at –100 mV, so the final sweep will
have a step level of 80 mV, reported below the table.
Now set the First duration at 100 milliseconds.
Again, this is reported below the table, in milliseconds as well
as in samples.
We will not set a delta duration, which would alter the length of
the epoch from sweep to sweep, so this completes our
waveform definition for the voltage clamp protocol.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p52
Axopatch Protocol 10
Click Update Preview in the bottom right corner of
the protocol editor.
This opens the Waveform Preview window shown
at right, where you can see a graphical
representation of the waveform you defined.
This window can be kept open while you
experiment with different epoch settings — click
Update whenever you need to update the display.
Note: The Waveform Preview opens with panes for all analog output channels — right-click in the upper
pane and select Maximize Signal from the popup menu to get the display shown above.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p53
Axopatch Protocol 11
We have completed the setup of the voltage clamp protocol — close the
protocol editor with the OK button.
The new protocol is loaded, still labeled “(untitled)”, and we could
acquire data under it if we wanted, but it is not saved for future use.
Go to Save Protocol As in the Acquire menu. This opens a standard
file-saving dialog. Name the protocol Voltage Clamp 1, and click Save.
The protocol is now saved and can be loaded whenever needed,
with the Open Protocol command in the Acquire menu, or button:
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p54
Axopatch Protocol 12
Setup of the current clamp protocol follows similar lines to that for the voltage clamp protocol.
Open the protocol editor again with New Protocol, in the
Acquire menu.
We again accept the default settings in the Mode/Rate tab,
go to the Inputs tab.
This time select Vm_scaled for Channel #0.
Recall that the second, current-monitoring signal that we
want to read in current clamp (“I_Output”) is setup for the
connection from the I OUTPUT port on the amplifier to
digitizer channel Analog IN #2 (Connections).
Check Channel #2 and select I_Output.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p55
Axopatch Protocol 13
On the Outputs tab, select the current clamp command signal we
configured for Analog OUT channel #0: I_clamp.
Leave the holding level at the default zero setting for current clamp.
Create your own command waveform on
the Waveform Channel #0 tab.
Experiment with the different waveform
options, viewing these in the
Waveform Preview window.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p56
Axopatch Protocol 14
Finally, close the protocol editor by clicking OK, and save the protocol
(Save Protocol As in the Acquire menu), calling it Current Clamp 1.
You will see on exit that the scope window is set up in preparation to
receive the two input signals configured for this protocol.
To change from current clamp to voltage clamp you need only load the appropriate protocol, then start
acquisition. Alternatively, you can link each protocol to a sequencing key, so that you only have to click
one button, or use one keyboard combination, to load each protocol. The sequencing keys setup dialog is
in the Configure menu.
For more detailed information, consult the Clampex software online Help.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p57
Axopatch Protocol 15
When you use Gap-free mode in the Real Time Controls panel, open the pre-programming dialog by clicking the
< button. You can pre-program voltage level and holding duration values for each channel, as well as turning the digital
bit on or off. You can pre-program up to 50 epochs. You can also manually change values during a recording.
Finish
This completes the Axopatch section of the guide.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p58
MultiClamp
MultiClamp Sequence
This sequence describes how to set up two distinct data-acquisition protocols for use in
whole-cell recording with a MultiClamp 700B amplifier.
After we create the protocols, as an optional final step, we will integrate these with the
MultiClamp amplifier mode telegraph so that Clampex software automatically loads the
appropriate protocol when you shift between current and voltage clamp in MultiClamp
Commander software.
Move through the sequence page by page, or skip sections with the links below—but note
that the discussion assumes the setup from earlier sections:
Digitizer–Amplifier Connections
Configure Telegraphs
Create Signals
Configure Protocols
Configure Sequencing Keys
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p59
MultiClamp
Digitizer – Amplifier Connections
In this section we put in the cabling between the digitizer and MultiClamp amplifier. We will use just
one MultiClamp channel (i.e. headstage) in this configuration.
Connect MultiClamp
If you have not already done so, switch on your MultiClamp 700B amplifier and open MultiClamp
Commander software.
If Commander opens in demo mode (reported in the title bar), you need to connect the amplifier to
the software.
In Commander, click the Select Device button:
Select MultiClamp Hardware.
700B: Click Scan—Commander displays the amplifier
serial number when the amplifier is found.
Click OK to exit.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p61
MultiClamp Connections 1
We want the following signals:
Voltage Clamp
Digitizer Analog Inputs
Digitizer Analog Output
 Membrane current—primary output
 Command potential
 Membrane potential—secondary output
Current Clamp
Digitizer Analog Inputs
Digitizer Analog Output
 Membrane potential—primary output
 Command current
 Membrane current—secondary output
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p62
MultiClamp Connections 2
Clampex software allows for more than one signal to be sent, at different times, on each channel (the
relationship between signals and channels is more fully explained in the Create Signals section).
Because we are never in current clamp and voltage clamp at the same time, signals associated with these
modes can share channels.
Specifically, the following signals can share channels:
 the primary input signals for current and voltage clamp
 the secondary input signals for current and voltage clamp
 the command signals for current and voltage clamp
The six signals from the previous slide, then, require only three digitizer-to-amplifier connections, as shown
on the next slide.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p63
MultiClamp Connections 3
700B
(Channel 1)
COMMAND
PRIMARY
OUTPUT
SECONDARY
OUTPUT
Digitizer
ANALOG
OUT 0
ANALOG
IN 0
ANALOG
IN 1
Finish
With the three cables connected, we are ready to configure Clampex software , starting with telegraphs.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p64
MultiClamp
Configure Telegraphs
MultiClamp 700B telegraphs are software messages sent from MultiClamp Commander software to
Clampex software, registering key amplifier settings.
As well as simply reporting the settings in Clampex software, the telegraphs are integrated into Clampex
software so that the greater proportion of signal setup is done automatically (as we will soon see).
The most important telegraph remains the gain telegraph, used to automatically rescale the Clampex
Scope window as gains settings are changed, and to ensure recorded data files are correctly scaled.
Lowpass filter and whole-cell capacitance compensation settings are reported in the Real Time Controls
and written into recorded file headers—as is the output gain.
In addition, the MultiClamp amplifier has telegraphs for amplifier mode, and for the units and scale factors
for command and acquisition signals. We will use these telegraphs in our setup in the following slides.
MultiClamp Telegraphs 1
Open Telegraphed Instrument from the
Configure menu.
All telegraphs must be configured for a specific digitizer input
channel. We will first enable telegraphs for the channel receiving
the amplifier primary output. We have connected the MultiClamp
Primary Output to Analog IN #0 on the Digidata digitizer
(Connections), so select this from the Input Channels list.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p66
MultiClamp Telegraphs 2
Select Axon MultiClamp 700B amplifier from the
Telegraphed Instrument list.
When you have made this selection note the options
with respect to linking protocols to amplifier modes—
we will use this functionality later in the guide
(Configure Sequencing Keys).
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p67
MultiClamp Telegraphs 3
The first enabled section on the dialog is Amplifier Configuration.
Identify the amplifier channel (i.e. headstage) and
signal type for the selected digitizer channel:
Primary output 1
Next click Scan — the MultiClamp amplifier serial number is shown when the amplifier is found.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p68
MultiClamp
Create Signals
In this section we name the signals we require, assigning these to input and output channels.
Before starting it is important to be clear on what signals and channels are:
 Signal: a set of name, unit, scale factor and offset, by means of which the voltage inputs and
outputs at the digitizer are represented in Clampex software as the parameter being read at,
or delivered to, the preparation.
 Channel: a cable connection to the digitizer, identified by the name of the BNC port where
connection is made, e.g. Analog IN #0, Digital OUT #2.
As already noted, analog channels can be configured for different signals at different times, which is
what we do in this section.
MultiClamp Signals 1
Open the Lab Bench from the Configure menu
or use the button:
The Lab Bench opens with the
Input Signals tab on top, and
digitizer channel Analog IN #0
selected. We have the amplifier’s
primary output connected to this
channel, so we need to create two
signals—one each for voltage and
current clamp—for this channel.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p70
MultiClamp Signals 2
Click Add in the Signals section, opening the Add
Signal dialog.
Type Im_prime — the name we will give the scaled
membrane current signal for voltage clamp, on
headstage 1, which will on the primary output.
Notice that the entire Scaling section is
grayed, as it is not used. This is
because signal scaling is now under
the control of the telegraphs we set up
in the last section. Do not worry if the
units and scale here are incorrect –
they are overridden.
Click OK. With the new signal selected in the Signals
list, the rest of the tab shows options and settings for
that signal.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p71
MultiClamp Signals 3
The telegraphs are reported at the bottom
of the Lab Bench. The screenshots show
the telegraphs in the Lab Bench with
Commander at default settings.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p72
MultiClamp Signals 4
Change settings in Commander and see the telegraphs update in the Lab Bench. The filter, gain, and
capacitance compensation telegraphs are also reported in the Real Time Controls.
Notice that the scale factor reported in the
Lab Bench does not change as you alter the
output gain. Clampex software reports the
unity gain scale factor,
i.e. the scale factor for an output gain of one.
Of course, the scale factor applied to the
signal takes the gain into account —
e.g. in these screenshots, Clampex software
will apply a scale factor of
10 x 0.5 V/nA = 5 V/nA,
as reported in Commander.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p73
MultiClamp Signals 5
This completes the creation of our first signal. All we actually did was to create a signal name.
Following that, with MultiClamp telegraphing enabled, the remainder of the signal configuration was
handled automatically.
Before we proceed to the next signal, note
the possibility of additional signal filtering
in Clampex software.
The Hardware Signal Conditioning section
has configuration options for Axon
Instruments’ CyberAmp signal conditioner.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p74
MultiClamp Signals 6
Now to the next signal— the amplifier
primary output signal for reading membrane
potential in current clamp.
We are using the same digitizer channel for
both the current and voltage clamp primary
output signals, so still with digitizer channel
Analog IN #0 selected,
click Add. This time type “Vm_prime” for
the name of the new signal.
Again, because we have enabled telegraphs for the
channel that this signal is associated with, signal units and
scale factor are set automatically from MultiClamp
Commander software. When you change Commander to
current clamp mode and membrane potential is measured,
Clampex software will update appropriately, if the
telegraphs were configured to start a new trial.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p75
MultiClamp Signals 7
Now we create current and voltage clamp signals for the amplifier secondary output.
We have the secondary output
connected to Analog IN #1
(Connections). Select this as the
digitizer channel, and then click Add.
Type Vm_sec, for the signal we will use to monitor
membrane potential in voltage clamp.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p76
MultiClamp Signals 8
Now for the other secondary output signal
from the MultiClamp, on digitizer channel
Analog IN #1. This will monitor membrane
current in current clamp.
Add Im_sec in the Add Signal dialog, as for
previous signals.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p77
MultiClamp Signals 9
Signal Im_sec will be used when Commander
is in current clamp with “Membrane Current”
as the secondary output signal.
Notice that the scale factor for current-reading output
signals is affected by the choice of headstage resistor.
This can be adjusted in Commander’s Options dialog:
in the Gains tab, Feedback Resistor sections.
We will use the default 500 MW setting.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p78
MultiClamp Signals 10
Now we create signals for the
command waveforms.
Go to the Output Signals tab in the
Lab Bench. Analog OUT #0 is
selected. This is the channel we have
connected for both voltage and
current clamp commands
(Connections).
Click Add in the Signals section, and type V_clamp
into the Add Signal dialog — for the command signal
for voltage clamp.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p79
MultiClamp Signals 11
We enabled telegraphs for Analog OUT #0
in association with both Analog IN #0 and
Analog IN #1, so the signal units and scale
factor are simply reported from Commander.
MultiClamp can output command signals in voltage
clamp at two scale factors—20 mV/V and 100 mV/V.
Select the command scale factor from the
Commander Options dialog:
We will use the 20 mV/V setting.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p80
MultiClamp Signals 12
Under default settings the command
signal holding level cannot be set in the
Lab Bench. We do not need to worry
about the holding level reported in the
field because we will set this when we
incorporate the signal into a protocol.
It only remains to create the current clamp command signal.
Click Add again, calling the new signal I_clamp.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p81
MultiClamp Signals 13
Set the amplifier mode in
Commander to current clamp:
In the Lab Bench, the scale factor units change to
express current.
Again, Commander has two scaling settings for the
command signal, 400 pA/V and 2 nA/V, selected in the
Options dialog Gains tab.
Check that you have 400 pA/V selected.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p82
MultiClamp Signals 14
Finish
In this section we played with settings in Commander in order to see how the telegraphs work, but
in the normal course of events you only need to add appropriately named signals in the Lab Bench,
ensuring these are associated with the correct digitizer channels. Then, each time you run a
protocol with one of the signals, Clampex software uses the units and scale factors telegraphed
from Commander at that time.
We created six signals:
Voltage clamp
Current clamp
 Im_prime
 Vm_prime
 Vm_sec
 Im_sec
 V_clamp
 I_clamp
This completes the creation and configuration of all our signals. We now proceed to the creation of
protocols, where these signals are built into a broader set of acquisition parameters.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p83
MultiClamp
Configure Protocols
Protocols in Clampex software are complete sets of acquisition parameters, including options for command
waveforms and preliminary data analysis. Particular signals, defined in the Lab Bench, are specified for
each protocol.
In this section we create two simple protocols, one each for current and voltage clamp, incorporating the
signals we have just defined.
MultiClamp Protocol 1
Open the protocol editor by selecting New Protocol
from the Acquire menu.
Note: If a previously saved protocol is not loaded in
Clampex software, it uses a place-holder protocol,
labeled “(untitled)”. If this is currently loaded you can
open the editor to create a new protocol by selecting
Edit Protocol, or by clicking the button:
The currently loaded protocol is reported in the status
bar at the bottom of the main Clampex software window.
We will begin by setting up the protocol for
voltage clamp.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p85
MultiClamp Protocol 2
The front tab of the protocol editor
has controls for, amongst other
things, acquisition mode, sampling
rate, and trial hierarchy.
The default acquisition mode is
episodic stimulation—the only mode
that allows a command waveform to
be generated. We want to generate
a command, so leave this setting. In
fact, all the default settings on this
tab can be left as they are, but take
time to note key parameters such as
the Sampling Rate (10 kHz), the
number of samples per sweep, and
the number of sweeps per run.
The sweep start-to-start interval is set at Minimum, so each
new sweep starts as soon as the previous one is finished.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p86
MultiClamp Protocol 3
Next go to the Inputs tab.
Here you select digitizer input channels for the
protocol, as well as the signals that you want to be
conveyed on these.
For voltage clamp, we want two input signals —
one scaled signal for membrane current, and a
second signal to monitor membrane voltage. We
created these in the Lab Bench — “Im_prime” and
“Vm_sec” — associating them with digitizer IN
channels 0 and 1. Now we incorporate them into
the voltage protocol.
Channel #0 should be already checked. Open the
list box beside it and select Im_prime.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p87
MultiClamp Protocol 4
Then check Channel #1 and select Vm_sec.
This completes the Inputs tab.
Next, go to the Outputs tab.
We created the signal “V_clamp” to deliver the voltage
clamp command waveform, on digitizer output channel #0.
Select V_clamp from the Channel #0 list box.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p88
MultiClamp Protocol 5
This tab is also used to set the holding level for the signal. There may be some confusion over the signal units for
this, due to telegraphs.
With telegraphs enabled, Clampex software shows command signal
units appropriate for the amplifier mode, so if the MultiClamp
amplifier was in Current Clamp mode (IC) when you opened the
protocol editor, “V_clamp” shows with units for current.
This is no cause for alarm, since “V_clamp” will only be used in voltage clamp, and Clampex software will
telegraph the correct units at that time. It does mean that in order to set a holding level we need to recall the
units used for the command signal in voltage clamp. These were millivolts. Alternatively, close the protocol
editor with the OK button, switch to Voltage Clamp mode (VC) in Commander, and reopen the protocol editor.
“V_ clamp” now shows the units that will be used when “V_clamp” is output — i.e. millivolts.
We want to set a holding level of –50 mV, so
type –50 in the holding level field.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p89
MultiClamp Protocol 6
Although we will not make any changes for the purposes of our protocol, it is worth taking a quick look at
the trigger settings.
Go to the Trigger tab.
Default settings give “Immediate” trial starts. This
means Clampex software is armed for data acquisition
as soon as you select Record, or View Only, from the
Acquire menu—or click the buttons:
Record
View Only
The default trigger source is “Internal Timer”. This triggers the command waveform and data acquisition
immediately after the trial is started, continuing through to the end of the trial automatically.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p90
MultiClamp Protocol 7
Now go to the Waveform Channel #0 tab, where outputs
are defined for digitizer output channel Analog OUT #0.
A default waveform is already defined—we will delete this
and create our own simple stimulus, but first familiarize
yourself with some key settings on this tab.
The Analog Waveform check box enables analog
command definition. Selecting Epochs means we define
the waveform using the table in the middle of the tab.
In this, the sweep can be divided into up to 50 sections
(epochs) A–AX, and a waveform defined for each of these.
The Epoch Description table in the Waveform tab includes
cut and paste functionality.
Notice the confirmation that “V_clamp” is the signal carrying the output
waveform. The units shown for it — in the “Info” message box and in the
epoch description table—are again derived from the amplifier mode
telegraph from MultiClamp Commander software.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p91
MultiClamp Protocol 8
Now to the definition of our waveform.
We will configure an output with a simple step,
increasing the amplitude of the step with each sweep.
In column A of the epoch description table, keep Step
in the Type row, but click in the First level row and
type in –50. This sets the output level for epoch A in
the first sweep of the run. Our entry of –50 mV
maintains the holding level.
Click in the next row, Delta level, and type in 0. This
keeps the first level setting for subsequent sweeps —
i.e. epoch A is maintained at –50 mV for each of the
10 sweeps in the trial.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p92
MultiClamp Protocol 9
Now to set the period for epoch A.
Click in the First duration row, and type in 50.
Our sampling interval is 10 kHz, so a 50 ms sample
duration equates to 500 samples. Shift focus to a
different cell in the table to see this reported below.
This completes epoch A.
Now we configure the step, in epoch B.
Click in the Type row in column B (currently set to “Off”).
Select Step from the popup menu.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p93
MultiClamp Protocol 10
Set the level for the first sweep at –100 mV.
For this epoch, because we want an incrementing step
level from sweep to sweep, we enter a delta level.
Click on the Delta level cell and type in 20. This forces the
step level up 20 mV with each successive sweep.
We have 10 sweeps starting at –100 mV, so the final
sweep will have a step level of 80 mV, reported below
the table.
Now set the duration, at 100 ms. Again, this is reported
below the table, in milliseconds as well as in samples.
We will not set a delta duration, which would alter the
length of the epoch from sweep to sweep, so this
completes our waveform definition.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p94
MultiClamp Protocol 11
Click Update Preview in the bottom right corner of
the protocol editor.
This opens the Waveform Preview window shown at
right, where you can see a graphical representation
of the waveform we have defined.
This window can be kept open while you experiment
with different epoch settings — click Update
whenever you want to update the display.
Note: The Waveform Preview opens with panes for all analog output channels—right-click in the upper pane and
select Maximize Signal from the pop-up menu to get the display shown.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p95
MultiClamp Protocol 12
We have completed the setup of the voltage clamp
protocol — close the protocol editor with the OK button.
The new protocol is loaded, still labeled “(untitled)”, and
we could acquire data under it if we wanted, but it is not
saved for future use.
Go to Save Protocol As in the Acquire menu. This opens
a standard file-saving dialog. Name the protocol
Voltage Clamp 1, and click Save.
The protocol is now saved and can be loaded whenever we
want, with Open Protocol in the Acquire menu,
or button:
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p96
MultiClamp Protocol 13
Setup of the current clamp protocol follows similar lines to the protocol for voltage clamp.
Open the protocol editor again with New
Protocol, in the Acquire menu.
We will again accept the default settings in
the Mode/Rate tab, so go straight to the
Inputs tab.
This time select Vm_prime for Channel
#0, and Im_sec for Channel #1.
Note: Be sure the MultiClamp amplifier is in IC Mode to see the
correct units when you configure the waveform.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p97
MultiClamp Protocol 14
On the Outputs tab, select the current clamp command
signal we configured for digitizer Analog OUT channel #0:
I_clamp.
Leave the holding level at the default zero setting for
current clamp.
Create your own command waveform
on the Channel #0 tab.
Experiment with the different
waveform options viewing these in
the Waveform Preview window.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p98
MultiClamp Protocol 15
Finally, close the protocol editor by clicking OK, and save
the protocol (Save Protocol As in the Acquire menu), calling
it Current Clamp 1.
You will see on exit that the Scope window is set up in
preparation for the two input signals configured for this
protocol, with units as currently telegraphed from the
MultiClamp amplifier.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p99
MultiClamp Protocol 16
When you use Gap-free mode in the Real Time Controls panel, open the pre-programming dialog by clicking the < button.
You can pre-program voltage level and holding duration values for each channel, as well as turning the digital bit on or
off. You can pre-program up to 50 epochs. You can also manually change values during a recording.
Finish
This completes the protocol tutorial and completes this guide. However, as a final, optional step we link the protocols to
MultiClamp Commander amplifier-mode telegraphs in the next section.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p100
MultiClamp
Configure Sequencing Keys
The amplifier-mode telegraphs sent by MultiClamp Commander software can be used to automatically
load protocols in Clampex software. This means that you can have an appropriate protocol load and run,
automatically, as soon as you change modes in Commander. This is done using sequencing keys.
In this section, as an optional final stage in the guide, we configure this linkage for the two protocols we
have created.
MultiClamp Sequencing Keys 1
Open the Sequencing Keys dialog box from
the Configure menu.
If the sequencing keys are at default
settings, the dialog opens with an
empty sequencing keys table, under
the title Startup Set.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p102
MultiClamp Sequencing Keys 2
Click Add and in the Add a key definition for list box select
V-Clamp IN 0.
“IN 0” refers to Analog IN #0, the digitizer input channel we configured Clampex software to receive MultiClamp
amplifier scaled output telegraphs on.
“V-Clamp” means that the sequencing key we are about to configure will be triggered by a change to voltage
clamp in the MultiClamp amplifier.
Click OK.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p103
MultiClamp Sequencing Keys 3
The Sequencing Keys Properties dialog opens with the
Operations tab uppermost. Select Protocol.
In the Action field, for the purposes of demonstration,
keep the View selection. This has the protocol run in
View Only mode when it is called;
in a real experiment you would choose Run.
Next we have to enter the protocol we want to run
when the MultiClamp amplifier is in voltage clamp.
Click Browse to open a file dialog.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p104
MultiClamp Sequencing Keys 4
The file dialog should open at the “Params” folder where we earlier saved the two protocols. We called
the voltage clamp protocol “Voltage Clamp 1”. Select this now and click Open.
The protocol is reported in the protocol
file field.
Leave the repetition count at one, to run
the protocol just once when it is called.
This completes the configuration of this sequencing key, however, before closing the Properties dialog
have a look at the Sequencing tab. Use this tab to link the current key to a second one so that it runs after
the current one is finished. You can create sequences of any number of operations using this functionality.
Click OK to return to the main Sequencing Keys dialog.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p105
MultiClamp Sequencing Keys 5
Our first sequencing key appears in the table.
Check its details before clicking Add to add a
second key, for current clamp.
This time select I-Clamp IN 0. The input channel
is the same, but we want this key to be triggered
by a change to current clamp.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p106
MultiClamp Sequencing Keys 6
As before, select Protocol and click Browse,
this time using the protocol we configured
for current clamp, “Current Clamp 1”.
Close the Properties dialog, and check the
details of the second sequencing key. It has
been added at the top of the list, following
the key order in the Add dialog list box.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p107
MultiClamp Sequencing Keys 7
Finally, click Options, at the bottom of the
Sequencing Keys command buttons. You are
offered three options for saving protocols in a
sequencing keys series.
Select the second option.
When you come to test the setup, this selection means that if you resize or rescale the Scope window
while a protocol is running, your new window settings are automatically saved with the protocol when
you switch over to the second protocol.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p108
MultiClamp Sequencing Keys 8
Finish
This completes the setup of the sequencing keys—the sequencing key set is automatically saved when you close
the dialog.
Test the Configuration
Connect the model cell to the Channel 1 headstage of the MultiClamp amplifier.
Make a final check on the I-Clamp and V-Clamp tabs in MultiClamp Commander software to ensure you have the
correct primary and secondary output signal types selected.
Now switch mode in Commander. The appropriate protocol should load and run in Clampex software, displaying
resultant data correctly labeled, and with the correct units, in the Scope window. You may need to scale the
window to see the signals clearly.
Switch over to the alternate mode. Again, the appropriate protocol should load and run.
This completes the MultiClamp section of the guide. For more detailed information on any of the matters covered,
use the online Help and consult the manual.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p109
Axoclamp 900A
Axoclamp Sequence
This sequence describes how to set up two distinct data-acquisition “protocols” for use in whole-cell
recording with an Axoclamp 900A amplifier.
Once we have created the protocols, as an optional final step, we will integrate these with the
Axoclamp amplifier’s mode telegraph so that Clampex software automatically loads the appropriate
protocol when you shift between current and voltage clamp in Axoclamp Commander software.
Move through the sequence page by page, or skip sections with the links below—but note that the
discussion assumes the setup from earlier sections:
Digitizer–Amplifier Connections
Configure Telegraphs
Create Signals
Configure Protocols
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p110
Axoclamp 900A
Digitizer – Amplifier Connections
In this section we put in the cabling between the digitizer and Axoclamp 900A amplifier.
Connect Axoclamp
If you have not already done so, switch on your Axoclamp 900A amplifier and open Axoclamp
Commander software. If Commander opens in demo mode (reported in the title bar), you will need to
connect the amplifier to the software.
In Commander, click Select Device button:
Select Axoclamp Hardware.
Click Scan — Commander displays the amplifier
serial number when the amplifier is found.
Click OK to exit.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p112
Axoclamp Connections 1
The signals we want to record depend on the Axoclamp 900A mode.
Current Clamp modes (IC , I=0, DCC, and HVIC)
Digitizer Analog Inputs
Digitizer Analog Output
 Membrane potential
 Command current
 Membrane current*
Voltage Clamp (TEVC and dSEVC)
Digitizer Analog Inputs
Digitizer Analog Output
 Membrane current
 Command potential
 Membrane potential
*Note:
Current can be monitored on the CURRENT OUTPUT of each channel.
This is optional.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p113
Axoclamp Connections 2
Clampex software allows for more than one signal to be sent, at different times, on each channel (the
relationship between signals and channels is more fully explained in the Create Signals section).
Because the Axoclamp 900A amplifier can only be in one mode at a given time, signals associated with
these modes can share digitizer Analog IN and Analog OUT channels.
Which signals can share channels depends on how the Axoclamp amplifier is going to be used:

For current clamp and TEVC, the input signals for current clamp (Vm) and voltage clamp (Im)
on Channel 2.

For current clamp and dSEVC, the input signals for current clamp (Vm) and voltage clamp (Im)
on Channel 1.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p114
Axoclamp Connections 3
900A
(Channel 1)
I-CLAMP
COMMAND
SCALED
OUTPUT
Digitizer
ANALOG
OUT 1
ANALOG
IN 0
900A
(Channel 2)
I-CLAMP
COMMAND
SCALED
OUTPUT
Digitizer
ANALOG
OUT 2*
ANALOG
IN 1
900A
V-CLAMP
COMMAND
ANALOG
OUT 0
*Note: To use both I-CLAMP COMMANDS and V-CLAMP COMMAND requires three
Analog Outputs, as available
on the Digidata 1440A. An alternative method when only two analog outputs are available is described in
the next slide.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p115
Axoclamp Connections 4
Alternative command connections
900A
(Channel 1)
STEPACTIVATE 1*
SCALED
OUTPUT
Digitizer
DIGITAL
OUT 0
ANALOG
IN 0
900A
(Channel 2)
STEP-ACTIVATE
2*
SCALED
OUTPUT
Digitizer
DIGITAL
OUT 1
ANALOG
IN 1
900A
V-CLAMP
COMMAND
ANALOG
OUT 0
*Note: STEP-ACTIVATE inputs are located on the rear of the unit.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p116
Axoclamp Connections 5
Alternative command connections – no voltage clamp
900A
(Channel 1)
I-CLAMP
COMMAND
SCALED
OUTPUT
Digitizer
ANALOG
OUT 0
ANALOG
IN 0
900A
(Channel 2)
I-CLAMP
COMMAND
SCALED
OUTPUT
Digitizer
ANALOG
OUT 1
ANALOG
IN 1
900A
V-CLAMP
COMMAND
(UNCONNECTED)
Note: This set-up does not allow external commands in dSEVC or TEVC modes.
It is only recommended if you do not plan to use either voltage clamp mode.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p117
Axoclamp Connections 6
Optional: Current monitor outputs for IC modes.
If you want to monitor the current while in IC modes on both channels, you can use the
dedicated CURRENT OUTPUT on each channel.
900A
(Channel 1)
CURRENT
OUTPUT
900A
(Channel 2)
CURRENT
OUTPUT
Digitizer
ANALOG IN
8
Digitizer
ANALOG IN
9
These outputs are not telegraphed to Clampex, so we will need to configure the scale
factor manually in the Lab Bench.
Details on this configuration can be found later in Axoclamp Signals.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p118
Axoclamp
Configure Telegraphs
Axoclamp 900A amplifier telegraphs are software messages sent from Axoclamp Commander software to Clampex
software, registering key amplifier settings.
As well as simply reporting the settings in Clampex software, the telegraphs are integrated into Clampex software
so that the greater proportion of signal setup is done automatically (as we will soon see).
The most important telegraph remains the gain telegraph, used to automatically rescale the Clampex Scope
window as gains settings are changed, and to ensure recorded data files are correctly scaled. The lowpass filter
settings are also reported in the Real Time Controls and written into recorded file headers—as is the output gain.
In addition, the Axoclamp amplifier has telegraphs for amplifier mode, and for the units and scale factors for
command and acquisition signals. We will use these telegraphs in our setup in the following slides.
We will assume that a voltage clamp mode (dSEVC or TEVC) will be used, and the connections shown in Axoclamp
Connections 3 are made. The changes necessary for other arrangements, e.g., the connections in Axoclamp
Connections 4 should be easy to make once the basic principles are understood.
Axoclamp Telegraphs 1
Open Telegraphed Instrument from the
Clampex software Configure menu.
All telegraphs must be configured for a specific digitizer input
channel. We will first enable telegraphs for the channel
receiving the amplifier Scaled Output from Channel 1. We
have connected the Axoclamp Scaled Output for Channel 1
to Analog IN #0 on the Digidata (Connections), so select this
from the Input Channels list.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p120
Axoclamp Telegraphs 2
Select Axon Axoclamp 900A from the Telegraphed Instrument list.
When you have made this selection notice the options with
respect to linking protocols to amplifier modes. You can learn
more about this in the MultiClamp section on Sequencing Keys
(Configure Sequencing Keys).
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p121
Axoclamp Telegraphs 3
The first enabled section on the dialog is Amplifier Configuration.
Identify the amplifier channel (i.e. headstage)
and signal type for the selected digitizer
channel (in this case, Analog In #0). In the
previous slides we chose Scaled output 1 to
connect to Analog In #0
Next click Scan — the Axoclamp serial number appears when the amplifier is found.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p122
Axoclamp Telegraphs 4
Axoclamp telegraphs scale factors for command signals as well as for its output signals. Enable
Clampex to receive these telegraphs in the bottom Output Channels section.
The digitizer input we are configuring
receives output from headstage 1 (i.e.
amplifier channel 1). The I-CLAMP
COMMAND input for this headstage
is fed from digitizer output Analog
OUT #1 (see Connections; Analog
Out #0 is connected to V-CLAMP
COMMAND). Select Analog OUT #1
in the Command field*.
*Note:
This completes telegraph setup for the Scaled Output of
Axoclamp channel 1.
(The CURRENT OUTPUT is not telegraphed.)
If you are using the STEP-ACTIVATE inputs instead of I-CLAMP COMMAND,
leave the Command field on (none).
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p123
Axoclamp Telegraphs 5
Open Telegraphed Instrument from the
Clampex Configure menu.
All telegraphs must be configured for a specific digitizer input
channel. Now we will enable telegraphs for the channel
receiving the Axoclamp Channel 2 Scaled Output. We have
connected the Axoclamp Scaled Output for Channel 2 to
Analog IN #1 on the Digidata (Connections), so select this
from the Input Channels list.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p124
Axoclamp Telegraphs 6
Select Axon Axoclamp 900A from the Telegraphed Instrument list.
When you have made this selection note the options with
respect to linking protocols to amplifier modes. You can
learn more about this in the MultiClamp section on
Sequencing Keys (Configure Sequencing Keys).
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p125
Axoclamp Telegraphs 7
The first enabled section on the dialog is Amplifier Configuration.
Identify the amplifier channel (i.e. headstage)
and signal type for the selected digitizer
channel (in this case, Analog In #1). In the
previous slides we chose Scaled output 2 to
connect to Analog In #1
Next click Scan — the Axoclamp serial number appears when the amplifier is found.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p126
Axoclamp Telegraphs 8
Axoclamp amplifier telegraphs scale factors for command signals as well as for its output signals.
Enable Clampex software to receive these telegraphs in the bottom Output Channels section.
The digitizer input we are configuring
receives output from headstage 2
(i.e. amplifier channel 2). The I-CLAMP
COMMAND input for this headstage is fed
from digitizer output Analog OUT #2
(see Connections; Analog Out #0 is connect
to V-CLAMP COMMAND).
Select Analog OUT #2 in the Command field.
This completes telegraph setup for the Scaled Output of Axoclamp Channel 2.
(The CURRENT OUTPUT is not telegraphed. See Axoclamp Connections 6 for
notes about these outputs.)
*Note:
If you are using the STEP-ACTIVATE inputs instead of I-CLAMP COMMAND, leave the
Command field on (none).
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p127
Axoclamp Telegraphs 9
Finish
This completes telegraph setup. We have configured Clampex software to receive telegraphs as follows:
● SCALED OUTPUT Channel 1 to Analog IN #0
● SCALED OUTPUT Channel 2 to Analog IN #1
● I-CLAMP COMMAND Channel 1 to Analog OUT #1*
● I-CLAMP COMMAND Channel 2 to Analog OUT #2*
● V-CLAMP COMMAND is not telegraphed, although connected to Analog OUT #0.
We now go to the Lab Bench for signal configuration.
*Note:
Assuming connections as shown in Axoclamp Connections 3.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p128
Axoclamp
Create Signals
In this section we name the signals we require, assigning these to input and output channels.
Before starting it is important to be clear on what signals and channels are:

Signal: a set of name, unit, scale factor and offset, by means of which the voltage inputs
and outputs at the digitizer are represented in Clampex software as the parameter being
read at, or delivered to, the preparation.

Channel: a cable connection to the digitizer, identified by the name of the BNC port
where connection is made, e.g. Analog IN #0, Digital OUT #2.
As already noted, analog channels can be configured for different signals at different times, which is
what we do in this section.
Axoclamp Signals 1
The configuration of the signals in the Lab Bench depends upon the modes in which you plan to use the
Axoclamp 900A amplifier.
Select a mode from the menu below, and complete the configuration for that mode.
Then return to this menu for other modes you plan to use.
Axoclamp 900A Modes
IC (both channels)
dSEVC
TEVC
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p130
Axoclamp Signals 2
Current Clamp (both channels)
Open the Lab Bench from the Configure menu — or
use the button:
The Lab Bench opens with the Input Signals
tab on top, and digitizer channel Analog IN #0
selected. We have the Scaled Output of
Channel 1 connected to this channel. In this
mode the Scaled Output we are interested in
is Membrane Potential, so we need to create
an appropriate signal.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p131
Axoclamp Signals 3
Current Clamp (both channels)
Click Add in the Signals section, opening the Add Signal dialog.
Type in Vm_1 — the name we will give the scaled membrane
potential signal for current clamp, on Channel 1.
Click OK. With the new signal selected in the Signals list, the
rest of the tab shows options and settings for that signal.
Notice that the entire Scaling section is
grayed, as it is not used. This is
because signal scaling is now under
the control of the telegraphs we set up
in the last section. Do not worry if the
units and scale here are incorrect –
they are overridden.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p132
Axoclamp Signals 4
Current Clamp (both channels)
The telegraphs are reported at the bottom
of the Lab Bench.
The screenshots show the telegraphs in
the Lab Bench with Commander at default
settings.
Axoclamp Commander 900A
does not report whole-cell
capacitance values, since it
has no whole-cell
capacitance compensation.
You may enter a value if you
know the capacitance.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p133
Axoclamp Signals 5
Current Clamp (both channels)
Change settings in Commander and see the telegraphs update in the Lab Bench. The filter, gain, and capacitance
compensation telegraphs are also reported in the Real Time Controls.
Notice that the scale factor reported in
the Lab Bench does not change as you
alter the output gain. Clampex software
reports the unity gain scale factor,
i.e. the scale factor for an output gain of
one (in this case, 10 mV/mV = 10 V/V).
Of course, the scale factor applied to
the signal takes the gain into account —
e.g. in these screenshots, Clampex
software will apply a scale factor of
10 x 10 mV/mV = 100 mV/mV,
as reported in Commander.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p134
Axoclamp Signals 6
Current Clamp (both channels)
This completes the creation of our first signal. All we actually did was to create a signal name. Following that,
with Axoclamp telegraphing enabled, the remainder of the signal configuration was handled automatically.
The signal we created was for Channel 1 in IC mode. To make a matching signal for Channel 2 the same steps
are followed.
Open the Lab Bench
Select the digitizer channel Analog IN #1
Add a new signal
This completes the configuration of the input signals used when the Axoclamp 900A amplifier channels are in
current clamp mode.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p135
Axoclamp Signals 7
Current Clamp (both channels)
Now we create signals for the current command waveform on Channel 1.
This only applies if the Axoclamp 900A amplifier is connected as in Axoclamp Connections 3.
If you are using the STEP-ACTIVATE inputs (Axoclamp Connections 4) to apply current commands, skip this part.
Go to the Output Signals tab in the Lab Bench.
Select Analog OUT #1. This is the channel we have
connected for current clamp commands to Channel 1
(Axoclamp Connections 3).
Click Add in the Signals section, and type IC_Cmd1 into the
Add Signal dialog — for the command signal for current clamp on
Channel 1.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p136
Axoclamp Signals 8
Current Clamp (both channels)
Now we create signals for the current command waveform on Channel 2.
This only applies if the Axoclamp 900A amplifier is connected as in Axoclamp Connections 3.
If you are using the STEP-ACTIVATE inputs (Axoclamp Connections 4) to apply current commands, skip this part.
Go to the Output Signals tab in the Lab Bench.
Select Analog OUT #2. This is the channel we have
connected for current clamp commands to Channel 2
(Axoclamp Connections 3).
Click Add in the Signals section, and type IC_Cmd2 into the
Add Signal dialog —f or the command signal for current clamp on
Channel 2.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p137
Axoclamp Signals 9
Current Clamp (both channels)
Now we create optional signals used to monitor the current while in current clamp.
Each channel has a dedicated CURRENT OUTPUT. Because its scale factor is not telegraphed to Clampex
software, we need to configure these signals manually in the Lab Bench.
Go to the Output Signals tab in the Lab Bench.
Select Analog OUT #8. This is the channel we have
connected to CURRENT OUTPUT on Channel 1
(Axoclamp Connections).
Click Add in the Signals section, and type I1 into the
Add Signal dialog—for the current on Channel 1.
We’ll do the scale factor and units on the next slide.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p138
Axoclamp Signals 10
Current Clamp (both channels)
Now we determine the scale factor and enter it.
First, select the units “nA” for this signal, by
choosing n from the drop-down prefix list
and typing A into the units field.
The Scale factor (V/nA) depends upon the headstage. The HS-9A and HS-2A headstages have an H value
corresponding to the value of the feedback resistor in the headstage.
The value of H is printed on the headstage. It is the number following the small “x”.
For example: HS-9A x1 indicates H=1.
The Scale factor to enter in the Lab Bench is given by this formula:
Scale factor (V/nA) = 0.01 / H
Therefore, for H=1, the scale factor is 0.01; if H=10, the scale factor is 0.001, and so on.
You should now be able to repeat these steps to create a matching signal on Analog IN #9, to be used
to monitor Channel 2 CURRENT OUTPUT.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p139
Axoclamp Signals 11
Current Clamp (both channels)
Finished!
In this section we created signals for the operation of Channel 1 and Channel 2 in current clamp mode. These signals
will work whether one or both channels is in current clamp mode. We created these signals:
Channel 1
Channel 2
Vm_1 for membrane potential
from SCALED OUTPUT
Vm_2 for membrane potential
from SCALED OUTPUT
IC_Cmd1 for current command
waveforms to I-CLAMP
COMMAND
IC_Cmd2 for current command
waveforms to I-CLAMP
COMMAND
I1 for membrane current from
CURRENT OUTPUT (optional)
I2 for membrane current from
CURRENT OUTPUT (optional)
As noted, the command signals may be created in alternative ways.
If you choose to use the STEP-ACTIVATE commands, then you need not create the IC_Cmd1 and IC_Cmd2 signals.
Click here to return to Modes Menu to create more
signals for use in other modes (e.g., TEVC, dSEVC,…)
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p140
Axoclamp Signals 12
dSEVC
Now we create signals for use when the Axoclamp 900A is in dSEVC mode. In this mode, only headstage 1 is
active. However, both SCALED OUTPUTS may carry signals from headstage 1.
We will configure Channel 1 SCALED OUTPUT to be Membrane Current of headstage 1; while Channel 2 SCALED
OUTPUT will be Membrane Potential, also of headstage 1.
We will then configure a voltage clamp command signal.
We have the SCALED OUTPUT of Channel 1 connected
to Analog IN #0 (Connections). Select this as the
digitizer channel, and then click Add.
Type in Im_dSEVC, for the signal we will use to monitor
membrane current in dSEVC.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p141
Axoclamp Signals 13
dSEVC
When a signal’s parameters are telegraphed, the Scaling
section on the Lab Bench is grayed. Don’t worry if the
grayed-out settings are incorrect; they are overridden.
Axoclamp Commander 900A does
not report whole-cell capacitance
values, since it has no whole-cell
capacitance compensation. You
may enter a value if you know the
capacitance.
The telegraphed parameters are reported at the
bottom of the Lab Bench. The screenshots show
the telegraphs in the Lab Bench with Channel 1 in
dSEVC mode, at default settings.
Note: You may need to select Headstage 1, Membrane Current on the Axoclamp 900A Commander
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p142
Axoclamp Signals 14
dSEVC
We have the SCALED OUTPUT of
Channel 2 connected to Analog IN #1
(Connections). Select this as the
digitizer channel, and then click Add.
Type in Vm_dSEVC, for the signal we will use to monitor
membrane potential in dSEVC.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p143
Axoclamp Signals 15
dSEVC
In dSEVC mode, only headstage 1 is operating. Therefore we may select signals from headstage 1 to be carried
on SCALED OUTPUT on Channel 1 and Channel 2.
In this case, we choose to monitor the “Headstage 1, Membrane Potential” on SCALED OUTPUT of Channel 2.
As before, the telegraphed parameters are reported at
the bottom of the Lab Bench. The screenshots show the
telegraphs in the Lab Bench with Channel 1 in dSEVC
mode, at default settings.
Axoclamp Commander 900A
does not report whole-cell
capacitance values, since it
has no whole-cell capacitance
compensation. You may enter
a value if you know the
capacitance.
Note:
You may need to select “Headstage 1, Membrane Potential” on the Axoclamp 900A Commander
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p144
Axoclamp Signals 16
dSEVC
Now we create a signal for the voltage command waveform on while in dSEVC mode.
Go to the Output Signals tab in the Lab Bench.
Select Analog OUT #0. This is the channel we
have connected to V-CLAMP COMMAND
(Axoclamp Connections 3).
Click Add in the Signals section, and type Vm_Cmd into
the Add Signal dialog as the name of the new voltage
command signal.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p145
Axoclamp Signals 17
dSEVC
Now we need to set the units and scale factor for this signal.
Since it is a voltage clamp command, choose m and type V, as shown below. This tells Clampex software that the
units are “mV”.
For the Axoclamp 900A (and other Axon CNS instruments), the usual voltage clamp command scale factor is
20 mV/V.
The scale factor of 20 mV/V tells Clampex software how to convert your desired command to the appropriate
voltage to produce from Analog OUT.
For example, if your desired holding potential is –50 mV, Clampex converts this to –50 mV / (20 mV/V) = -2.5 V at
the actual Digidata Analog OUT.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p146
Axoclamp Signals 18
dSEVC
Finished!
In this section we created signals for the operation of Channel 1 in dSEVC mode.
In this mode, headstage 2 does not operate and so SCALED OUTPUT on Channel 2 can be used
to carry a signal from headstage 1.
We created three signals:
●
Im_dSEVC for membrane current from Channel 1 SCALED OUTPUT
●
Vm_dSEVC for membrane potential from Channel 2 SCALED OUTPUT
(but measured at headstage 1)
●
VC_Cmd for voltage command waveforms to V-CLAMP COMMAND
Click here to return to Modes Menu to create more signals for
use in other modes (e.g., TEVC, dSEVC,…)
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p147
Axoclamp Signals 19
TEVC
Now we create signals for use when the Axoclamp 900A amplifier is in TEVC mode. In this mode, headstage 1
measures the membrane potential. Headstage 2 injects current.
Therefore, we will configure Channel 1 SCALED OUTPUT to be Membrane Potential of headstage 1.
Channel 2 SCALED OUTPUT will be Membrane Current, measured on headstage 2.
We will then configure a voltage clamp command signal.
Open the Lab Bench
We have the SCALED OUTPUT of Channel 1
connected to Analog IN #0 (Connections).
Select this as the digitizer channel, and then click Add.
Type Vm_1, for the signal we will use to monitor
membrane potential in TEVC.
Note: this is the same signal used when headstage 1 is
in IC mode. If you have already created this signal, it is
not necessary to do so again.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p148
Axoclamp Signals 20
TEVC
This completes the creation of our first TEVC signal. All we actually did was to create a signal name. Following that,
with Axoclamp amplifier telegraphing enabled, the remainder of the signal configuration was handled automatically.
The signal we created was for Channel 1, which measures Vm when the Axoclamp 900A amplifier is in TEVC mode.
Now we need to make a signal for Channel 2, which measures the membrane current. The same steps are followed.
From the Lab Bench, select the digitizer channel, Analog IN #1
Add a new signal
This completes the configuration of the input signals used when the Axoclamp 900A amplifier operates in TEVC
mode. Now let’s look at how the signals are telegraphed.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p149
Axoclamp Signals 21
TEVC
Reset the Axoclamp 900A amplifier to defaults, a put a model cell (in CELL position) on the headstages in
preparation for TEVC. Switch the instrument to TEVC mode. Don’t worry about getting the best clamp – we just want
to look at the operation of the telegraphs.
Open Lab Bench, and change settings in Commander. You see the telegraphs update in the Lab Bench:
Notice that the scale factor reported in
the Lab Bench does not change as you
alter the output gain. Clampex software
reports the unity gain scale factor,
i.e. the scale factor for an output gain of
one (in this case, 10 mV/mV = 10 V/V).
Of course, the scale factor applied to the
signal takes the gain into account —
e.g. in these screenshots, Clampex
software will apply a scale factor of
10 x 10 mV/mV = 100 mV/mV,
as reported in Commander.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p150
Axoclamp Signals 22
TEVC
Now we create a signal for the voltage command waveform on while in TEVC mode.
Go to the Output Signals tab in the Lab Bench.
Select Analog OUT #0. This is the channel we
have connected to V-CLAMP COMMAND
(Axoclamp Connections 3).
Click Add in the Signals section, and type Vm_Cmd into the
Add Signal dialog — the name of the new voltage command
signal. Click OK.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p151
Axoclamp Signals 23
TEVC
Now we need to set the units and scale factor for this signal.
Since it is a voltage clamp command, choose “m” and type “V”, as shown below. This tells Clampex software that
the units are “mV”.
For the Axoclamp 900A amplifier (and other Axon CNS instruments), the usual voltage clamp command scale
factor is 20 mV/V.
The scale factor of 20 mV/V tells Clampex software how to convert your desired command to the appropriate
voltage to produce from Analog OUT.
For example, if your desired holding potential is –50 mV, Clampex software converts this to
–50 mV / (20 mV/V) = -2.5 V at the actual Digidata digitizer Analog OUT.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p152
Axoclamp Signals 24
TEVC
Finished!
In this section we created signals for experiments in TEVC mode. In this mode, headstage 1
measures Vm and SCALED OUTPUT on Channel 1 carries this signal. Headstage 2 measures Im
and SCALED OUTPUT on Channel 2 carries this signal.
We created three signals:
●
Im_2 for membrane current from Channel 2 SCALED OUTPUT
●
Vm_1 for membrane potential from Channel 1 SCALED OUTPUT
●
VC_Cmd for voltage command waveforms to V-CLAMP COMMAND
Click here to return to Modes Menu to create
more signals for use in other modes (e.g., TEVC,
dSEVC,…)
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p153
Axoclamp Signals 25
TEVC
Now we create signals for use when the Axoclamp 900A is in TEVC mode.
In this mode, headstage 1 is measures the membrane potential.
Headstage 2 injects current.
Therefore, we will configure Channel 1 SCALED OUTPUT to be Membrane Potential of headstage 1.
Channel 2 SCALED OUTPUT will be Membrane Current, measured on headstage 2.
We will then configure a voltage clamp command signal.
Open the Lab Bench
We have the SCALED OUTPUT of Channel 1 connected to
Analog IN #0 (Connections). Select this as the digitizer
channel, and then click Add.
Type Vm_1, for the signal we will use to monitor membrane
potential in TEVC.
Note: This is the same signal used when headstage 1 is in
IC mode. If you have already created this signal, it is not
necessary to do so again.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p154
Axoclamp
Configure Protocols
Protocols in Clampex software are complete sets of acquisition parameters, including options for command
waveforms and preliminary data analysis. Particular signals, defined in the Lab Bench, are specified for
each protocol.
In this section we create two simple protocols, one each for current and voltage clamp, incorporating the
signals we have just defined.
Axoclamp Protocol 1
Open the protocol editor by selecting New Protocol
from the Acquire menu.
Note: If a previously saved protocol is not loaded in
Clampex software, it uses a place-holder protocol,
labeled “(untitled)”. If this is currently loaded you can
open the editor to create a new protocol by selecting
Edit Protocol, or by clicking the button:
The currently loaded protocol is reported in the status
bar at the bottom of the main Clampex software window.
We will begin by setting up the protocol for
voltage clamp (TEVC).
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p156
Axoclamp Protocol 2
The front tab of the protocol editor
has controls for, amongst other
things, acquisition mode, sampling
rate, and trial hierarchy.
The default acquisition mode is
episodic stimulation — the only
mode that allows a command
waveform to be generated. We want
to generate a command, so leave
this setting. In fact, all the default
settings on this tab can be left as
they are, but take time to note key
parameters such as the Sampling
Rate (10 kHz), the number of
samples per sweep, and the number
of sweeps per run.
The sweep start-to-start interval is set at Minimum, so each
new sweep starts as soon as the previous one is finished.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p157
Axoclamp Protocol 3
Next go to the Inputs tab.
Here you select digitizer input channels for the
protocol, as well as the signals that you want to be
conveyed on these.
For TEVC, we want two input signals — one scaled
signal for membrane current, and a second signal to
monitor membrane voltage. We created these in the
Lab Bench —“Vm_1” and “Im_2”— associating them
with digitizer Analog IN channels 0 and 1,
respectively (see Axoclamp Signals). Now we
incorporate them into the voltage protocol.
Channel #0 should be already checked. Open the
list box beside it and select Vm_1.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p158
Axoclamp Protocol 4
Then check Channel #1 and select Im_2.
This completes the Inputs tab.
Next, go to the Outputs tab.
We created the signal “Vm_Cmd” to deliver the voltage clamp
command waveform, on digitizer output channel #0
(Axoclamp Signals 22) .
Select Vm_Cmd from the Channel #0 list box.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p159
Axoclamp Protocol 5
We have already configured the
signal Vm_Cmd in the Lab
Bench (Axoclamp Signals 23), so
the units appear as we
designated. Notice that the scale
factor is also indicated, along
with the resulting range of
available commands.
This tab is also used to set the holding level for
the signal. Notice that the units are as we
designated in the Lab Bench
We want to set a holding level of –50 mV,
so type –50 in the holding level field.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p160
Axoclamp Protocol 6
Although we will not make any changes for the purposes of our protocol, it is worth taking a quick look at
the trigger settings.
Go to the Trigger tab.
Default settings give “Immediate” trial starts. This
means Clampex software is armed for data acquisition
as soon as you select Record, or View Only, from the
Acquire menu — or click the buttons:
Record
View Only
The default trigger source is “Internal Timer”. This triggers the command waveform and data acquisition
immediately after the trial is started, continuing through to the end of the trial automatically.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p161
Axoclamp Protocol 7
Now go to the Waveform Channel #0 tab, where outputs
are defined for digitizer output channel Analog OUT #0.
A default waveform is already defined — we will delete
this and create our own simple stimulus, but first
familiarize yourself with some key settings on this tab.
The Analog Waveform check box enables analog
command definition. Selecting Epochs means we define
the waveform using the table in the middle of the tab. In
this, the sweep can be divided into up to 50 sections
(epochs) A–AX, and a waveform defined for each of these.
The Epoch Description table in the Waveform tab includes
cut and paste functionality.
Confirm that Vm_Cmd is the signal carrying the output waveform. The
units shown for it — in the “Info” message box and in the epoch
description table — are again derived from the amplifier mode
telegraph from Axoclamp Commander software.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p162
Axoclamp Protocol 8
Now to the definition of our waveform.
We will configure an output with a simple step,
increasing the amplitude of the step with each sweep.
In column A of the epoch description table, keep Step
in the Type row, but click in the First level row and
type –50. This sets the output level for epoch A in the
first sweep of the run. Our entry of –50 mV maintains
the holding level.
Click in the next row, Delta level, and type 0. This
keeps the first level setting for subsequent sweeps —
i.e. epoch A is maintained at –50 mV for each of the 10
sweeps in the trial.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p163
Axoclamp Protocol 9
Now to set the period for epoch A.
Click in the First duration row, and type in 50.
Our sampling interval is 10 kHz, so a 50 ms sample
duration equates to 500 samples. Shift focus to a
different cell in the table to see this reported below.
This completes epoch A.
Now we configure the step, in epoch B.
Click in the Type row in column B (currently set to “Off”).
Select Step from the popup menu.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p164
Axoclamp Protocol 10
Set the level for the first sweep at –100 mV.
For this epoch, because we want an incrementing step
level from sweep to sweep, we enter a delta level. Click
on the Delta level cell and type in 20. This forces the step
level up 20 mV with each successive sweep.
We have 10 sweeps starting at –100 mV, so the final
sweep will have a step level of 80 mV, reported below
the table.
Now set the duration, at 100 ms. Again, this is reported
below the table, in milliseconds as well as in samples.
We will not set a delta duration, which would alter the
length of the epoch from sweep to sweep, so this
completes our waveform definition.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p165
Axoclamp Protocol 11
Click Update Preview in the bottom right corner of
the protocol editor.
This opens the Waveform Preview window shown at
right, where you can see a graphical representation
of the waveform we have defined.
This window can be kept open while you experiment
with different epoch settings — click Update
whenever you want to update the display.
Note: The Waveform Preview opens with panes for all analog output channels — right-click in the upper pane and
select Maximize Signal from the popup menu to get the display shown.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p166
Axoclamp Protocol 12
Finally, close the protocol editor by clicking OK.
You will see on exit that the Scope window is set up in
preparation for the two input signals configured for this
protocol, with units as currently telegraphed from the
Axoclamp 900A amplifier. Here the Axoclamp software
has been put into TEVC mode.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p167
Axoclamp Protocol 13
We have completed the setup of the voltage clamp
protocol — close the protocol editor with OK.
The new protocol is loaded, still labeled “(untitled)”,
and we could acquire data under it if we wanted, but it
is not saved for future use.
Go to Save Protocol As in the Acquire menu. This
opens a standard file-saving dialog. Name the
protocol Voltage Clamp 1, and click Save.
The protocol is now saved and can be loaded whenever
we want, with Open Protocol in the Acquire menu,
or button:
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p168
Axoclamp Protocol 14
Setup of the current clamp protocol follows similar lines to the protocol for voltage clamp.
Open the protocol editor again with
New Protocol, in the Acquire menu.
We will again accept the default
settings in the Mode/Rate tab, so go to
the Inputs tab.
This time select Vm_1 for Channel #0,
and Vm_2 for Channel #1.
Note:
Be sure both channels of the Axoclamp are in IC Mode to see the correct units when you
configure the waveform.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p169
Axoclamp Protocol 15
On the Outputs tab, select IC_Cmd1, the current-clamp
command signal we configured for digitizer Analog
OUT #1. Similarly, select IC_Cmd2 on Analog OUT #2*.
Leave the holding level at the default zero setting for
current clamp.
*Note: This applies only for the Digidata 1440A digitizer with four analog outputs.
For details on using the digital outputs to trigger STEP-ACTIVATE, see Axoclamp Protocols 18.
If you do not plan to use a voltage clamp mode, you can use OUT #0 and OUT #1 for the two current commands.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p170
Axoclamp Protocol 16
Create your own command waveform on the
Channel #1 tab, in this example.
Experiment with the different waveform options, and
display them in the Waveform Preview.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p171
Axoclamp Protocol 17
Finally, close the protocol editor by clicking OK, and save the protocol
(Save Protocol As in the Acquire menu), calling it Current Clamp 1.
You will see on exit that the Scope window is set up in preparation for
the two input signals configured for this protocol, with units as currently
telegraphed from the Axoclamp amplifier.
Finished!
This completes our two protocols, “Voltage Clamp 1” and
“Current Clamp 1”, and is the completion of the guide for
Axoclamp 900A amplifier setup.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p172
Axoclamp Protocol 18
When you use Gap-free mode in the Real Time Controls panel, open the pre-programming dialog by clicking the <
button. You can pre-program voltage level and holding duration values for each channel, as well as turning the
digital bit on or off. You can pre-program up to 50 epochs. You can also manually change values during a recording.
Finish
This completes the protocol tutorial and completes this guide.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p173
Keep Your Software up to Date
At Molecular Devices, we aim to make improvements to released software frequently, and to fix reported
problems quickly.
pCLAMP™ Electrophysiology Data Acquisition & Analysis Software version 11 and Axoclamp 900A
Commander software are both regularly updated. The programs have internal settings to remind you to
check our website for updates. Alternatively, you may force the program to update.
•
To update pCLAMP 11 software, open Clampex
software, and go to Help > About Clampex.
Note the complete version number, and close
the window.
•
Click here to check if a newer version of
pCLAMP software is available on the
Knowledge Base. If the version number on the
web page is newer than yours, download and
install the update.
•
Click here to check if a newer version of
Axoclamp software is available.
•
To update MultiClamp 700B Commander
software or Axoclamp 900A Commander
software, go to the Options > About dialog.
•
Remember the full version number displayed.
•
Click here to check if a newer version of
MultiClamp software is available on the
Knowledge Base.
For research use only. Not for use in diagnostic procedures.
© 2021 Molecular Devices, LLC. Trademarks are the property of Molecular Devices, LLC or their respective owners. | p174
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