MultiClamp 700B

MultiClamp 700B
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MultiClamp 700B
COMPUTER-CONTROLLED
MICROELECTRODE AMPLIFIER
Theory and Operation
Part Number 2500-0157 Rev D
March 2005 Printed in USA
Copyright 2005 Axon Instruments / Molecular Devices Corp.
No part of this manual may be reproduced, stored in a retrieval system, or
transmitted, in any form or by any means, electronic, mechanical, photocopying,
microfilming, recording, or otherwise, without written permission from Molecular
Devices Corp.
QUESTIONS? See Axon's Knowledge Base: http://support.axon.com
i
PLEASE READ!!!!!
SAFETY
There are important safety issues that you must take into account when using this
instrument. Please carefully read the safety warnings starting on page 159 before
you use this instrument.
VERIFICATION
This instrument is extensively tested and thoroughly calibrated before leaving the
factory. Nevertheless, researchers should independently verify the basic accuracy
of the controls using resistor/capacitor models of their electrodes and cell
membranes.
WARNING
If this equipment is used in a manner not specified by the manufacturer, the
protection provided by the equipment may be impaired.
DISCLAIMER
This equipment is not intended to be used, and should not be used, in human
experimentation or applied to humans in any way.
Verification, Warning, Disclaimer
Table of Contents • iii
Table of Contents
Chapter 1 Introduction .......................................................................................... 1
Chapter 2 Installation and Basic Operation ........................................................ 3
Installation............................................................................................................. 3
Check List ......................................................................................................... 3
Installing Hardware ........................................................................................... 4
Installing the MultiClamp 700B Commander ................................................... 4
Functional Checkout ............................................................................................. 6
Communication with the MultiClamp 700B ..................................................... 6
Setting Parameters in the MultiClamp 700B Commander ................................ 7
Toolbar Buttons in the MultiClamp 700B Commander .................................... 9
Test the Noise.................................................................................................. 10
Calibration....................................................................................................... 11
Getting Help in the MultiClamp 700B Commander ....................................... 13
Chapter 3 Tutorials .............................................................................................. 15
Check List ........................................................................................................... 15
Model Cell....................................................................................................... 16
Tutorial 1 – Electrode in the Bath: Voltage Clamp............................................. 17
Tutorial 2 – Electrode in the Bath: Current Clamp ............................................. 19
Tutorial 3 – Giga Seal Configuration .................................................................. 21
Tutorial 4 – Whole-Cell Configuration: Voltage Clamp..................................... 26
Tutorial 5 – Whole-Cell Configuration: Current Clamp ..................................... 31
Tutorial 6 – Whole-Cell Configuration: Automatic Mode Switching................. 37
Table of Contents
iv • Table of Contents
Chapter 4 Guide to Electrophysiological Recording......................................... 43
General Advice.................................................................................................... 44
Chamber Design .............................................................................................. 44
Perfusion.......................................................................................................... 44
Mechanical Stability........................................................................................ 44
Optics .............................................................................................................. 45
Bath Electrode ................................................................................................. 45
Interfacing a Computer.................................................................................... 46
Computer Noise............................................................................................... 47
Patch Clamping ................................................................................................... 47
Headstage and Holder Considerations............................................................. 47
Forming a Gigaseal ......................................................................................... 49
Whole-cell Voltage Clamp Recording ............................................................ 51
Perforated-patch Recording............................................................................. 54
Low Noise Techniques.................................................................................... 55
Sharp Microelectrode Recording......................................................................... 59
Sharp Microelectrode or Patch Electrode? ...................................................... 60
Microelectrode Properties ............................................................................... 60
Filling Solutions .............................................................................................. 64
Impaling Cells ................................................................................................. 64
Chapter 5 Reference Section ............................................................................... 67
Audio Monitor..................................................................................................... 68
Bath Headstage and Electrodes ........................................................................... 72
Rb Minimization .............................................................................................. 72
Use of a Bath Headstage ................................................................................. 74
Bridge Balance .................................................................................................... 75
Bridge Balance in the Bath.............................................................................. 76
Bridge Balance in the Cell............................................................................... 77
Buzz..................................................................................................................... 78
Capacitance Compensation ................................................................................. 78
Electrode Capacitance Compensation ............................................................. 79
Whole-Cell Capacitance Compensation.......................................................... 80
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Table of Contents • v
Auto Button..................................................................................................... 83
Manual Adjustment of Capacitance Compensation ........................................ 83
Filtering the Command Stimulus .................................................................... 84
Capacitance Neutralization ................................................................................. 84
Input Capacitance............................................................................................ 84
Adjusting Capacitance Neutralization............................................................. 86
Limitations of Capacitance Neutralization...................................................... 86
Clear .................................................................................................................... 87
Electrochemistry.................................................................................................. 87
External Command Inputs................................................................................... 89
External Command Sensitivity........................................................................ 89
Additivity of Commands................................................................................. 90
Command Filter Frequency............................................................................. 90
Feedback Resistor ............................................................................................... 91
V-Clamp Mode................................................................................................ 91
I-Clamp Mode ................................................................................................. 93
Filters................................................................................................................... 93
-3 dB Frequency .............................................................................................. 94
Types of Filters ................................................................................................... 95
Bessel Filter..................................................................................................... 95
Butterworth Filter............................................................................................ 95
Choosing the Cutoff Frequency ...................................................................... 95
High-pass Filter ............................................................................................... 97
Command Filter Frequency............................................................................. 97
Grounding and Hum............................................................................................ 97
Headstage ............................................................................................................ 98
Voltage Clamp Circuit .................................................................................... 99
Current Clamp Circuit................................................................................... 101
Mounting the Headstage................................................................................ 102
Bath Connection............................................................................................ 102
Cleaning ........................................................................................................ 102
Static Precautions .......................................................................................... 102
Acoustic Pick-up ........................................................................................... 102
Table of Contents
vi • Table of Contents
Help ................................................................................................................... 103
Holders .............................................................................................................. 103
Holder Design ............................................................................................... 103
Optional Ag/AgCl Pellets.............................................................................. 105
Holder Use..................................................................................................... 105
Holder Maintenance ...................................................................................... 107
Adapters ........................................................................................................ 107
Input/Output Connections ................................................................................. 107
Front Panel .................................................................................................... 107
Rear Panel ..................................................................................................... 109
Leak Subtraction................................................................................................ 110
Mode.................................................................................................................. 111
Model Cell......................................................................................................... 112
Noise.................................................................................................................. 113
Noise.................................................................................................................. 114
Measurement of Noise................................................................................... 114
Sources of Noise............................................................................................ 114
Oscilloscope Triggering .................................................................................... 116
Output Zero ....................................................................................................... 117
Overload ............................................................................................................ 118
Polarity Conventions ......................................................................................... 118
Biological Polarity Conventions ................................................................... 118
MultiClamp Polarity Conventions................................................................. 119
Polarity Summary for Different Recording Configurations .......................... 120
Power Supply .................................................................................................... 120
Supply Voltage Selection .............................................................................. 121
Changing the Fuse ......................................................................................... 121
Glitches.......................................................................................................... 121
Select Device..................................................................................................... 122
Series Resistance Compensation ....................................................................... 122
Introduction to Rs Compensation .................................................................. 122
Is Rs Compensation Necessary? .................................................................... 124
Adjusting Rs Compensation .......................................................................... 125
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Table of Contents • vii
Theory of Rs Compensation .......................................................................... 129
The ‘Prediction’ Control ............................................................................... 130
The ‘Prediction’ Control ............................................................................... 131
Saturation Effects .......................................................................................... 132
Readjustment of Whole Cell Compensation with ‘Prediction’ ..................... 133
The ‘Correction’ Control............................................................................... 134
Readjustment of Whole Cell Compensation with ‘Correction’ .................... 135
Setting ‘Prediction’ and ‘Correction’ Values ................................................ 135
Oscillations.................................................................................................... 135
Limitations of Rs Compensation ................................................................... 136
SoftPanel Configuration.................................................................................... 137
Status ................................................................................................................. 138
Zap..................................................................................................................... 138
Chapter 6 Troubleshooting................................................................................ 139
Chapter 7 Specifications .................................................................................... 141
References ............................................................................................................ 147
Technical Assistance............................................................................................ 149
Warranty and Repair Service ............................................................................ 151
Circuit Diagrams Request Form........................................................................ 155
Declaration of Conformity.................................................................................. 157
Important Safety Information............................................................................ 159
Index ..................................................................................................................... 163
Table of Contents
Introduction • 1
Chapter 1
Introduction
The MultiClamp 700B is a computer-controlled microelectrode current and voltage
clamp amplifier for electrophysiology and electrochemistry. It is a versatile
instrument capable of single-channel and whole-cell voltage patch clamping, highspeed current clamping (patch or sharp electrode), ion-selective electrode recording,
amperommetry / voltammetry and bilayer recording (optional headstage).
The MultiClamp 700B was designed to support one or two primary headstages
(CV-7), in addition to two auxiliary headstages (optional HS- or VG-type,
purchased separately). Each CV-7 headstage contains a current-to-voltage
converter for voltage (patch) clamp and a voltage follower for current clamp. This
allows the user to conveniently switch between low-noise patch-clamp recording
and high-speed current-clamp recording. Also, an optional CV-7 headstage will
allow bilayer recording.
The MultiClamp 700B is essentially an analog input / output instrument, similar to
conventional amplifiers by Axon Instruments. Thus, BNC-type input and output
connections are necessary to communicate with a digitizing interface, oscilloscope
or other recording device. The MultiClamp 700B contains no front panel knobs
and switches. Instead, the instrument is operated using a control panel program, the
MultiClamp 700B Commander, which runs on a host computer and communicates
with the amplifier via a USB cable.
Chapter 1
2 • Introduction
Computer control permits “smart” automatic features, such as capacitance
compensation, bridge balance and offsets. Telegraph information, performed
through software messaging, includes Gain, Filter and Capacitance, as well as
input/output Scaling Factors and recording Mode.
The MultiClamp 700B Commander interface is completely independent of
acquisition software. Thus, the MultiClamp 700B can be used with any data
acquisition package. It is, of course, compatible with the Digidata series (1200A or
later) digitizers and pCLAMP 7 (or later) software. (Note: However, telegraphing
is only supported in pCLAMP versions 9 and higher.) Regarding third-party
software, see our webpage “Developer Info” for a detailed Software Development
Kit that describes how to read telegraph information.
We recognize that software control of an amplifier is an unusual step forward for
some users. If computer mouse control is unsettling, consider the optional
SoftPanel device to control the MultiClamp 700B. The SoftPanel is essentially a
hardware extension of the MultiClamp 700B Commander software. Knobs and
buttons replace mouse or keyboard control. For more information, visit our website
or call Axon Technical Support.
The MultiClamp 700B is a sophisticated instrument. Experienced and
inexperienced researchers alike are advised to read this manual thoroughly and to
familiarize themselves with the instrument. The Functional Checkout and
Tutorials sections of the following chapter provide step-by-step instructions using
the PATCH-1U model cell, which provides resistors and parallel RC circuits to
mimic the pipette, patch and whole-cell recording conditions.
We will be pleased to answer any questions regarding the theory and use of the
MultiClamp 700B. Any comments and suggestions on the use and design of the
MultiClamp 700B will be much appreciated. We welcome reprints of papers
describing work performed with the MultiClamp 700B. Keeping abreast of your
research helps us to design our instruments for maximum usefulness.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Installation and Basic Operation • 3
Chapter 2
Installation and Basic Operation
Installation
Carefully unpack all parts, and use the enclosed shipping list to verify that all parts
have been received. Retain packing materials in case the instrument needs to be
returned to the factory at a later date.
For the initial checkout, the MultiClamp 700B should be situated on a bench top
away from other equipment. Do not install it in a rack until the checkout is
complete.
Check List
These installation and checkout procedures require the following:
1. MultiClamp 700B main unit with power cord.
2. CV-7 headstage(s) with PATCH-1U model cell(s).
3. USB (A/B-type) control cable.
4. MultiClamp 700B Commander host software (from CD or website).
Chapter 2
4 • Installation and Basic Operation
5. A PC running Windows operating system (version 95 and NT not
supported), or Mac OS 10.2 or higher with the display set to at least 800 x
600. The PC should have one spare USB port. In order to use on-line
Help, the PC should have Internet access and a web browser with
JavaScript (Internet Explorer v. 4 or later, or equivalent).
6. External oscilloscope.
Installing Hardware
1. Connect the appropriate end of the USB cable to the USB connector on the
MultiClamp 700B rear panel, and the other end to a free USB port on your
PC.
2. Connect the CV-7 headstage(s) to HEADSTAGE #1 and HEADSTAGE #2
rear panel connectors, respectively. THE AMPLIFIER SHOULD BE
TURNED OFF WHENEVER HEADSTAGES ARE CONNECTED. Note
a small white cap covering one of the headstage input pin sockets, and a
corresponding missing pin on the headstage connector. This is normal.
3. Connect the power cable, and turn on the MultiClamp 700B. The front
panel POWER light should illuminate, as well as the VOLTAGE CLAMP
light for each channel. Windows operating system will automatically
recognize the new USB hardware as a Human Interface Device (HID).
4. If you are using the optional SoftPanel device, connect it to a different
computer USB port using the USB cable supplied with the SoftPanel.
Installing the MultiClamp 700B Commander
1. Run the MultiClamp 700B Commander installer from the enclosed CD, or
from the installation file downloaded from the Axon website. This will
install all necessary files and generate a shortcut for MultiClamp 700B on
your desktop.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Installation and Basic Operation • 5
2. Run MultiClamp 700B Commander by double-clicking on the MultiClamp
700B desktop icon. The first time the program is run, you will be asked to
update MultiClamp 700B Commander. If you’ve just installed the software
from the CD, we suggest that you download the latest update. Axon
Instruments is very responsive to customer feedback, thus the website will
likely contain a newer, updated version. We also recommend that you
choose to be reminded every 30 days to check for a new download.
3. Next you will see the Device Selection dialog. Select MultiClamp
Hardware, then click the Scan button. The amplifier Serial Number will be
identified in the list when the instrument is successfully recognized.
Figure 2.1
If the program is unable to find a valid Serial Number, check that the
MultiClamp 700B is switched on and that the USB cable is connected
properly.
4. If you are using the optional SoftPanel device, click on the SoftPanel tab
and click the Scan button. After this device is recognized, click the OK
button.
5. The main MultiClamp 700B Commander window should appear. If
installed correctly, the MultiClamp Serial Number will appear in the 700B
Chapter 2
6 • Installation and Basic Operation
Commander window heading. (And, if the optional SoftPanel is configured
correctly, the Configure SoftPanel icon
will be colored.
Functional Checkout
The purpose of this section is to quickly check the correct operation of the
MultiClamp 700B and to briefly describe the basic controls of the MultiClamp
700B Commander. This information, in addition to the extensive 700B
Commander online Help, should enable you to work comfortably with the features
of the amplifier. Finally, the following chapter Tutorials will guide you step-bystep through the various recording configurations.
Communication with the MultiClamp 700B
1. Check that the STATUS light on the front of the MultiClamp 700B is
flashing. This indicates that the MultiClamp 700B Commander is polling
the MultiClamp 700B, updating its meter displays.
2. Toggle the Channel 1 and Channel 2 Mode buttons, switching repeatedly
between Voltage Clamp (VC) and Current Clamp (I=0, IC) modes:
Figure 2.2
The tabs immediately below the mode switches will change appropriately.
Also, the VOLTAGE CLAMP (blue) and CURRENT CLAMP (green)
indicator lights on the front panel of the MultiClamp 700B should also
confirm that the amplifier is changing modes.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Installation and Basic Operation • 7
Setting Parameters in the MultiClamp 700B Commander
Many parameter fields in the MultiClamp 700B Commander can be set in three
different ways. To demonstrate this, press the V-Clamp 1 tab and try the following.
1. Glider control
•
Position the cursor over the parameter field to the right of Holding,
noting that the cursor changes to a vertical double-headed arrow
(
). Hold down the left mouse button and drag the mouse up and
down; the holding potential changes in 1 mV steps.
•
Press the Shift key while dragging the mouse; the holding potential
changes in 5 mV steps.
•
Press the Ctrl key while dragging the mouse; the holding potential
changes in 20 mV steps.
•
Position the cursor over the button with the black dot (dual control) to
the right of Cp Fast, noting that the cursor changes to crossed doubleheaded arrows ( ). Holding down the left mouse button and dragging
the mouse vertically changes the capacitance parameter (pF), while
dragging horizontally changes the time constant parameter (τs).
Simultaneously pressing the Shift or Ctrl key respectively magnifies
the effect (however, in a non-linear manner for this particular control).
2. Entering text directly
•
Position the cursor over the parameter field to the right of Holding and
double click. Type a number, and then press Enter.
Figure 2.3
Chapter 2
8 • Installation and Basic Operation
3. Selecting from a list
•
Position the cursor over the frequency parameter to the right of Seal
Test and press the right mouse button. A list of possible frequencies is
displayed, one of which can be selected by a mouse click.
Figure 2.4
•
Repeat for the Primary (or Secondary) Output field. In this case a
right-click of the mouse will display a list of output signals.
Figure 2.5
•
Right-clicking the mouse over most other 700B Commander glider
fields will display a menu to select the sensitivity of the glider. For
example, right-click the mouse while over the Holding glider, and you
will see the following menu.
Figure 2.6
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Installation and Basic Operation • 9
Toolbar Buttons in the MultiClamp 700B Commander
At the top of the MultiClamp 700B Commander main window is a row of toolbar
buttons that provide access to a number of special features.
Figure 2.7
Positioning the mouse cursor over each button will, after a short delay, display a
Tool Tip for the button. This Tool Tip will identify the alternative keyboard
shortcut that will also activate the feature. For example, the "Resize Window"
button is associated with the <F2> key. This feature will toggle the size of the
Commander window between full-size, meters-only, or the user-adjusted size.
Drag the lower-right corner of the Commander window to change the size, then
click the <F2> key to toggle between window sizes.
Most other buttons are self-explanatory, with the possible exception of the Save
Configuration ( ), Load Configuration ( ) and Quick Select buttons
). These buttons allow the user to store and retrieve parameter settings
(
for the MultiClamp 700B Commander. The Quick Select buttons can be assigned
to a particular set of parameter settings to facilitate rapid loading, or, alternatively,
to run an executable command line. This might be useful for experiments that
require different configurations, or when several users share the same recording
setup, or when an external command is desired (for example, starting a custom
script to initiate a software-controlled perfusion device).
Quick Select buttons are assigned as follows.
1. After setting the MultiClamp 700B Commander parameters to the desired
values, press the Save Settings toolbar button. Enter a file name and
directory (the file name is given the extension MCC, for MultiClamp 700B
Commander file).
Chapter 2
10 • Installation and Basic Operation
2. Press the Options toolbar button ( ) and then press the Quick Select tab.
Click in the name field for the Quick Select Button you wish to assign (1
through 3). Then use the Browse button to choose the name of the MCC
file containing the desired parameter settings. (Note: if any executable file
other than a MultiClamp configuration file is chosen for this button
assignment, then that executable command will be run when this button is
clicked.)
3. Back in the main MultiClamp 700B Commander panel, positioning the
mouse over the assigned Quick Select button now displays the name of the
assigned MCC file. Press the Quick Select button to load the parameter
settings. Alternatively, the Load Configuration button ( ) can be pressed
to load any previously stored MCC file.
Test the Noise
All electronic equipment generates some amount of thermal noise. Follow these
steps to measure the intrinsic MultiClamp 700B current noise (“Irms”, or the rootmean-square of the current noise):
1. Leave the CV-7 headstage in an “open circuit” configuration (i.e., nothing
should be attached to the input of the CV-7).
2. To reduce extraneous noise, the CV-7 must be shielded. This can be
accomplished using aluminum foil, which should be loosely but completely
wrapped around the headstage. A great alternative to foil shielding is a
metal container, such as a coffee can. Most importantly, the input of the
CV-7 should not make contact with the shield.
3. The shield must now be grounded to the CV-7. Connect the small, black
grounding wire provided with your MultiClamp hardware to the gold,
1 mm input at the rear of the headstage case. Connect the other end of the
ground wire to the foil or metal container using an “alligator” clip or other
appropriate connection.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Installation and Basic Operation • 11
4. In the MultiClamp 700B Commander, check the “Irms” box beneath the
corresponding Channel meter for the CV-7 headstage (test one headstage at
a time). Compare the value indicated by the meter to that listed in the table
below (*5 kHz, 4-pole Butterworth measurement).
Feedback Resistor
Noise*
50 MΩ
2.0 pA rms
500 MΩ
0.8 pA rms
5 GΩ
0.5 pA rms
50 GΩ
0.15 pA rms
5. Repeat the Irms noise measure for each Feedback Resistor selected from
the MultiClamp 700B Commander Options menu.
6. If your MultiClamp has more than one CV-7 headstage, repeat steps 1-5 for
the second headstage.
Calibration
The steps below provide a quick check of the calibration of the MultiClamp 700B.
It is assumed that appropriate shielding (as described in “Test the Noise”, above)
will be used during these tests.
1. Connect an oscilloscope to the front panel PRIMARY or SCOPE Output
BNC.
2. Synchronize the oscilloscope by connecting to the rear panel SYNC
OUTPUT BNC.
Press the “Reset to Program Defaults” button in the MultiClamp 700B Commander
to standardize the MultiClamp 700B.
Chapter 2
12 • Installation and Basic Operation
50 G Range
1. Press the “Options” button, choose the Gains tab, and select the 50 G
feedback resistor in the Voltage Clamp pane. Return to the main
MultiClamp 700B Commander window.
2. Plug the PATCH connector of the PATCH-1U model cell into the
CV-7 headstage.
3. Set Seal Test amplitude to 100 mV and frequency to 50 Hz, then check
the box to make it active.
4. Press Auto Cp Fast to remove the bulk of electrode capacitance
transient.
5. The resulting waveform should be square, except for an initial
overshoot (possibly twice the size of the steady-state response) that
settles to the baseline in about 1 to 2 ms. The rise time to the peak of
the overshoot should be about 50 µs. The steady-state amplitude
following the transient should be ~500 mVp-p (±50 mV).
5 G Range
1. Change the feedback resistor to 5 G in the Options / Gains menu.
2. Press Auto Cp Fast.
3. The step response should be ~50 mVp-p (±5 mV).
500 M Range
1. Press the “Reset to Program Defaults” button. (By default, the 500 M
range is selected.)
2. Check Seal Test and set the amplitude to 25 mV.
3. Plug the CELL connector of the PATCH-1U model cell into the CV-7
headstage.
4. Press the Auto Whole Cell button.
5. Press Auto Cp Fast button.
6. The step response should be ~25 mVp-p.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Installation and Basic Operation • 13
50 M Range
1. Change the feedback resistor to 50 M.
2. Increase Output Gain to 10.
3. Press Auto Whole Cell and Auto Cp Fast.
4. The step response should be ~25 mVp-p.
Getting Help in the MultiClamp 700B Commander
First, ensure that your computer is connected to the Internet and has a correctly
configured web browser with JavaScript (such as Internet Explorer v. 4 or later.).
Pressing the
button at the top of the MultiClamp 700B Commander will connect
you to the On-line Help, which describes many of the functions of the MultiClamp
700B Commander.
This manual is designed to be used in conjunction with the On-line Help. This
manual does not, for example, describe all the buttons and windows in MultiClamp
700B Commander, because this information is better provided in an interactive way
using the On-line Help. Rather, the purpose of this manual is to provide tutorials
and detailed information about the design and operation of the MultiClamp 700B
amplifier as a whole. Therefore, the On-line Help and this manual complement
each other. If you have suggestions for improving this manual or On-line Help, we
encourage you to submit them to Axon Technical Support.
Chapter 2
Tutorials • 15
Chapter 3
Tutorials
The purpose of this chapter is to lead the user through the basics of patch clamping
and ‘sharp’ microelectrode recording, using the PATCH-1U model cell that comes
with the MultiClamp 700B. The tutorials are designed to illustrate the operation of
the MultiClamp 700B and associated Commander control software. Although this
chapter is directed primarily at inexperienced electrophysiologists, it may also be
useful for experienced researchers who desire a simple introduction to the features
of the MultiClamp 700B.
We recommend that you perform the Tutorials in order to avoid confusion.
Check List
These tutorials require the following:
1. MultiClamp 700B main unit, and at least one CV-7 headstage. The
tutorials address Headstage #1, but of course you should repeat the tests for
Headstage #2 if you have a second.
2. PATCH-1U model cell.
Chapter 3
16 • Tutorials
3. Piece of aluminum foil or a metal container, such as a coffee can (in which
to place the model cell) grounded to the CV-7 headstage or 4 mm Signal
Ground plug on the rear of the MultiClamp 700B.
4. Oscilloscope to monitor the output of the MultiClamp 700B. Alternatively,
pCLAMP / Digidata acquisition system could be used to monitor the
output.
Model Cell
All of these tutorials use the PATCH-1U model cell, which contains simple circuits
of resistors and capacitors designed to simulate three patch clamp recording
conditions: (1) Pipette in the bath (Connector labeled BATH on the model cell), (2)
Gigaseal (PATCH), and (3) Whole-cell (CELL). The circuit for each of these is as
follows. (Also see MODEL CELL in Chapter 5.)
BATH:
10 MΩ "pipette" resistor to ground.
PATCH: 10 GΩ "patch" resistor to ground.
Approximately 5 pF stray capacitance to ground.
CELL:
10 MΩ "pipette" resistor.
500 MΩ "cell membrane" resistor in parallel with 33 pF cell membrane
capacitor.
Approximately 5 pF stray capacitance to ground.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 17
Tutorial 1 – Electrode in the Bath: Voltage Clamp
1. Switch on the MultiClamp 700B and run the MultiClamp 700B Commander by
double-clicking on the shortcut icon on the desktop of the PC. Press the Reset
to Program Defaults toolbar button, or press the F6 key.
Figure 3.1
This puts the MultiClamp 700B in V-Clamp mode and directs the Membrane
Current (0.5V/nA) signal to the Primary Output BNC connector on the front
panel of the amplifier.
2. Plug the BATH connector of the model cell into the white Teflon input
connector of the Channel 1 headstage. Connect the 2 mm gold socket on the
side of the model cell to the 1 mm gold socket on the rear of the CV-7
headstage, using the short black wire provided with the model cell. Shield the
headstage and model cell with the aluminum foil or metal box. Ground the
shield by connecting (using an “alligator” clip) to the 1 mm plug inserted
previously into the rear socket of the CV-7.
3. Connect a BNC cable from the Channel 1 Primary Output on the front panel of
the MultiClamp 700B to the oscilloscope. The oscilloscope display should be
set initially at 0.5 V/division and 2 ms/division. Triggering should be set to
Line. Alternatively, connect a BNC cable from the Channel 1 Primary Output
to and Analog Input on the front panel of a Digidata digitizer for monitoring on
the Scope Window of Clampex.
Chapter 3
18 • Tutorials
4. Press the Pipette Offset button while looking at the oscilloscope.
Figure 3.2
After making a brief series of steps (due to the MultiClamp 700B’s algorithm for
finding the offset) the Membrane Current is zeroed. Note also that the Pipette
Offset button is grayed out and the padlock icon to the left appears locked.
5. Check the Seal Test checkbox.
Figure 3.3
A repetitive pulse appears on the Primary output signal. (The trace can be
triggered on the oscilloscope screen by making a connection from the SYNC
output on the rear of the MultiClamp 700B to the External Trigger input on the
oscilloscope. See Options / General tab.) The amplitude of the Seal Test pulse
is 10 mV. The amplitude of the Membrane Current output pulse is 0.5 V,
which corresponds to 1 nA at the default gain of 0.5 V/nA (shown under
Primary Output section).
Figure 3.4
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 19
Therefore, the resistance of the model electrode is calculated from Ohm’s Law
to be R = V/I = 10 mV/1 nA = 10 MΩ. Alternatively, check the Resistance
checkbox under the Channel 1 meters.
Figure 3.5
The resistance is displayed on the meter. Uncheck the box when done. (DC
fluctuations in the signal are due to pulses from the MultiClamp 700B
Commander for calculating meter resistance values.)
6. Try changing the Seal Test amplitude and frequency by using the glider control
with the mouse. (See SETTING PARAMETERS IN THE MULTICLAMP 700B
COMMANDER in Chapter 6.)
Figure 3.6
Note how the Primary Output signal changes on the oscilloscope as the test
pulse parameters are changed.
Tutorial 2 – Electrode in the Bath: Current Clamp
Note that the model cell used in this tutorial is designed to simulate a patch pipette,
rather than a typical intracellular electrode, which generally has a higher
resistance. However, the principles illustrated are the same.
1. Set up the MultiClamp 700B and the MultiClamp 700B Commander as in Steps
1-3 of Tutorial 1.
Chapter 3
20 • Tutorials
2. Press the Mode button labeled IC. The tab labeled I-Clamp 1 will move to
front, and the Current Clamp light (green) on the front panel of the MultiClamp
700B unit will illuminate.
Figure 3.7
Note that the Primary Output signal displayed on the oscilloscope is now
Membrane Potential (10 mV/mV).
3. Press the Pipette Offset button. This operates exactly like in Voltage Clamp
mode. (See Tutorial 1, Step 4.) Note how the Primary Output signal changes
on the oscilloscope.
4. Check the Tuning checkbox.
Figure 3.8
A repetitive pulse appears on the Membrane Potential output. The amplitude of
the Tuning pulse is 1 nA. The amplitude of the Membrane Potential output
pulse is 100 mV, which corresponds to 10 mV at the default gain of 10 V/mV.
Therefore, the resistance of the model electrode is calculated from Ohm’s Law
to be R = V/I = 10 mV/1 nA = 10 MΩ. Alternatively, the resistance can be
directly displayed by checking the Resistance checkbox under the Channel 1
meters.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 21
5. Try changing the Tuning amplitude and frequency by using the glider control
with the mouse.
Tutorial 3 – Giga Seal Configuration
1. Set up the MultiClamp 700B and the MultiClamp 700B Commander as in Steps
1-3 of Tutorial 1, except that the PATCH connector on the model cell should be
plugged into the headstage of the MultiClamp 700B. This connects a 10 GΩ
resistor to ground, simulating a gigaseal.
2. One of the advantages of a gigaseal is that the recording noise is dramatically
reduced, enabling single-channel measurements. However, to facilitate singlechannel recording it is necessary to change the feedback resistor in the
headstage of the patch clamp amplifier. For illustration, look at the Channel 1
Primary Output after turning up the vertical gain on the oscilloscope. The noise
on Primary Output should be about 5 mV peak-to-peak (p-p), which
corresponds to 10 pA (p-p) at the default scale factor of 0.5V/nA. 10 pA is too
noisy for most single-channel recording.
3. Press the Options toolbar button at the top of the MultiClamp 700B
Commander.
Figure 3.9
This opens the Options panel. Select the Gains tab. You will note that the
default Feedback Resistor under Channel 1 Voltage Clamp is 500 MΩ.
Increasing the size of the feedback resistor, which is located in the headstage,
increases the gain of the headstage. As a rule of thumb, the larger the value of
the feedback resistor, the smaller the noise of the headstage but the smaller the
range of the output. For this reason, larger feedback resistors are usually
selected for patch recording, where low noise is more important than range.
Chapter 3
22 • Tutorials
(Note the information provided under Experiment Type and Range in the Gains
panel.)
Figure 3.10
Select 50 GΩ feedback resistor and then close this panel.
4. Note that the noise trace on the oscilloscope is now about 150 mVp-p. However,
the Primary Output gain is now 0.05 V/pA, so the noise is 3 pAp-p, a 3-fold
reduction compared with before. This is still quite noisy for recording singlechannel currents of a few picoamps. To clearly see small currents, it is
necessary to filter the Primary Output.
5. Locate the Primary Output section in the main window of the MultiClamp
700B Commander and position the mouse cursor over Bessel: 10 kHz. Using
the glider control (see Chapter 2) examine the effect of filtering the Primary
Output.
Figure 3.11
Note that with a filter setting of 2 kHz the peak-to-peak noise on Primary
Output is about 0.5 pA, which is adequate for most single-channel recording.
(See Chapter 4 for practical hints on how to reduce the noise further.)
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 23
6. This section of the MultiClamp 700B Commander displays three other
adjustable parameters: Output Gain, AC and Scope.
•
Use the glider to adjust Output Gain. Note the changes in the scaling factor
at Primary Output: Membrane Current, as well as the change in signal
amplitude on the oscilloscope. Unlike changing the feedback resistor
range, altering the Output Gain has no effect on the relative amplitude of
the (current) noise.
•
AC: allows you to send the Primary Output through a high-pass filter. This
may be desirable if you wish to remove a DC offset or low-frequency
component in the signal output.
•
Scope is used to filter the signal provided by the SCOPE BNC on the front
panel of the MultiClamp 700B. In the default configuration, this BNC
simply duplicates the signal available at the Primary Output BNC.
However, in some circumstances you may wish to filter the SCOPE signal
(normally viewed on an oscilloscope) more heavily than the PRIMARY
Output signal being sent to a computer. The Scope parameter in the
MultiClamp 700B Commander allows you to do this.
Chapter 3
24 • Tutorials
7. Open the Options panel and set the feedback resistor to 500M. Close this
panel, then reset the Bessel filter to 10 kHz, the Output Gain to 1 and the Seal
Test frequency to 200 Hz. Check the Seal Test checkbox; a train of ~1 Volt
transients will appear on the Primary Output trace. (These are more easily seen
if the oscilloscope is triggered using the SYNC output of the MultiClamp 700B,
as described in Tutorial 1.)
Figure 3.12
The transients result from the charging of the 5 pF capacitance of the model
cell, which simulates the capacitance of a patch electrode. In a real experiment
these transients are undesirable because they may saturate the amplifier, leading
to distortions in the measured currents. They can be eliminated by using the Cp
Fast and Cp Slow controls in the main window of the MultiClamp 700B
Commander.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 25
8. Place the mouse cursor over the button (dual control) opposite Cp Fast. The
cursor changes to crossed arrows. (See the figure below.) While holding down
the Shift key (to magnify the movement; see Chapter 2) use the glider, sliding
the mouse horizontally and vertically, to change the values of the time constant
and capacitance, respectively. Alternatively, you can place the mouse cursor
over each parameter display in turn, and use the glider to adjust each
individually.
Figure 3.13
Notice that you can change the amplitude and, to a lesser extent, the decay time
constant of the transients on the oscilloscope. With Cp Fast capacitance set to
about 5 pF the transients should be minimized.
9. An alternative way to cancel the transients is by pressing the Auto button
opposite Cp Fast. The algorithm should find optimum values of about 5 pF and
1 µs. In experiments with real cells you may need to make manual fine
adjustments for optimal cancellation.
10. Sometimes an additional, slower capacitance transient is visible after canceling
the fast transient in the PATCH configuration (not to be confused with the very
slow transient that appears in the CELL configuration, discussed in Tutorial 4.)
This can be compensated using the Cp Slow controls. The PATCH setting on
the model cell has only a very minor slower transient.
Chapter 3
26 • Tutorials
11. Now that the capacitance transients are compensated, it will be possible to
increase the amplitude of the Seal Test pulse without overloading the
MultiClamp 700B. Set the Seal Test amplitude to 100 mV by placing the
cursor over the display (10 mV), double clicking and typing 100 <Enter>.
Clear steps should now be visible on the oscilloscope, with amplitudes of about
5 mV.
Figure 3.14
With the Primary Output: Membrane Current gain set at 0.5 V/nA, this is
equivalent to 10 pA. Hence the resistance of the model patch is calculated from
Ohm’s Law to be R = V/I = 100 mV/10 pA = 10 GΩ. Alternatively, check the
Resistance checkbox under the Channel 1 meters.
Tutorial 4 – Whole-Cell Configuration: Voltage Clamp
1. Reset to Program Defaults and set Seal Test frequency to 200 Hz. Plug the
CELL connector on the model cell into the CV-7 headstage.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 27
2. Check the Seal Test checkbox; a train of ~0.5 Volt transients decaying over
~2 ms will appear on the Primary Output trace. (These are more easily seen if
the oscilloscope is triggered using the SYNC output of the MultiClamp 700B.)
Figure 3.15
The fast component of the transients reflects the simulated electrode
capacitance (5 pF), while the slow component reflects the capacitance of the
simulated cell (33 pF). Following the 10 mV Seal Test step the transients
decay to a plateau of 10 mV, equivalent to a current of 20 pA. This yields a
resistance of 10 mV/20 pA = 500 MΩ, which is the “input resistance” of the
model cell. This can also be found by checking the Resistance checkbox under
the meters.
3. In a real cell, the holding potential would have been set prior to going to wholecell mode. Set the holding potential now by checking the Holding checkbox
and using glider control to apply a negative holding potential (e.g. –60 mV).
Figure 3.16
Chapter 3
28 • Tutorials
4. We now wish to cancel the slow component of the transient, because (a) it may,
like the fast transient, saturate the headstage amplifier, and (b) this cancellation
is necessary for proper series resistance compensation (see step 8, this
Tutorial). Check the Whole Cell checkbox and use the toggle button to adjust
the capacitance (pF) and series resistance (MΩ) parameters. It will be easier to
do this while holding down the Shift key to accelerate the effect of mouse
movement.
Figure 3.17
It should be possible to compensate completely the slow transient. The optimal
values will be around 33 pF (the model cell capacitance) and 10 MΩ (the model
electrode resistance). Note that a small, fast transient may reappear after the
slow one is canceled. This can be removed by again pressing the Cp Fast Auto
button.
5. An alternative way to cancel the slow transient is by pressing the Auto button.
Try this, after first using glider control to set the pF and MΩ values to “wrong”
values, such as 100 pF and 100 MΩ. After imposing a series of voltage steps
on the model cell, the algorithm should converge on about 33 pF and 10 MΩ.
In real experiments it may be necessary to make manual adjustments for
optimum cancellation of the slow transient.
6. Press the Auto button opposite Cp Fast. This will cancel the fast component of
the transient.
The residual step, due to current flow through the “input resistance” of the
model cell, can be canceled using the Leak Subtraction feature of the
MultiClamp 700B. This subtracts from Primary Output a current that is scaled
linearly from the voltage command. (See Chapter 5, LEAK
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 29
SUBTRACTION). Check the Leak Subtraction checkbox and press the button
(or use the glider to obtain a flat trace).
Figure 3.18
The optimum value is about 500 MΩ, the “input resistance” of the model cell.
Manual adjustments of Whole Cell and Cp Fast may be necessary to perfectly
compensate the response.
Directly to the left of the Leak Subtraction button is the Output Zero button,
which provides a slightly different way of removing offsets in the Primary
Output trace. Output Zero acts like a high-pass filter, subtracting a constant DC
offset without regard to the voltage command. To illustrate its use, switch off
Leak Subtraction, and check and press Output Zero (with Holding set to a large
negative value, as described in step 3 of this Tutorial). The Primary Output
trace is baselined but unlike with Leak Subtraction, the step due to Seal Test is
not subtracted.
7. The series resistance (Rs), which typically originates near the tip of the
recording electrode, can be thought of as an unwanted resistance that is
interposed between the headstage circuitry and the membrane of the cell. Since
Rs can cause serious errors in voltage clamp mode, it needs to be reduced as
much as possible. This can be done both mechanically (e.g. by using lowerresistance electrodes) and electronically. Full details are given in Chapter 5,
but the following exercise gives a foretaste of electronic Rs compensation.
Ensure that Seal Test is running (10 mV, 100 Hz) and both Cp Fast and Whole
Cell compensation have been adjusted as at the end of step 6 above. Switch off
Output Zero and Leak Subtraction and increase Seal Test amplitude to 50 mV.
The relatively slow rise in the Primary Output current trace (~1 ms) is a
manifestation of series resistance error. The goal is to speed up this risetime
using Rs compensation.
Chapter 3
30 • Tutorials
8. Check the Rs Compensation checkbox, set Bandwidth to 5 kHz, and ensure that
the Prediction and Correction controls change together.
Figure 3.19
Using glider control, slowly advance the percent setting under Prediction or
Correction while watching the Primary Output trace on the oscilloscope. The
trace becomes noisier, the rising edge is speeded up, and a transient develops at
the rising edge. As the settings are increased beyond about 80% the transients
become larger, and then rapidly escalate into a full-blown oscillation. The art
of Rs compensation is to choose a combination of Bandwidth, Prediction and
Correction that provides maximal compensation without oscillation. Full
details are given in SERIES RESISTANCE COMPENSATION in Chapter 5.
9. The MultiClamp 700B is designed to be used with an external pulse generator
or computer to provide voltage-clamp (and current-clamp) command steps.
However, the Pulse button in the MultiClamp 700B Commander allows you to
apply simple, on-off steps with a selectable amplitude and duration.
Figure 3.20
Experiment with different pulse settings, monitoring the Primary Output trace
while repeatedly pressing the Pulse button. Note that only a discrete list of
pulse durations is allowed (seen by positioning the mouse over the duration
field and clicking the right button).
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 31
Tutorial 5 – Whole-Cell Configuration: Current Clamp
1. Reset to Program Defaults. In the Gains tab of the Options menu, select the
50 MΩ range in the Current Clamp section.
2. With the model cell in the CELL position, click Auto Pipette Offset.
3. Under Channel 1 Mode: press the button labeled IC. The tab labeled I-Clamp 1
appears, the Current Clamp light (green) on the front panel of the MultiClamp
700B unit illuminates, and Primary Output displays Membrane Potential.
4. Check the box next to “Holding”and, using glider control, vary the holding
current (pA) while viewing the Primary Output signal on the oscilloscope and
on the MultiClamp 700B Commander voltage meter. The model membrane
potential varies smoothly with Holding current.
Figure 3.21
5. Switch off Holding and check the Tuning checkbox while monitoring Primary
Output on the oscilloscope. This injects a repetitive square current pulse into
the current clamp circuit.
Figure 3.22
Chapter 3
32 • Tutorials
A sawtooth pattern appears on Primary Output (Figure 3.23). Each segment of
the sawtooth is actually an incompletely relaxing exponential.
Figure 3.23
6. Set the Tuning frequency to 50 Hz and note that, on an expanded oscilloscope
timebase, a step is visible at the beginning of each segment of the sawtooth.
This step is due to the resistance of the model “electrode”. As in the case of
whole-cell voltage clamp, electrode series resistance can introduce errors to
current-clamp recordings and needs to be compensated electronically. In
current-clamp mode, Rs is compensated using Bridge Balance.
Figure 3.24
Check the Bridge Balance checkbox and, using glider control, vary the MΩ
value until the step is eliminated. Alternatively, press the Auto Bridge Balance
button for automatic adjustment. The electrode resistance of the model cell is
10 MΩ, but in the CELL position you may record slightly higher values (near
14 MΩ) because the electrode resistance is mixed with the cell capacitance and
resistance components.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 33
To the left of Bridge Balance is the Output Zero button. This works exactly
like the corresponding button in voltage clamp, removing constant DC offsets.
7. In current-clamp mode the stray electrode capacitance can cause additional
errors, acting to filter the membrane potential signal. This error can be reduced
by using electronic compensation of the pipette capacitance.
While holding down the Ctrl key to magnify mouse movement, use glider
control to increase the Pipette Capacitance Neutralization (pF) value while
monitoring Primary Output on the oscilloscope.
Figure 3.25
Note that, as you increase the value beyond about 3 pF, damped oscillations
start to appear at the beginning of each sawtooth.
Figure 3.26
If you go further still, full-scale oscillations develop. Increase the value until
you see full oscillations, then reduce the value until your output again looks like
Figure 3.26.
Chapter 3
34 • Tutorials
8. As in the case of Rs compensation in voltage-clamp mode, the art of pipette
capacitance neutralization is to increase the neutralization as far as possible
without provoking oscillations that may be harmful to your cell (see further
details in the Reference section). But no matter how carefully you compensate
capacitance to begin your experiment, it is still possible to experience
oscillations later in an experiment because electrode properties (e.g. resistance,
junction potential) may change over time. The MultiClamp 700B provides
you with an option to protect your cell from harmful oscillations during a
current-clamp experiment by automatically disabling (or alternatively,
reducing) Capacitance Neutralization. (In a similar manner, Series Resistance
compensation can be disabled or reduced in Voltage Clamp.)
Check the box beneath Pipette Capacitance Neutralization labeled, “Disable if
oscillation detected”.
Figure 3.27
9. Now, increase the Pipette Capacitance Neutralization until you reach a value
that evokes full-scale oscillations. The automatic protection circuit will work
quickly to disable the Pipette Capacitance Neutralization, and several things
will happen:
•
To the right of the “Disable…” field, a small icon will appear briefly to
display repeated images of a sine wave that is reduced to a flat line.
•
The Pipette Capacitance Neutralization feature will be disabled (box
will become unchecked).
•
You will hear an audible tone.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 35
•
A warning message will appear to indicate the detection of oscillations
and the disabling of Pipette Capacitance Neutralization.
Figure 3.28
10. You can choose to prevent the warning message from appearing. Go to the
Options / Auto menu, and disable (uncheck) the “Display warning” feature.
Figure 3.29
Also in the Options / Auto menu, you can alternatively choose to reduce
instead of disable Pipette Capacitance Neutralization in IC mode.
Neutralization will be iteratively reduced by 1 pF steps until oscillations are no
longer detected. (Note also that this menu applies a similar reducing effect to
Series Resistance compensation if oscillations are detected in VC mode.)
11. Check on the “Reduce Rs Compensation…” radio button in the Options / Auto
menu. Close this menu, then repeat step #8 above to evoke oscillations. (Note
that the warning dialog will not be shown this time after the automatic
reduction of Pipette Capacitance Neutralization, because you have disabled
this feature in the Options menu.)
Chapter 3
36 • Tutorials
12. Turn off the Tuning pulse. Set Holding to 0 pA. Set your oscilloscope time
scale to view at least 2 seconds per sweep.
13. Below the Holding / Tuning section, see the feature labeled, “Inject slow
current to maintain potential at:” Set the potential to –100 mV, and the time
constant to 500 ms.
Figure 3.30
14. Check the box to activate the slow current injection feature, and monitor the
Primary Output Membrane Potential on the oscilloscope. You should note a
voltage deflection from 0 V to –1 V (since the scale factor is 10 mV/mV). The
deflection will require approximately 500 ms to reach steady state.
Figure 3.31
The time required to reach the selected voltage depends upon the feedback
resistor and headstage load. See the MultiClamp 700B Commander on-line
Help for more detail.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 37
15. The Pulse button in current clamp allows you to apply single current steps of
variable amplitude and duration. Experiment with different settings for Pulse
amplitude and duration while monitoring the effect on Primary Output.
Figure 3.32
16. Switch on both Holding and Tuning features. Observe Primary Output on the
oscilloscope while pressing the I=0 button.
Figure 3.33
I=0 is a special mode of current clamp in which all command inputs are
disconnected. With the model cell, the Primary Output Membrane Potential
signal returns to near 0 mV when I=0 is pressed. In a real cell the Membrane
Potential would return to the resting potential of the cell. See IMPALING
CELLS in Chapter 4 for detailed information on current clamp experiments
with real cells.
Tutorial 6 – Whole-Cell Configuration: Automatic Mode
Switching
1. Set up MultiClamp 700B as follows: Reset to Program Defaults, connect CELL
position of Patch-1U model cell (shielded and grounded) to CV-7 headstage.
2. Make the following changes in VC mode:
a. Click Auto Pipette Offset.
b. Turn on Seal Test (check box).
c. Click Auto Cp Fast.
Chapter 3
38 • Tutorials
d. Set Holding value to 20 mV, and check box to activate.
e. Change Primary Output signal to read Membrane Potential
(10 mV/mV). In a real experiment, you might consider simultaneously
recording Membrane Current on a different channel.
3. Change Mode to Current Clamp by clicking IC button. Set Tune pulse for
100 pA @ 2 Hz, then check box to activate.
4. Click on the Options / Auto tab. In the Switch to voltage clamp section, click
the radio button for On positive-to-negative Vm threshold crossing. Set Delay
change to voltage clamp = 0 ms, and Membrane Potential (Vm)
threshold = 20 mV. Next, click the radio button for Return to current clamp:
After:, and set this value to 500 ms. Close the Options menu to return to the
main IC window.
Figure 3.34
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 39
5. Monitor Primary Output on external oscilloscope. Set display for at least 2 full
seconds per sweep. You should observe a slowly charging and discharging
voltage response to the Tune current step.
Figure 3.35
6. Now check the Auto checkbox next to the Mode buttons. Note that the VC, I=0
and IC buttons are now greyed out, since they are under automatic control.
Figure 3.36
7. Monitor the Primary Output on the oscilloscope. You should now observe the
following events (see figure below):
a. A switch from IC to VC when the negative-going deflection of the
membrane potential reaches 20 mV.
Chapter 3
40 • Tutorials
b. MultiClamp 700B will remain in VC for 500 ms, then switch back to
IC. A transient due to this mode change will appear, then the potential
will begin to decay.
c. When the potential again reaches 20 mV (going from positive to
negative direction), the MultiClamp 700B will again switch from IC to
VC. This latter process will continue until the Auto mode switch is unchecked.
Switch to VC at 20 mV
Auto ON
SYNC output monitoring Mode
Figure 3.37
8. The lower trace in the figure above shows a recording from the SYNC output
on the rear of the MultiClamp 700B. In the Options / General tab, select
“Mode on channel 1” to follow the mode switching event. A deflection to 5 V
indicates VC mode, while 0 V is the output during IC mode.
9. Note: During a real experiment, if you are using an external command input to
the MultiClamp 700B (such as the output of a Digidata), then you must be
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Tutorials • 41
careful to turn OFF this external command during the Auto Mode switch. If
you do not, then the incoming command will conflict with the Auto Mode
switch settings. To disable the external commands, go to the Options / Gains
tab, and click the OFF radio button in the VC and IC External Command
Sensitivity sections.
Figure 3.38
10. Experiment with different settings (Threshold crossing, Delay, Vm, and
Return) in the Options / Auto tab, in order to appreciate the flexibility of
this automatic Mode-switching feature.
Chapter 3
Guide to Electrophysiological Recording • 43
Chapter 4
Guide to Electrophysiological Recording
The purpose of this chapter is to provide practical advice on patch clamping and
sharp microelectrode recording, both of which are possible using the MultiClamp
700B. It includes both tutorial-style guidance and technical details for reference.
This information has been distilled from textbooks on the subject (see References at
the end of this manual) and from experienced researchers working in laboratories
around the world. However, as is the case for all advice (and particularly that
pertaining to research), the suggestions given here should be taken as provisional
until they have been tested in your own circumstances.
This chapter has been divided into three parts: (1) general advice for in vitro
electrophysiology, (2) patch clamping, and (3) sharp microelectrode recording.
Chapter 4
44 • Guide to Electrophysiological Recording
General Advice
Chamber Design
The tissue chambers used in many in vitro electrophysiological experiments usually
have four main requirements:
•
•
•
•
a perfusion system for keeping the tissue alive and applying drugs
a method for keeping the tissue mechanically stable
optical properties suitable for observing the tissue and positioning
electrodes
an electrically stable bath (reference) electrode
Perfusion
Normally the external solution used in in vitro experiments is a pH-buffered salt
solution that mimics the extra- or intracellular composition of the cells under study.
Sometimes the solution is bubbled with CO2 (to maintain the pH of bicarbonatebuffered solutions) and/or O2 (to maintain the metabolic viability of the cells).
Some cells (e.g. those in retinal slices) have unusually high metabolic rates and
require fast perfusion with high-O2 solution to remain viable. Other cells (e.g.
neurons in dissociated cell culture) may not need any perfusion or bubbling at all.
Because the health of the cells is the single most important factor in determining the
success of your experiments, it is worth spending some time establishing the
optimal conditions for cell survival.
Mechanical Stability
Patch clamp recordings can be surprisingly robust in the presence of vibrations.
However, sharp microelectrode recordings are not so robust in the presence of
vibrations. Neither type of experiment is tolerant of large drifts in the tissue or
electrode that tend to pull the electrode out of the cell. For this reason, it is
important to use a good, drift-free micromanipulator for the electrode, and to secure
the tissue or cells in the chamber so they cannot move very far. Tissue slices are
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Guide to Electrophysiological Recording • 45
commonly held in place in the chamber by a weighted “net” or “grid” of fine
threads.
A grid is easily made as follows. Bend a piece of 0.2-0.4 mm diameter platinum
wire into a ring small enough to fit in the bottom of your chamber, then flatten the
wire in a vise. Using a pair of fine forceps, pull a single strand of nylon thread off a
~1 m length of unwaxed nylon dental floss. (It is very wispy but remarkably
strong.) Wrap the thread tightly in a spiral around a strip of thin black card about 3
x 10 cm, securing each end with sticky tape. Bending the card slightly, slip the
flattened platinum ring under the threads, and adjust its position and the spacing of
the threads until the optimal grid pattern is obtained. Finally, add a tiny spot of
cyanoacrylate glue to each thread crossing point and, after it is dry, cut the
completed grid free.
Optics
Again, it is difficult to generalize about the optical requirements of the chamber,
since the optical technology in use may range from a simple dissection scope to a
multiphoton microscope. In general, however, it is probably best to build a
chamber with a glass microscope coverslip forming the bottom, to ensure the best
possible optical clarity.
Bath Electrode
The simplest kind of bath electrode is a chlorided silver wire placed in the bath
solution. However, if the chloride ion concentration of the bath is altered by
perfusion during the experiment, this kind of electrode will introduce serious
voltage offset errors. In this case it is essential to use a salt bridge for the bath
electrode. (See BATH HEADSTAGE AND ELECTRODES in Chapter 5.) In any
case, it is good practice, at the end of every experiment, to check for drift in
electrode offsets. This is easily done by blowing out the patch and pressing the I=0
button on The MultiClamp 700B Commander. This will display on the meter the
pipette voltage required for zero current through the electrode. If, for instance, the
meter shows 2 mV, there has been a 2 mV drift since the electrodes were nulled at
Chapter 4
46 • Guide to Electrophysiological Recording
the beginning of the experiment, and your voltage values may be in error by at least
this amount. Large offset errors may indicate that your electrode wires need
rechloriding, or a fluid leak has developed in your chamber, causing an electrical
short circuit to the microscope.
Note: If you use both headstages on the MultiClamp 700B (e.g. for making
simultaneous recordings from pairs of cells) you may wonder whether one
or both headstage ground sockets need to be connected to the bath
electrode. We have found empirically that the noise in the recordings
depends on which headstage is grounded and what mode it is in (V-Clamp
or I-Clamp). It is helpful to have a wire connected from each headstage to
the bath electrode, with the connection able to be switched off by a toggle
switch without bumping the electrode. In this way the best grounding
configuration can be established during the experiment.
Interfacing a Computer
Because the MultiClamp 700B is a computer-controlled instrument, the installation
of a computer in your electrophysiology rig is obligatory. The minimum computer
configuration requires a USB port for communicating with the MultiClamp 700B.
However, in order to make full use of the power and convenience of your computer,
it is recommended that you also attach a digitizing interface, such as the Digidata
1322A. An interface allows you to generate command signals and save the data in
a very flexible manner, without the cost and complexity of a conventional system
based on stimulators, digital oscilloscopes, laboratory tape recorders and chart
recorders. Digitizing interfaces are typically connected to the computer via a card
(i.e., a SCSI card) that is provided with the interface. Finally, it is necessary to
install software to control the interface. Software is available from Axon
Instruments (e.g. pCLAMP) or other vendors, or you can write your own. The
beauty of the MultiClamp package is that you are not tied to any particular PC data
acquisition software. Any PC-based software that is able to control the digitizing
interface is acceptable, while the MultiClamp 700B Commander runs in the
background controlling the MultiClamp 700B.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Guide to Electrophysiological Recording • 47
Computer Noise
Digital computers can generate considerable electrical noise, both via the power
ground and via radiative interference from the monitor. For optimal noise
performance of the MultiClamp 700B, careful attention should be paid to the
placement of the computer. For example, the monitor should not be placed
immediately above or below the MultiClamp 700B in the instrument rack. Other
advice on noise reduction is given in the NOISE section of Chapter 5.
Patch Clamping
The patch clamp technique enables stable, low-resistance access to the interior of
many cell types. Once this access is established, it is up to the experimenter, of
course, whether to record in V-Clamp or I-Clamp mode. However, the discussion
in this Part will assume that V-Clamp mode is being used, at least for the initial
steps of seal formation and gaining whole-cell access. Once in the whole-cell
configuration, you can switch to I-Clamp mode. Advice on recording in I-Clamp
mode is given in the following section, “Sharp Microelectrode Recording”.
Headstage and Holder Considerations
Ensure the headstage is securely attached to the micromanipulator using one of the
mounting plates on the headstage case. Before attaching the pipette holder, or
inserting a pipette in the holder, be sure to touch grounded metal to discharge any
static charge that may have inadvertently built up on you or on the holder. Attach a
piece of flexible tubing to the suction port on the side of the holder, arranging the
tubing in such a way that it will not pull on the holder, even if you unintentionally
tug on the tubing while applying suction.
Before using the holder in a real experiment, check for leaks. Insert an unfilled
patch pipette in the holder, apply moderate suction by mouth, and then allow the
end of the tube to seal against your upper lip. The tube should remain stuck to your
lip indefinitely, were you prepared to wait. If it falls off in a few seconds, check
that the cone-washers (or O-rings) in the holder are tight.
Chapter 4
48 • Guide to Electrophysiological Recording
In patch clamping, and particularly if you are a beginner, it is very useful to have a
means of calibrating the amount of pressure or suction that is applied. This allows
you to reproducibly apply successful patch clamping strategies, or to systematically
alter unsuccessful ones. Ideally, you would attach a manometer to your suction
system. A less accurate but cheaper way is to use a 10 cc syringe. Set the syringe
at the 5 cc mark and attach it to the headstage suction tubing. The pressure in the
tubing (in millibars) is then given approximately by the formula:
Pressure (mbar) ≈ -70*x + 350
where x is the mark on the syringe to which the plunger is depressed or withdrawn.
For example, depressing the syringe to 4 (cc) will give about 70 mbar of pressure.
This formula assumes about 2 m of 1/16″ i.d. tubing is attached to the headstage
holder. Be aware that any air leaks in your system will nullify this estimate. If you
do not explicitly check for leaks, the only indication that a leak exists may be an
inability to get seals.
Some researchers prefer to apply pressure and suction by mouth. In this case, it
might be useful to roughly “calibrate your mouth” using the syringe method.
Note the following pressure conversion factors:
1 psi ≡ 70 mbar
100 mbar ≡ 75 mm Hg
The pipette holder is a potential source of electrical noise if it becomes moist. For
this reason, electrodes should be filled with solution only far enough that the end of
the holder wire or pellet is immersed. Further details are given under “Low Noise
Techniques”, below.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Guide to Electrophysiological Recording • 49
Forming a Gigaseal
Start with the MultiClamp 700B in voltage clamp mode (VC). Fill a patch pipette
with internal solution and secure it firmly in the pipette holder (fill the patch pipette
with external solution if cell-attached recording is the goal). Be sure to support the
headstage with your other hand so that the micromanipulator will not have to
absorb your force. Apply about 30 mbar of positive pressure to the holder tubing,
then lower the pipette tip into the bath. Any voltage offset between the bath
electrode and the patch electrode will show up as a non-zero tracking voltage on the
I (nA) meter of the MultiClamp 700B Commander. Press the Pipette Offset button
to null the offset. Remember that the Pipette Offset does not permanently remove
liquid junction potentials in whole-cell recordings (the liquid junction potential
returns after the whole-cell configuration is achieved).
Note: Check the stability of your bath (ground) and patch (recording) electrodes.
Drifting electrodes will cause a continual current drift off zero, indicating
that the electrodes probably need to be rechlorided.
Check the Seal Test checkbox and observe the “Primary output: Membrane
Current” on a scope; the trace should resemble the top trace in Figure 4.1. Note the
electrode resistance by checking the Resistance checkbox. Lower resistances
(2-4 MΩ) are preferred for whole-cell recording (to minimize series resistance), but
if the resistance is too low it can be difficult to obtain a gigaseal. Higher resistances
(>5 MΩ) are obviously necessary for sealing onto smaller cells or processes. Apart
from these basic rules, choice of the appropriate electrode resistance is largely a
matter of experience and experimental design.
The method of approaching the cell depends upon whether it is in a “clean”
environment (cell culture) or “dirty” environment (intact tissue). For a cell in
culture, you can maintain the positive air pressure at about 30 mbar. Lower the
pipette until it just touches the cell. As you press harder, causing dimpling of the
surface of the cell, you will see the electrode resistance increase, appearing as a
decrease in the size of the current pulse (Figure 4.1, three upper traces).
Chapter 4
50 • Guide to Electrophysiological Recording
For a cell in a piece of tissue (e.g. a brain slice) you should increase the air pressure
to about 80-120 mbar before the electrode tip touches the surface of the tissue. This
is to help the electrode punch through the surface debris. Once inside the tissue, it
may help to reduce the pressure to 30-50 mbar, so you are not simply blowing cells
away from the tip of the electrode. If you are “blind” patch clamping in a slice,
slowly advance the electrode while looking for a sudden increase in resistance,
indicating that you have encountered a cell. A slow increase probably means the tip
is becoming clogged, in which case you can try blowing it out with high pressure
before advancing again at lower pressure.
IN BATH
PUSHED AGAINST CELL
MORE PRESSURE
AGAINST CELL
GIGOHM SEAL
250 pA
2 ms
Figure 4.1 Change in resistance while forming a seal.
When you are pushed up against a cell, apply 50-100 mbar of suction (negative
pressure) to the pipette holder. At the same time, steadily increase the holding
potential towards –60 or –70 mV; doing this usually helps seal formation. There
should be a rapid increase in the resistance. Release the suction when the resistance
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Guide to Electrophysiological Recording • 51
reaches a gigohm. The resistance often continues to increase slowly over the next
several minutes.
The best gigaseals are those that form nearly instantaneously. If a seal does not
form within about a minute, continued suction is usually pointless. It is best to
change electrodes and try again.
Once the gigohm seal is established, the rectangular current pulse will disappear
entirely and be replaced by capacitance transients in synchrony with the rising and
falling edges of the command pulse (Figure 4.1, lowest trace). These can be
canceled by pressing the “Cp Fast: Auto button”. You may need to manually adjust
the capacitance (pF) and time constant (µs) parameters for optimal cancellation.
(See Chapter 3, TUTORIAL 3.) A slower component of the transients may be
reduced using the Cp Slow controls.
If you wish to remain in cell-attached mode (for example, to record single-channel
currents) you should increase the value of the feedback resistor in the headstage in
order to reduce instrument noise. (See Chapter 3, TUTORIAL 3.) This is done
under the Options button at the top of the MultiClamp 700B Commander. After
changing the feedback resistor you may need to readjust the Cp Fast and Cp Slow
settings.
If you intend to apply voltage steps to the patch, you may wish to use the Leak
Subtraction feature of the MultiClamp 700B. This subtracts a scaled (divided by
the resistance) version of the command pulse from the membrane current signal,
and is particularly intended for use at high gains where the interesting singlechannel currents are sitting on top of a leak current that may saturate the digitizing
interface. The operation of this feature is described in Chapter 3, TUTORIAL 4.
Whole-cell Voltage Clamp Recording
Obtain a gigaseal as described above. The electrode should contain a low Ca2+
solution (i.e., buffered with EGTA to ~ 100 nM) that mimics the intracellular
milieu, and the electrode resistance should be low (~3-4 MΩ). During or
immediately after seal formation, set the holding potential (Holding:) in the
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52 • Guide to Electrophysiological Recording
MultiClamp 700B Commander to the anticipated resting potential of the cell
(typically ~ –60 or –70 mV). Alternatively, the holding potential can be set in
Clampex.
A pulse of strong suction is applied to rupture the cell membrane. This can again
be done by mouth suction or by a syringe. Mouth suction tends to give the best
control. Apply brief (~0.5 s) pulses of suction, starting gently (e.g. ~80 mbar) and
increasing the suction after every couple of pulses until a large capacitance transient
suddenly appears (Figure 4.2). If you are using a 10 cc syringe, draw back on the
plunger until the capacitance transient appears, but be prepared to quickly release
the suction as soon as this occurs so the cell is not sucked up into the electrode.
The MultiClamp 700B contains a Zap circuit to aid in breaking into the cell. This
circuit delivers a pulse of 1 V DC to the patch for variable durations ranging from
0.1 to 10 ms. Start with the Zap duration at 1 ms then depress the Zap button in the
MultiClamp 700B Commander. A successful break-in will again look like that in
Figure 4.2. If the patch is not disrupted, the Zap duration can be increased and the
Zap applied a second time, and so on. Some investigators have found that the
application of moderate suction while the Zap pulse is given results in a higher
incidence of successful patch disruption. The reappearance of the original
rectangular pulse either means that you have lost the seal or that the cell does not
have a large input resistance. It is not unusual for small cells to have an input
resistance of several gigohms but with active conductances it might be as low as a
few tens of megohms.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Guide to Electrophysiological Recording • 53
SEAL
ADJUSTFASTMAG
AND FASTτ
GO WHOLE CELL
300 pA
2 ms
Figure 4.2. Going whole-cell: capacity transients observed when rupturing the patch.
After achieving stable whole-cell access, press the Auto button in the Whole Cell
section of the MultiClamp 700B Commander to compensate the whole-cell
capacitance transient. It may be necessary to manually adjust the Whole Cell pF
and MΩ values for optimal compensation, and to readjust the Cp Fast values
slightly. You should end up with a reasonably square current step, the amplitude of
which reflects the input resistance of the cell. (See Chapter 3, TUTORIAL 4.) The
Whole Cell pF and MΩ values are estimates of, respectively, the cell’s membrane
capacitance and the access resistance due to the electrode plus any resistive
contribution from the cell’s contents. The access resistance is typically about 3
times the electrode resistance, if a clean “break-in” has been achieved. Access can
sometimes be improved by applying further pulses of suction or, more dangerously,
by brief pulses of pressure.
Whenever voltage clamping in whole-cell mode, it is advisable to use Rs
compensation to minimize the voltage drop across the access resistance. A
Chapter 4
54 • Guide to Electrophysiological Recording
common mistake is to assume that this Rs error is small, so as to avoid the fiddly
process of setting Rs compensation. This is false economy. Rs errors can be
surprisingly large and can easily render your hard-won data meaningless. We
strongly recommend that Rs compensation be used, at least to convince yourself
that its use is unnecessary in your particular case. The theory and practice of Rs
compensation are described in Chapter 5, SERIES RESISTANCE
COMPENSATION.
The Leak Subtraction feature of the MultiClamp 700B allows you to subtract linear
leak currents from the membrane current traces. Generally speaking it is not a good
idea to do this in the whole-cell configuration, because whole cells may contain
background currents that have some dependence on voltage. Software packages
like pCLAMP allow a user-specified after-the-fact leakage correction, which is a
much safer procedure.
Perforated-patch Recording
With some cells it has proven nearly impossible to go whole cell without loss of
seal. If you have one of those cells, you might consider the “perforated patch”
technique. In this approach, the very tip of the pipette is filled with a normal filling
solution and the rest of the pipette is backfilled with the same filling solution to
which 120-150 µg/ml of the pore-formers Nystatin, Amphotericin B or Gramicidin
[from a stock solution of 30 mg/ml in DMSO] has been added (Rae et al., 1991;
Yawo & Chuhma, 1993). Gramicidin has lower conductance than the other two,
but it offers the advantage that it is impermeable to chloride ions, which may be
important in some applications (Ebihara et al., 1995). A cell-attached seal is then
formed on the cell. Over a 5-30 minute time period, myriad tiny cation-selective,
voltage-independent channels are inserted in the membrane patch. These channels
allow small ions to equilibrate between the cell and the pipette allowing the cell to
be voltage clamped through the open channels. Since substances as large as, or
larger than, glucose will not permeate these channels, cell contents are not washed
out as in standard whole-cell techniques. This is an advantage or a disadvantage,
depending on the experiment. A distinct advantage is the maintenance of the
intracellular environment that might influence conductances. With the perforated
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Guide to Electrophysiological Recording • 55
patch technique, a rise in whole-cell capacity transients will be observed as the
compound partitions into the cell, as shown in Figure 4.3. The Membrane Test
feature of Clampex (v. 7 and higher) allows graphically monitoring the gradual rise
in capacitance (and decrease in Rs) as pores are formed in the patch membrane.
1 MIN
2 MIN
3 MIN
4 MIN
5 MIN
400 pA
3 ms
Figure 4.3. Going whole-cell: capacity transients observed during amphotericin partitioning.
Low Noise Techniques
The MultiClamp 700B is capable of producing stable, low-noise recordings. To
realize this performance the user must pay close attention to other sources of
noise. This is because the total rms noise of a patch clamp recording is the square
root of the sum of the individual squared rms noise sources. This means that any
particular noise source that is large will dominate the total noise and make other
noise sources insignificant. Therefore, all potentially contributing noise sources
must be minimized. Specifically, the headstage, the pipette glass, the holder, and
Chapter 4
56 • Guide to Electrophysiological Recording
the seal contribute significantly even under circumstances where extraneous noise
pickup from the environment is negligible. It is absolutely crucial that the entire
preparation be properly shielded, and that hum from power supply, mains, and other
sources be negligible, i.e., < 0.1 pAp-p. (Actually, < 0.01 pAp-p is possible with some
effort.) In this section, we suggest some approaches to low-noise recording of
single channels. While these same approaches are a good idea for whole-cell
recording, they are less important since in whole-cell recording the dominant noise
source comes from the access resistance in series with the whole-cell capacitance.
Glass Type and Coating
The noise from pipette glass itself arises from the lossy characteristics of its
walls1. Therefore, it is expected that glasses with the lowest inherent dielectric
loss will have the lowest noise. Generally, the thicker the wall is, the lower the
noise will be. These expectations have been largely borne out by actual
experiments. Pipette glass can be obtained from specialty glass houses such as:
•
Clark Electromedical Instruments
P.O. Box 8, Pangbourne, Reading, RG8 7HU, England, (073) 573-888
•
Garner Glass
177 S. Indian Hill Road, Claremont, CA 91711, USA, (909) 624-5071
•
Jencons Scientific
Cherycourt Way Industrial Estate, Stanbridge Road, Leighton Buzzard
LU7 8UA, UK, (0525) 372-010
•
Sutter Instrument Company
51 Digital Drive, Novato, CA 94949, USA, (415) 883-0128
Each type of glass has unique advantages and disadvantages. Aluminosilicate
glasses have lower loss factors, but are hard to pull because of their high
1 When a sine voltage is applied across a perfect dielectric, the alternating current should be 90° out of phase with the voltage. The
deviation from 90° is the "loss factor". The loss factor is related to the power dissipated in the dielectric. Since energy is lost in the
dielectric, dielectrics (e.g., glasses) are commonly referred to as "lossy".
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Guide to Electrophysiological Recording • 57
softening temperature. High lead glasses are easier to pull, but have been
reported to modify channel currents (e.g. see Cota and Armstrong, Furman and
Tanaka, Biophysical J. 53:107-109, 1988; Furman and Tanaka, Biophysical J.
53:287-292, 1988). Since any glass may potentially modify channel currents,
one must be aware of this fact and control for it regardless of the glass one uses.
We recommend two glasses for noise-critical work: Corning #7052 and quartz.
Both have been successfully sealed to many different cell types. Quartz, with
its significantly lower loss factor, has produced the lowest noise recordings
known to us. However, because of its extremely high-softening temperature,
quartz requires a special puller like the P-2000 from the Sutter Instrument
Company.
Even if one uses electrically superior glasses, low noise will not be obtained
unless the outer surface of the glass is coated with a hydrophobic substance,
such as Dow Corning Sylgard #184. This substance prevents the bathing
solution from creeping up the outer wall of the pipette glass. This is important
since a thin film of solution on the outer surface of the glass produces a
distributed resistance that interacts with the glass capacitance to produce a noise
source that rises with frequency. Since it becomes the dominant noise source, it
must be eliminated. While many other hydrophobic substances have been used,
none, to the best of our knowledge, produces as low noise as does Sylgard
#184. Sylgard also decreases the capacitance of the pipette wall and so reduces
the lossiness of the wall as well. It has been shown experimentally that Sylgard
will improve the noise of any glass but it will not turn a poor electrical glass
into a good one. Low-loss glasses coated with Sylgard give significantly less
noise than poor glasses coated with Sylgard.
Obviously, the closer to the tip that the Sylgard can be applied, the lower the
noise. However, under some conditions a thin film of Sylgard may flow right
to the tip of the electrode, interfering with seal formation. This problem can be
reduced by using partially-cured, thickened Sylgard for coating. Alternatively,
or in addition, the tip of the electrode can be gently “polished” using a
microforge to burn off the contaminating Sylgard.
Chapter 4
58 • Guide to Electrophysiological Recording
Sylgard can be obtained from:
•
Dow Corning
2200 Salzburg, Midland, Michigan 48611, USA, (517) 496-6000
•
K. R. Anderson
2800 Bowers Avenue, Santa Clara, CA 95051, USA (800) 538-8712
•
UTSU SHOJI
Tokyo, Japan (03) 3663-5581
Headstage
The noise of the current-to-voltage circuit in the headstage depends on the
value of the feedback resistor. Larger feedback resistors generate less noise.
(See Chapter 3, TUTORIAL 3; and Chapter 5, FEEDBACK RESISTOR.) The
noise can be reduced still further by replacing the feedback resistor with a
feedback capacitor, as is done in the integrating headstage circuit of the
Axopatch 200B. This circuit was not used in the CV-7 headstage of the
MultiClamp 700B (because of technical limitations with the digital circuitry).
Therefore, for the most demanding low-noise applications it is recommended
that an Axopatch 200B is used.
Electrode Holder
The holders supplied with the MultiClamp 700B are made of polycarbonate.
Polycarbonate was experimentally found to produce the lowest noise among ten
substances tested. It was only slightly better than polyethylene, polypropylene,
and Teflon, but was much better than nylon, Plexiglass, and Delrin. Axon
holders avoid metal and shielding, which are noise sources. Holders, however,
do become a significant noise source if fluid gets into them. Therefore, great
care must be taken in filling pipettes with solution. They should be filled only
far enough from the tip so that the end of the internal chlorided silver wire or
silver/silver chloride pellet is immersed. Any solution that gets near the back of
the pipette should be dried with dry air or nitrogen to keep it from getting into
the holder. Holders that become contaminated with solution should be
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Guide to Electrophysiological Recording • 59
disassembled and sonicated in ethanol or pure deionized water, and allowed to
dry thoroughly before being used again. It is also a good idea to periodically
clean the holders by sonication even if no fluid has been observed in them.
Seal
The seal will usually be the dominant noise source if it is only a few gigohms.
Seal resistances in excess of 20 GΩ must be obtained if exceptionally low noise
single-channel recordings are to be routinely achieved. The noise depends also
on the depth of the pipette tip below the surface of the bathing solution since
the effective pipette capacitance increases as the depth of immersion increases.
The voltage noise of the headstage interacts with the pipette capacitance to
produce a noise source that rises with frequency. In order to minimize noise
when recording from excised membrane patches, the electrode tip should be
lifted until it is just under the surface of the bathing solution.
Signal Generator
One last potential noise source to consider is the noise in the signal generator
that provides the command. In the MultiClamp 700B we have succeeded in
minimizing this noise by heavily attenuating the external command. However,
it is possible for this noise source to be significant, particularly if the command
signal comes from a D/A converter.
Sharp Microelectrode Recording
The CV-7 headstage of the MultiClamp 700B contains both an Axopatch-like
current-to-voltage converter and an Axoclamp-like voltage follower circuit. The
former is activated when VC (V-Clamp) mode is selected in the MultiClamp 700B
Commander, the latter when I=0 or IC (I-Clamp) mode is selected. Although the
I-Clamp circuit is designed to be used with high-resistance sharp microelectrodes, it
can also be used with lower-resistance patch electrodes, which in some cases offer
advantages. (See next paragraph.) In this chapter it will be assumed for the most
part that sharp microelectrodes are being used for the I-Clamp recording. However,
Chapter 4
60 • Guide to Electrophysiological Recording
some of the general advice about I-Clamp recording applies equally well to patch
electrodes.
Sharp Microelectrode or Patch Electrode?
The type and resistance of the electrode will depend on the particular application,
and ultimately on personal preference, but there are a few points that should be
considered.
Patch pipettes offer some advantages over intracellular micropipettes. First, the
recording configuration is often more mechanically stable. Second, stable
recordings can be obtained with patch pipette resistances one to two orders of
magnitude lower than those of micropipettes.
This second point is most important and a number of benefits accrue. Due to its
low resistance, a patch pipette used for voltage recording will have a better
frequency response and lower noise level than a micropipette. Furthermore, the tip
potential of high resistance intracellular micropipettes is often unstable and can
change erratically as the cell is penetrated. In contrast, the tip (or junction)
potential of patch pipettes is stable and can be accurately measured and corrected
for electronically.
There are some instances where micropipettes may be more useful. If your study
requires that the contents of the cell remain relatively intact (second messenger
systems, for example), then patch pipettes may not be appropriate since the
diffusible cellular components will eventually become diluted. In such cases you
may wish to consider the “perforated patch” technique that prevents the loss of
large intracellular molecules to the patch pipette (see Patch Clamping, above).
Finally, for some cell types (e.g. those tightly wrapped in glial cells or connective
tissue) it simply may not be possible to obtain gigohm seals with patch electrodes.
Microelectrode Properties
Users of sharp microelectrodes spend far more time than patch clampers worrying
about the properties of their electrodes. This is because the higher resistance of
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Guide to Electrophysiological Recording • 61
sharp microelectrodes may introduce a number of undesirable properties. For best
results, the microelectrode voltage must settle rapidly after a current pulse, and the
microelectrode must be able to pass current without large changes in resistance.
The important factors that need to be considered are discussed below.
Electrode Glass
Borosilicate glass is often used; however, through trial and error one type of
glass supplied by a specific glass manufacturer may have been shown to yield
the best results. It is suggested that the literature be consulted prior to selecting
glass for recording.
Tip Resistance
Tip resistance (Re) should be as low as possible and consistent with good
impalements of the cell. Low values of Re allow for greater stability and faster
settling time of the microelectrode.
Stability
Re of most microelectrodes changes with time and with current passing. Re
is affected not only by the magnitude of the current but also by its polarity.
In general, microelectrodes of lower resistance are more stable during
current passing than those of higher resistance.
Settling time
The decay time constant of the microelectrode voltage after a current pulse
depends strongly on Re. Thus, lower Re values produce faster settling
times. As well, high Re values are sometimes associated with a slow final
decay even after the electrode capacitance has been eliminated. (See next
page.)
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62 • Guide to Electrophysiological Recording
Microelectrode Capacitance
The settling time of a microelectrode depends not only on Re but also on the
transmural capacitance (Ct) from the inside of the microelectrode to the external
solution. For fastest settling, Ct must be as small as possible. Ct is usually 1-2 pF
per mm of immersion. In order to reduce the effect of Ct, two approaches may be
taken. One is to electronically compensate Ct using the Pipette Capacitance
Neutralization control in the MultiClamp 700B Commander. This is discussed
below, in the section on “Impaling Cells”. The other approach is to minimize the
problem by careful experimental design, as follows.
In an isolated preparation, lowering the surface of the solution as far as possible
can reduce Ct. For a long slender microelectrode, 200 µm or less is regarded as
a low solution level; 500 µm is tolerable. Deep is regarded as 1 mm or more.
For a microelectrode that tapers steeply (i.e. a stubby microelectrode) deeper
solutions can be used with less loss of performance. When working with very
low solution levels there is a risk of evaporation exposing the cells to the air
unless a continuous flow of solution is provided across or through the
preparation. If evaporation is a problem, try floating a layer of mineral oil on
the surface of the solution. If used, this layer of oil will have the additional
advantage of automatically coating the microelectrode as it is lowered into the
solution.
Precautions must be taken to prevent surface tension effects from drawing a
thin layer of solution up the outer wall of the microelectrode. If this film of
saline is allowed to develop, Ct will increase substantially. Because the film of
saline has axial resistance the contribution to Ct will be very nonlinear, and the
voltage decay after a current pulse will either be biphasic or slow, even when
capacitance neutralization is used. To prevent the saline film from developing,
the microelectrode should be coated with a hydrophobic material. This can be
done just before use by dipping the filled microelectrode into a fluid such as
silicone oil or mineral oil. Another method is to coat the microelectrode with
Sylgard #184 or Q-dope (model airplane glue). The selected material should be
painted onto the electrode to within 100 µm of the tip.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Guide to Electrophysiological Recording • 63
Tip Potentials
During the passage of current, a slowly changing voltage may be generated at the tip
of a microelectrode. Changes in this tip potential are indistinguishable from changes
in the membrane potential and can therefore be a serious source of error.
Identifying Tip Potentials
•
While the microelectrode is outside the cell, press the Pipette Offset
button to zero the offset. In IC mode, pass a constant current into the
bath for about 10 seconds; this can be done by setting a Holding current
in the MultiClamp 700B Commander and checking the Holding
checkbox. The current magnitude should be the same as the maximum
sustained current likely to be passed during the experiment. When the
current is switched off the recorded potential should return to zero
within a few milliseconds at most. Some microelectrodes either return
very slowly to zero potential, or not at all. These micropipettes should
be discarded.
•
While the experiment is in progress, occasionally check the resistance
of the microelectrode. Changes in tip potential are usually
accompanied by changes in microelectrode resistance.
Preventing Tip Potentials
Not much can be done to prevent tip potentials from changing but the
following may be helpful.
•
Sometimes the slow changes in tip potentials are worse when a AgCl
pellet is used instead of a Ag/AgCl wire. Some holders are acceptable
while other, ostensibly identical, holders are not. Therefore holders
should be tested and selected.
•
The variability of the tip potentials may be related to pressure
developed when the microelectrode is pressed into an unvented holder.
Chapter 4
64 • Guide to Electrophysiological Recording
The suction port on the HL-U series holders provided with the
MultiClamp 700B should therefore be left open.
•
Using filling solutions with low pH, or adding small concentrations of
polyvalent cations like Th4+, may reduce the size of the tip potential and
therefore the magnitude of any changes (Purves, 1981).
Filling Solutions
The best filling solution to use depends on the preparation under investigation and
the experience of the investigator. Although KCl gives one of the lowest tip
resistances for a given tip diameter, a KCl-filled electrode is not necessarily the
fastest to settle after a current pulse. K-citrate is sometimes faster.
It is important to be aware that during current-passing, large amounts of ions from
inside the microelectrode can be ionophoresed into the cell. For example, if current
is passed by the flow of ion species A from the microelectrode into the cell, then
after 50 seconds of current at 1 nA (or 1 s of current at 50 nA) the change in
concentration of A inside a cell 100 µm in diameter is 1 mM. If A is an
impermeant ion, the cell may swell due to the inflow of water to balance the
osmotic pressure. The injection of a permeant ion, such as chloride, can
significantly alter the equilibrium potential for that ion.
Impaling Cells
Start with the MultiClamp 700B in IC mode (I-Clamp). Fill a microelectrode with
internal solution and secure it firmly in the pipette holder. Be sure to support the
headstage with your other hand so that the micromanipulator will not have to
absorb your force. Advance the electrode until its tip enters the bath. Press the
Pipette Offset button to null the offset.
Note: Check the stability of the bath electrode and microelectrode. Drifts in
Primary output: Membrane Potential indicates that the electrode wires
probably need to be rechlorided. Also check for a changing tip potential by
passing a steady current, as described above.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Guide to Electrophysiological Recording • 65
Check the Tuning checkbox and observe the Primary Output: Membrane Potential
on a scope. Move the electrode tip close to where cells are likely to be encountered,
and then increase Pipette Capacitance Neutralization in the MultiClamp 700B
Commander to give the fastest step response. It is advisable to adjust the
capacitance neutralization with the microelectrode as close as possible to the final
position, since moving the electrode can change Ct and invalidate the setting. It
may be wise to slightly under-compensate, because changes in the solution level
could lead to oscillations that may destroy the cell.
Press the Bridge Balance button. The value (MΩ) found for optimal balance gives
the resistance of the electrode. See Chapter 5, BRIDGE BALANCE, for further
details.
Sometimes the cell is impaled as soon as the microelectrode is pressed against the
cell surface. More often the microelectrode is advanced until there is a slight
deflection in the tip potential. At this point the cell can be impaled by pressing the
Buzz button or the Clear +/Clear - buttons. If these fail, vibrating the
microelectrode tip by lightly tapping on the micromanipulator sometimes works.
When the electrode penetrates the cell there is a sudden change in the Membrane
Potential trace, reflecting the intracellular potential. The voltage response to the
Tuning steps will be slower and much larger, reflecting the membrane time constant
and input resistance. After impaling the cell, it is often helpful to back off the
microelectrode slightly and allow the penetration to stabilize for a few minutes. For
some cells it may help to apply a small DC current to the electrode (enough to
produce several mV hyperpolarization) during the penetration. Selecting the
Holding checkbox and slowly increasing the Holding value can apply this DC
current.
Once the penetration has stabilized, you should recheck the Bridge Balance and
Pipette Capacitance Neutralization. Further details on this are given in Chapter 5.
It is sometimes useful to inject a small, brief current pulse at the start of each sweep
of data collection in order to continually check the Bridge Balance setting during
the course of an experiment.
Chapter 4
Reference Section • 67
Chapter 5
Reference Section
It is expected that the MultiClamp 700B Commander On-line Help will answer
many questions about the operation of the MultiClamp 700B. This chapter
provides details of the theory and operation of the MultiClamp 700B, beyond what
is available in the On-line Help. The information in this section is gathered under a
number of broad topics, arranged in alphabetical order. Because the MultiClamp
700B is effectively two instruments in one (an Axopatch-1D and an Axoclamp 2B),
the topics are sometimes divided into two sections, or refer to only voltage clamp or
current clamp mode.
Please consult the Index if you are having trouble locating a particular item.
Note:
Before using this chapter, it may be helpful to first read the entry under
“Polarity Conventions”. This summarizes the conventions used for the
polarities of currents and voltages in all amplifiers manufactured by Axon
Instruments.
Chapter 5
68 • Reference Section
Audio Monitor
•
Used for audio monitoring of an electrical signal.
•
The Audio control panel is accessed via the toolbar button
.
The Audio Monitor provides auditory feedback for a user-selectable signal
(Membrane Current or Potential on Channel 1 or 2). This is sometimes useful
while attempting to seal onto or impale a cell, since it obviates the need to look at
an oscilloscope while manipulating the electrode.
One of two Audio Modes can be selected.
•
Direct Signal Monitoring. The selected signal is relayed directly to the output
speaker. This mode is especially useful for monitoring spikes, which are heard
as audible clicks.
•
Voltage Controlled Oscillator (VCO). The voltage of the signal determines the
frequency of a sine wave that is then directed to the output speaker. This is
useful if the signal of interest is a DC signal, e.g. the membrane potential. The
default setting for the VCO is 2200 Hz at 0 V ranging to 300 Hz at -100 mV.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 69
Audio output can be monitored by making connections to the MultiClamp 700B in
one of three different ways:
1. Connect the rear panel AUDIO OUTPUT to the Line IN connector of your
computer sound card. This allows the MultiClamp 700B to use the computer’s
speaker.
Figure 5.1. Possible Audio configuration #1.
Chapter 5
70 • Reference Section
2. Connect headphones or remote powered speakers to the front panel PHONES
output or the rear panel AUDIO OUTPUT. This allows dedicated use of the
headphones or external speakers by the MultiClamp 700B.
Figure 5.2. Possible Audio configuration #2.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 71
3. Connect the Line OUT of your computer sound card to the rear panel AUDIO
INPUT of the MultiClamp 700B, and the rear panel AUDIO OUTPUT to
external powered speakers. This is the same as option 2, except that now the
MultiClamp 700B audio output is mixed with the computer’s audio output to
external speakers.
Figure 5.3. Possible Audio configuration #3.
Never connect the computer’s microphone jack to Audio
connectors on the MultiClamp 700B. This could lead to large voltages being
sent to the MultiClamp 700B, with the possibility of causing damage to its
circuitry.
WARNING:
Chapter 5
72 • Reference Section
Bath Headstage and Electrodes
The Bath Headstage is used when recording from cells with a large conductance, in
order to minimize errors due to current flow through the bath electrode. The VG-2
series Bath Headstage is optional hardware that can be used with the MultiClamp
700B for this purpose.
In most experiments, the bathing solution is grounded by a solid grounding
electrode (such as an agar/KCl bridge) and all measurements are made relative to
the system ground (on the assumption that the bath is also at ground). This
assumption may not be true if the Cl- concentration or the temperature of the
bathing solution is significantly changed, if there is restricted access from the
extracellular space to the grounding point, or if the membrane current is sufficiently
large as to cause a significant voltage drop across the resistance of the grounding
electrode. The latter circumstance, which normally arises only when voltage
clamping large cells with large membrane currents, is the situation for which the
bath headstage is intended.
In a simple voltage clamp setup, the voltage drop across the resistance of the bath
grounding electrode (Rb) is indistinguishable from the membrane potential. That is,
the potential recorded by the microelectrode is the sum of the transmembrane
potential (Vm) and the voltage drop across Rb. Problems arise if the product of the
clamp current (I) and Rb is significant. For example, for I = 5 µA and Rb = 2 kΩ,
the voltage error is 10 mV.
To minimize this problem with the MultiClamp 700B, the following two strategies
can be adopted.
Rb Minimization
There are three main contributors to Rb:
•
The cell access resistance from the membrane surface to the bath
•
The resistance of the grounding pellet
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 73
•
The resistance of the agar bridge (if used)
Typical values of the access resistance of a 1 mm diameter sphere in Ringer's
solution (such as an oocyte) are on the order of 150-200 Ω. This is a given, and no
amount of manipulation can alter this for a given set of experimental conditions;
fortunately it is relatively small. On the other hand, the combined resistance of the
grounding pellet and agar bridge are larger, but one can take precautions to
minimize them. A 1 mm diameter Ag/AgCl pellet in Ringer's solution has a
resistance of 300-600 Ω, depending on how much of the surface is in contact with
the saline. The larger the surface area in contact with the saline, the smaller the
resistance.
The resistance of an agar bridge depends on the length and diameter of the bridge,
as well as its contents (i.e. Ringer's Solution versus 3 M KCl). For a 1 cm long
bridge:
Ringer's
3 M KCl
1 mm diameter
10.2 kΩ
510 Ω
2 mm diameter
2.6 kΩ
130 Ω
Therefore, to minimize Rb, it would be best to eliminate the agar bridge and ground
the preparation directly with a Ag/AgCl pellet. The pellet should be as large as
practical, and the area of it in contact with the solution should be maximized. With
this kind of bath electrode, you should avoid perfusing the bath with solutions
containing different chloride activities. The DC offset of an Ag/AgCl pellet
changes with chloride activity.
In order to minimize Rb when using an agar bridge, it is best to fill the bridge with
3 M KCl instead of Ringer's solution. When the agar bridge is filled with 3 M KCl,
the sum of all components of Rb will be approximately 1-2 kΩ. If leakage of KCl
from the agar bridge is a problem, it may be necessary to fill the agar bridge with
Ringer. In this case, Rb will be several kilohms.
Chapter 5
74 • Reference Section
Use of a Bath Headstage
Another method for minimizing the effect of the voltage drop across Rb is to
actively control the bath potential, measured near the outside surface of the cell.
This is achieved using a virtual-ground circuit, the bath headstage.
The MultiClamp 700B is compatible with one of the following bath headstages
from Axon Instruments: VG-2-x0.1 and VG-2A-x100. These headstages attach to
the MultiClamp 700B via the rear-panel 15-pin D connector.
The basic design of both types of headstage is illustrated in Figure 5.4.
Figure 5.4. Bath headstage.
One electrode (SENSE) is a voltage-sensing electrode. It is placed in the bath near
the cell surface. It is connected to the virtual-ground circuit by an agar bridge or
similar, of resistance Rb2. Since there is no current flowing through this electrode,
there is no voltage drop across Rb2. The other electrode (IBATH), with resistance
Rb1, is also placed in the bath. This electrode carries the ionic current. The
feedback action of the operational amplifier ensures that the potential at the SENSE
electrode is equal to the potential at the positive input, i.e. 0 mV, irrespective of the
voltage drop across Rb1.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 75
Bridge Balance
•
Used to subtract voltage drops across the microelectrode when in I-Clamp
mode.
•
button in the Bridge
Bridge balance is activated by pressing the
Balance box in the I-Clamp pane or by checking the checkbox and using
manual glider control.
•
See also Capacitance Neutralization.
In some experiments it may be desired to inject a current (I) into a cell in currentclamp mode, e.g. to depolarize the cell and evoke action potentials. The flow of I
through the microelectrode produces a voltage drop across the electrode that
depends on the product of I and the microelectrode resistance (Re). This unwanted
IRe voltage drop adds to the recorded potential. The Bridge Balance control can be
used to balance out this voltage drop so that only the membrane potential is
recorded. The term “Bridge” refers to the original Wheatstone Bridge circuit used
to balance the IR voltage drop and is retained by tradition, even though operational
amplifiers have replaced the original circuitry.
The technique is illustrated schematically in Figure 5.5A. A differential amplifier
is used to subtract a scaled fraction of the current I from the voltage recorded at the
back of the microelectrode, Vp. The scaling factor is the microelectrode resistance
(Re). The result of this subtraction is thus the true membrane potential, Vm.
Chapter 5
76 • Reference Section
Figure 5.5B shows how bridge balance is done in practice. When the current is
stepped to a new value (top), there is a rapid voltage step on Vp due to the ohmic
voltage drop across the microelectrode (middle). Following this instantaneous step,
there is a slower rise in Vp largely due to the membrane time constant of the cell.
Correct adjustment of the bridge amplifier removes the instantaneous step, leaving
the corrected Vm trace (bottom). Although this adjustment is done with a step
current injection, the correction remains valid for any arbitrary waveform of
injected current, provided the microelectrode maintains a constant resistance.
Figure 5.5. Schematic bridge balance circuit and adjustment procedure.
Bridge Balance in the Bath
Some investigators like to set Bridge Balance in the bath, before attempting to
impale cells. This is to make it easier to see when a cell has been penetrated.
Check the Tuning checkbox and set the parameters to -1 nA and 50 Hz. Observe
the Membrane Potential on Primary Output. Press the Auto Bridge Balance button;
the fast voltage steps seen at the start and finish of the current step should be
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 77
eliminated. You may need to manually adjust the Bridge Balance MΩ value for
optimum balance. The MΩ value is the resistance of the electrode.
Bridge Balance in the Cell
The Bridge Balance should be frequently checked when inside a cell, because the
electrode resistance can drift. While setting Bridge Balance, Pipette Capacitance
Neutralization should also be set. (See Capacitance Neutralization.) Both settings
can be monitored continuously through the experiment by injecting a small current
step near the beginning of each data sweep.
It is recommended that Pipette Capacitance Neutralization be set at the same time
as Bridge Balance, because both the electrode capacitance and the electrode
resistance cause errors if left uncompensated. Also, it is easier to correctly balance
the bridge when electrode capacitance is minimized, because the “break” between
the rapidly decaying voltage across the microelectrode and the slowly decaying
voltage across the cell’s membrane resistance is more distinct.
The balancing procedure is the same as in the bath, except that the trace appears
more rounded because of the time constant of the cell membrane. Because the
Tuning pulse width is typically brief compared with the membrane time constant,
the voltage response looks like a series of ramping straight lines. To make it easier
to see the fast voltage step in Vp on an oscilloscope (Figure 5.5B), it is
recommended that the scope input be AC coupled to remove the resting membrane
potential from the signal. The scope gain can then be turned up without the
annoying offset. The MΩ value found by Bridge Balance is the resistance of the
electrode, which may be slightly higher than the value in the bath because of partial
blockage of the tip during penetration.
The residual transient at the start and finish of the current step is due to the finite
response speed of the microelectrode, which is determined in part by the
capacitance of the electrode. The transient can be minimized by correctly setting
the Pipette Capacitance Neutralization control. (See Capacitance Neutralization.)
Adjust Pipette Capacitance Neutralization for the most rapid decay without causing
an overshoot. (See Figure 3.27, Chapter 3.)
Chapter 5
78 • Reference Section
Buzz
•
Used as an aid for cell impalement or for clearing electrodes.
•
Buzz is activated by pressing the
•
See also Clear.
button in the I-Clamp pane.
Buzz works by briefly applying a 15 Vp-p 10 kHz filtered square wave to the
neutralization capacitor.
Depending on the microelectrode and the preparation, this method can aid in
clearing blocked electrode tips. When used while the tip of the microelectrode is
pressing against the membrane, Buzz may also cause the micropipette to penetrate
the cell. The exact mechanism is unknown, but it may involve attraction between
the charge at the tip of the electrode and bound charges on the inside of the
membrane.
The duration of the Buzz oscillation is set by the user (50 µs-500 ms). The
frequency of the oscillation is 10 kHz. For some small cells a long duration Buzz
can be deadly. An appropriate duration can be found for most cells that is
sufficiently long to allow penetration of the membrane but short enough that the
cell is not damaged after penetration.
Capacitance Compensation
•
Used to compensate electrode and cell capacitance when in V-Clamp mode.
•
Electrode capacitance is compensated using the
controls in the
V-Clamp pane.
•
Cell capacitance is compensated by checking the
using the associated controls in the V-Clamp pane.
•
See also External Command Inputs, Series Resistance Compensation.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
checkbox and
Reference Section • 79
Electrode Capacitance Compensation
When a voltage-clamp step is applied to an electrode, the clamp must provide a
spike of current at the start (and finish) of the step to charge (and discharge) the
capacitance of the electrode (Cp). The main problem with these spikes is that they
may saturate the headstage circuit or later circuits, leading to distortion of the
signals of interest. Injecting into the input of the headstage a current that directly
charges the electrode capacitance, bypassing the normal voltage clamp circuitry,
solves this problem. Thus, when the compensation is correctly adjusted, the charge
The MultiClamp 700B Commander provides two electrode compensation controls,
and discharge of the electrode capacitance is invisible to the user.
I C1
C1 = 1 pF
Rf
OPEN
CIRCUIT
Rp
-
I
Cp
Ip
+
Vp
FAST τ
10 Vp
+
FAST MAG
SLOW τ
+
+
+
SLOW MAG
Figure 5.6. Pipette capacitance compensation circuit.
Chapter 5
80 • Reference Section
Cp Fast and Cp Slow. Cp Fast compensates that part of the electrode capacitance
that can be represented by a lumped capacitance at the headstage input. This is the
major part of Cp. A small amount of Cp can only be represented as a capacitor with
a series resistance component. This takes longer to charge to its final value and is
compensated by the Cp Slow controls.
A simplified description of the fast and slow compensation circuitry is shown in
Figure 5.6. When the pipette command potential (Vp) changes, current Ip flows into
Cp to charge it to the new potential. If no compensation is used, Ip is supplied by
the feedback element (Rf) resulting in a large transient signal on the output (I). By
properly setting the fast and slow magnitude and τ controls, a current (IC1) can be
induced in capacitor C1 (connected to the headstage input) to exactly equal Ip. In
this case Rf needs to supply no current and there is no transient on the output.
Whole-Cell Capacitance Compensation
When in whole-cell mode, a voltage-clamp step must charge not only the electrode
capacitance, but also the capacitance of the cell (Cm). The decay time constant of
the whole-cell capacitance transient is determined by the product of Cm and the
resistance in series (Rs) with Cm. If Rs and Cm are both reasonably large, the
resultant capacitance transient can last for several milliseconds, perhaps distorting
the rising phase of biologically interesting currents. Furthermore, as in the case of
the electrode capacitance transient, the whole-cell transient may saturate the
circuitry of the MultiClamp 700B or downstream instruments if left
uncompensated. Finally, whole-cell capacitance compensation is necessary for
series resistance compensation. For all of these reasons, it is desirable to
electronically compensate the capacitance of the cell.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 81
Like electrode capacitance compensation, whole-cell compensation uses a circuit to
inject current directly into the input of the headstage. Figure 5.7 shows a simplified
schematic of this circuit.
Figure 5.7. Whole-cell capacitance compensation circuit.
Assume that the fast and slow electrode compensation controls have already been
set to compensate for Cp. By appropriately adjusting the SERIES RESISTANCE
and WHOLE CELL CAP values in this circuit, the current injected through C2 will
supply the transient membrane current (I). These adjustments do not alter the time
constant for charging the membrane. Their function is to offload the burden of this
task from the feedback resistor, Rf. In many cells, even a small command voltage
(Vc) of a few tens of millivolts can require such a large current to charge the
membrane that it cannot be supplied by Rf. The headstage output saturates for a
few hundred microseconds or a few milliseconds, thus extending the total time
Chapter 5
82 • Reference Section
necessary to charge the membrane. This saturation problem is eliminated by
appropriate adjustment of whole-cell capacitance compensation. This adjustment is
particularly important during series resistance correction since it increases the
current-passing demands on Rf . By moving the pathway for charging the
membrane capacitance from Rf to C2, the series resistance compensation circuitry
can operate without causing the headstage input to saturate. (See also Chapter 5,
SERIES RESISTANCE COMPENSATION.)
The effect of transferring the current-passing burden from Rf to C2 is illustrated in
Figure 5.8.
Figure 5.8. Using the injection capacitor to charge the membrane capacitance.
After perfect whole-cell compensation is applied, the current to charge the
membrane capacitor is removed from the IRf trace and only the steady state current
remains. All of the transient current appears in the IC2 trace. (The IC2 trace in the
figure was recorded using an oscilloscope probe connected to the internal circuitry).
The Membrane Current and Command Potential outputs on the MultiClamp 700B
would look like the IRf and Vc traces, respectively (Figure 5.8). It is easy to
mistakenly think that the time course for charging the membrane is very fast but
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 83
this is clearly not the case. Use of an independent electrode in the cell would show
that the cell charging rate is not affected by these adjustments.
The pF and MΩ values found by the MultiClamp 700B Commander for optimal
whole cell compensation provide estimates of the cell capacitance and the series
resistance, respectively. However, these estimates are accurate only if the cell input
resistance is significantly greater than Rs.
Auto Button
When the Auto button is pressed to automatically compensate Cp or Whole Cell
capacitance, the MultiClamp 700B Commander applies a series of brief voltage
pulses to the electrode and uses the Membrane Current response to optimize the
compensation. The parameters used in this optimization can be set in the
Options/Advanced pane. We recommend setting the pulse amplitude to be as large
as possible without causing damage to the cell. The amplitude can be positive or
negative (default is –50 mV).
The Whole Cell Window Width is the duration of the window (in multiples of Tau,
the fitted time constant of the whole cell transient) over which the algorithm
optimizes whole cell compensation. The best setting depends on the cell type and is
best found by trial and error. As a general rule of thumb, 1 x Tau works best for
large cells with a highly distributed capacitance and 10 x Tau works best for small,
compact cells (default 8 x Tau).
Manual Adjustment of Capacitance Compensation
Although the algorithm used by the Auto button is reasonably robust, and is likely
to work under most circumstances, it may sometimes be necessary to manually
adjust the Cp Fast/Slow or Whole Cell compensation. This is done by using the
dual controls,
, or by entering values directly. It is recommended that you
practice using these controls with the PATCH-1U model cell. The best strategy is
to first set the capacitance (pF) value to roughly what is expected (i.e. ~5 pF for
electrode capacitance, ~30-100 pF for whole-cell capacitance) and then to adjust the
time constant (µs) or resistance (MΩ) values, respectively, for optimal
Chapter 5
84 • Reference Section
compensation. After these approximate values have been established, iterative
adjustment using
becomes easier.
Filtering the Command Stimulus
Under some conditions, such as when very large voltage clamp steps are applied,
the capacitance transients cannot be fully compensated and the amplifier may still
saturate. Under these conditions it may be helpful to reduce the size of the
capacitance transient by slowing down the voltage clamp command step. This can
be achieved by filtering the command stimulus before it is applied to the cell. This
filtering can be done within the MultiClamp 700B. (See Chapter 5, EXTERNAL
COMMAND INPUTS.)
Capacitance Neutralization
•
Used to partially cancel the effects of microelectrode capacitance in I-Clamp
mode.
•
This control is adjusted in the
I-Clamp pane.
•
See also Bridge Balance.
field in the
Input Capacitance
The capacitance (Cin) at the input of the headstage amplifier is due to the
capacitance of the amplifier input itself (Cin1) plus the capacitance to ground of the
microelectrode and any connecting lead (Cin2). Cin combined with the
microelectrode resistance (Re) acts as a low-pass filter for signals recorded at the tip
of the microelectrode. For optimal performance at high frequencies this RC time
constant must be made as small as possible.
Two techniques may be used to increase the recording bandwidth.
•
Use microelectrodes with the lowest possible resistance compatible with stable
recording, and take steps to minimize the contribution to Cin by the capacitance
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 85
of the microelectrode. In practice, this means using patch electrodes where
possible, or using sharp microelectrodes with minimal capacitance. (See
Chapter 4, SHARP MICROELECTRODE RECORDING).
•
Electronically neutralize Cin.
The second approach has been implemented in the MultiClamp 700B in two ways.
Primary Method for Neutralizing Cin
A special technique is used in the CV-7 headstage to keep the contribution to
Cin from the input amplifier as small as possible. The technique is known as
“bootstrapping”. Unity gain feedback is used to reduce the component of stray
capacitance that exists between the amplifier input and its power supplies and
case. Sophisticated circuitry is used to superimpose the unity-gain output of the
buffer amplifier back onto its own power supplies and the headstage case,
fixing the voltage drop across Cin1 to a constant value, thereby preventing
current flow through Cin1. The effective value of Cin1 is thus reduced to well
below its real value. This eliminates the high-frequency current loss through
the power supply capacitance, thereby increasing the bandwidth. Since the
power supply capacitance is present whether or not the power supply is
bootstrapped, there is no noise penalty due to implementing this technique.
Secondary Method for Neutralizing Cin
In some cases the steps discussed above may not be sufficient to decrease the
RC time constant of the voltage-recording microelectrode, particularly in
situations where high resistance microelectrodes must be used. For this reason
an effective, though less desirable, technique is provided that can electrically
reduce the effective magnitude of Cin2. The technique is known as “capacitance
compensation”, “negative capacitance” or “capacitance neutralization”. A
compensation amplifier at the output of the unity gain buffer drives a current
injection capacitor connected to the input. At the ideal setting of the
compensation-amplifier gain, the current injected by the injection capacitor is
exactly equal to the current that passes through Cin2 to ground.
Chapter 5
86 • Reference Section
Adjusting Capacitance Neutralization
Check the Tuning checkbox and choose amplitude (nA) and frequency (Hz)
parameters that result in a sawtooth pattern of about 10 mV amplitude on “Primary
Output: Membrane Potential”. Carefully increase the Pipette Capacitance
Neutralization value until overshoot just starts to appear in the step response. This
is easiest to see if you have already adjusted Bridge Balance. (See Chapter 5,
BRIDGE BALANCE.) If you go too far the overshoot may become a damped
oscillation, which may escalate into a continuous oscillation, killing the cell.
Sometimes the overshoot is difficult to see. In this case, you may prefer to look at
the “Primary Output: Membrane Potential” trace at high gain on an oscilloscope,
advancing the Pipette Capacitance Neutralization value until the trace becomes
noisy and oscillations seem imminent. It is usually prudent to reduce the Pipette
Capacitance Neutralization setting slightly from the optimal, in case the capacitance
changes during the experiment.
Limitations of Capacitance Neutralization
Use of capacitance neutralization is less desirable than physically minimizing Cin2,
since the neutralizing circuit adds noise to the voltage signal. This noise has been
minimized in the CV-7 headstage of the MultiClamp 700B by using low-noise
amplifiers and small injection capacitors, but it is still significant.
It is important to recognize that the capacitance neutralization circuit is not more
than 90% effective even for ideal microelectrodes. This is because of the finite
frequency responses of the headstage amplifiers and the capacitance neutralization
circuit, and also because Cin2 does not behave ideally as a linear lumped capacitor.
Consequently, the amount of Cin2 that the circuit must neutralize should be kept as
small as possible. (See Chapter 4, SHARP MICROELECTRODE RECORDING.)
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 87
Clear
•
Used to clear blocked microelectrodes and to assist in impaling cells in I-Clamp
mode.
•
Clear is operated by alternately pressing the
the I-Clamp pane.
•
See also Buzz.
and
buttons in
Clear is used to pass large amounts of current down the microelectrode. Plus (+)
and minus (-) correspond to depolarizing and hyperpolarizing currents, respectively.
Clear is used for two purposes:
•
Clearing blocked microelectrodes. If the microelectrode resistance in the bath
seems much higher than it should be, the electrode can often be cleared by
rapidly toggling the Clear switch from plus to minus. Because of the large
current passed this should only be done extracellularly.
•
Penetrating cells. Sometimes microelectrode tips press against the cell
membrane but fail to penetrate. A quick press on the Clear buttons will often
force the electrode to penetrate. Whether to use a hyperpolarizing or
depolarizing current depends on the preparation and must be determined by trial
and error. Like Buzz, the mechanism for impalement is unknown.
Electrochemistry
•
Using the MultiClamp 700B for electrochemistry.
•
See also electrochemistry application notes under ‘Technical Support’ at
http://www.axon.com
Electrochemistry, with the meaning intended here, is the use of an electrochemical
sensor to record signals that reflect the presence of electro-active chemicals in
biological tissue. For biological applications, the sensor is typically a carbon-fiber
microelectrode. Examples of electro-active biological chemicals are dopamine and
Chapter 5
88 • Reference Section
norepinephrine. The MultiClamp 700B, like the Axopatch 200B, can be used to
measure the electrical signals generated by the presence of these chemicals.
To make electrochemical measurements, a voltage is typically applied to the sensor.
This results in the oxidation or reduction of the electro-active species in solution
near the tip of the sensor. The current that is derived from the measurement is a
complex combination of chemical kinetics and molecular diffusion that is relatively
specific for different chemical classes of compounds. In short, the technique
generates a chemical fingerprint for each compound of interest. Furthermore, the
current that is derived from the oxidation (or reduction) of these compounds is
directly proportional to the concentration.
Two methods are used for making electrochemical measurements, cyclic
voltammetry and amperometry.
Cyclic voltammetry typically involves applying an episodic voltage ramp to the
sensor while the resultant current is measured under voltage clamp. The potential
at which dopamine (and other catechol-containing species such as epinephrine and
norepinephrine) oxidizes is approximately 0.15 V cf. the silver/silver chloride
reference potential. In order to accurately measure the voltammetric response of
dopamine in solution, the sensor is poised at a reducing potential between
measurements and ramped to more oxidizing potentials to generate the
electrochemical fingerprint. In a typical experiment, the ramp may last 100 ms;
this, then, is the resolution of the measurement. Cyclic voltammetry is most often
used to make relatively slow, volume-averaged measurements of the concentrations
of electro-active compounds.
Amperometry involves voltage clamping the sensor at the oxidation/reduction
potential of the compound of interest while measuring the resultant current. Sudden
changes in the concentration of the compound are registered as blips of current.
Amperometry is typically used for measuring quantal release of electro-active
chemicals from vesicles. The temporal resolution is determined only by the
response times of the sensor and the voltage clamp.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 89
Both cyclic voltammetry and amperometry can be performed by the MultiClamp
700B without modifications. Such modifications are necessary for some other
Axon amplifiers because electrochemistry typically requires larger voltage
commands than is usual for patch or intracellular recording. However, the
MultiClamp 700B was designed with these larger commands in mind, providing
±1000 mV range.
External Command Inputs
•
External command stimuli are applied to the COMMAND BNC on the front
panel of the MultiClamp 700B.
•
External Command Sensitivity is set in the Gains tab under the Options button
( ).
•
Command Filter Frequency is set in the General tab under
•
See also Capacitance Compensation, Feedback Resistor, Filter, and Mode.
.
Although the MultiClamp 700B Commander provides some simple built-in
command stimuli (e.g. via the Pulse button), it is expected that most experiments
will require more complex stimulus protocols. These must be supplied by an
external pulse generator or a computer program like pCLAMP. External stimulus
commands are supplied to the MultiClamp 700B via the COMMAND BNC on the
front panel (one BNC for each Channel). Note that this is a DC-coupled input, so
be sure that the external pulse generator is correctly calibrated so that zero volts
really correspond to zero.
External Command Sensitivity
External Command Sensitivity is a scaling parameter that is set in the Gains tab
under the Options button.
In V-Clamp mode, the purpose of External Command Sensitivity is to scale down
the command signal in order to reduce the effect of noise in the external pulse
Chapter 5
90 • Reference Section
generator. There are three settings: 20 mV/V, 100 mV/V and OFF. For example,
20 mV/V means that a 1 Volt step applied to the COMMAND BNC will appear to
the cell as a 20 mV step; i.e. external commands are divided down by 50-fold. This
setting should be used when you wish to minimize noise as much as possible. The
100 mV/V setting (10-fold dividing down) should be used when you wish to apply
larger command stimuli to the cell and noise is less of a concern.
In I-Clamp mode, the purpose of External Command Sensitivity is to scale a
voltage COMMAND signal into current. For example, 0.4 nA/V means that a
1 Volt step applied to the COMMAND BNC will appear to the cell as a 0.4 nA step
injection of current. The three Sensitivity settings change as the value of the
Current Clamp Feedback Resistor is changed, since the amount of current that can
be injected by the headstage depends on this resistor. (The maximum current
possible with each resistor is listed in the Gains tab under Current Clamp.)
Additivity of Commands
All command stimuli applied by the MultiClamp 700B are additive. That is, the
external command is algebraically added to Holding, Pulse and Seal Test or Tuning
commands before the sum is applied to the cell.
Command Filter Frequency
Prior to being applied to the cell, the summed commands can be low-pass filtered at
a selectable frequency. The Command Filter Frequency is set in the General tab
under the Options button. The selectable frequency is the –3 dB cutoff frequency
of a 4-pole Bessel filter. Two filter settings are provided for each Channel, one for
V-Clamp, the other for I-Clamp.
This feature is provided because sometimes is desirable to round off the commands
applied to a cell. For example, a large voltage step in V-Clamp mode may produce
a large capacitance transient that cannot be fully compensated by Capacitance
Compensation and which still saturates the amplifier. Lightly filtering the
command signal solves this problem by slowing down the charging of the cell
capacitance. The tradeoff, of course, is that fast kinetic processes in the cell will
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 91
not be so accurately resolved. Another application might be to smooth a sine wave
stimulus that is generated by a digital pulse generator. Lower-resolution digital
devices may produce an output composed of distinct steps. By using the command
filter, these steps can be effectively smoothed before the stimulus is applied to the
cell.
Feedback Resistor
•
The feedback resistor determines the gain of the headstage in V-Clamp mode
and the amount of current that can be passed in I-Clamp mode.
•
The value of this resistor is set in the Gains tab under the Options button (
•
See also External Command Inputs, Headstage, Noise, Overload.
).
V-Clamp Mode
In V-Clamp mode, changing the value of the feedback resistor (Rf) in the headstage
provides a method of changing the gain of the headstage. Choice of the appropriate
Rf involves a tradeoff between two competing factors. (See Chapter 5,
HEADSTAGE, for a technical discussion of these factors.)
•
Larger Rf means smaller current noise due to the headstage circuitry.
•
Smaller Rf means a larger range of membrane currents can be measured
without saturating the headstage circuitry.
Chapter 5
92 • Reference Section
Thus, larger values of Rf are more suited to patch recordings, where the noise is
more critical and the currents are smaller, whereas smaller values of Rf are more
suited to whole-cell recordings, with their larger currents. The following table
summarizes these properties for different values of Rf.
Feedback Resistor
50 MΩ
Experiment Type
Whole Cell
Range
1-200 nA
Noise*
3.0 pA rms
500 MΩ
Whole Cell
0.1-20 nA
1.4 pA rms
5 GΩ
Patch
10-2000 pA
0.9 pA rms
50 GΩ
Patch
0.2-200 pA
0.28 pA rms
*Bandwidth 10 kHz using an 8-pole Bessel filter. Noise is measured with the headstage open-circuit; i.e. it
represents the best possible intrinsic noise of the headstage circuitry.
Note: Vcmd is limited to 10 V in the MultiClamp 700B, which in turn limits the
maximum amount of current that can be injected through the headstage resistor into
the electrode. For example, with Rf = 500 MΩ, the maximum current that can be
injected is 10 V/500 MΩ = 20 nA. These current limits are listed in the
Options/Gains panel of the MultiClamp 700B Commander.
Figure 5.9
As a rule of thumb, it is best to use the largest possible value of Rf without risk of
saturation. Be aware that incompletely compensated capacitance transients, which
are brief and often hard to see, may saturate before ionic currents. The
OVERLOAD LED on the front panel of the MultiClamp 700B will assist you in
judging when saturation has occurred.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 93
Note that Rf can be changed safely “on the fly” with a cell or patch at the end of the
electrode. Under some conditions a small switching transient is generated at the
input of the headstage, and the cell sees this transient. However, after extensive
tests on many types of cells in all recording configurations, we have concluded that
these switching transients are too small to cause any damage to the cell membrane.
I-Clamp Mode
In I-Clamp mode, Rf determines the maximum amount of current that can be
injected into the cell without saturating the headstage circuitry. To enable optimal
neutralization of input capacitance, Rf values should be selected to match the
resistive load of the cell. If possible, the load should be in the range Rf /10 to Rf x
10. For example, for a typical hippocampal pyramidal cell with an input resistance
of 150 MΩ, R Rf = 50 MΩ is suitable.
Note that changing Rf in I-Clamp mode changes the External Command Sensitivity
for I-Clamp.
Filters
•
Low-pass and high-pass filters can be chosen to condition the Primary Output
and Scope outputs. The -3 dB frequency is selectable from a list in the Output
Signals section of the main MultiClamp 700B Commander window.
•
The type of low-pass filter (4-pole Bessel or Butterworth) is selected in the
General tab under the Options button (
).
•
The command stimulus can be low-pass filtered (with a 4-pole Bessel filter) at
.
a –3 dB frequency set in the General tab under
•
See also External Command Inputs, Headstage, and Noise.
The theory behind the design and choice of appropriate filters is very extensive, as
you will see from any book on signal processing. Here we provide just a few basic
Chapter 5
94 • Reference Section
principles that will assist you in choosing the filter type and cutoff frequency that
are most suited to your experiments.
-3 dB Frequency
The –3 dB, or cutoff, frequency (fc) of a filter is the frequency at which the output
signal voltage (or current) is reduced to 1/√2 (i.e. 0.7071) of the input.
Equivalently, fc is the frequency at which the output signal power is reduced to half
of the input. These terms arise from the definition of decibel (dB):
Voltage: dB = 20 log(Vout/Vin)
Power: dB = 10 log(Pout/Pin)
For a low-pass filter, the frequency region below fc is called the pass band, while
that above fc is called the stop band. In the stop band, the signal attenuates (or ‘rolls
off’) with a characteristic steepness. (See Figure 5.10, noting the logarithmic
frequency axis.) The steepness of the roll-off at higher frequencies is determined
both by the type of filter (see below) and the number of poles of the filter: the larger
the number of poles, the faster the roll-off. The low-pass on the Primary Output of
the MultiClamp 700B are 4-pole filters. Filters with more poles can be constructed,
but they are more complex to implement and yield diminishing returns.
Figure 5.10. Filter characteristics, illustrated for a single-pole, low-pass filter. The spectrum
has been normalized so that the signal magnitude in the pass band is 0 dB. The –3 dB frequency
has been normalized to unity.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 95
Types of Filters
There are many types of filters, distinguished by their effects on both the amplitude
and phase of the signal. The two most common filters used in electrophysiology
are the Bessel filter and the Butterworth filter, both of which are implemented in the
MultiClamp 700B.
Bessel Filter
This is the analog filter used for most signals for which minimum distortion in the
time domain is required. The Bessel filter does not provide as sharp a roll-off as the
Butterworth filter, but it is well behaved at sharp transitions in the signal, such as
might occur at capacitance transients or single-channel current steps.
Butterworth Filter
This is the filter of choice when analyzing signals in the frequency domain, e.g.
when making power spectra for noise analysis. The Butterworth filter has a sharp,
smooth roll-off in the frequency domain, but introduces an overshoot and “ringing”
appearance to step signals in the time domain.
Choosing the Cutoff Frequency
In practice, there are two important considerations when selecting the filter cutoff
frequency.
Aliasing
If the digitizing interface samples at 2 kHz, for example, any noise in the
sampled signal that has a frequency greater than 1 kHz will appear in the
digitized trace as extra noise in the range 0 to 1 kHz. In other words, higherfrequency noise (>1 kHz) will appear under the alias of lower-frequency noise
(<1 kHz). This error is called aliasing. A fundamental principle of signal
analysis, called the Nyquist Principle, therefore states that, in order to avoid
Chapter 5
96 • Reference Section
aliasing, the digitizing frequency (fd) should be at least twice the filter
cutoff frequency (fc):
fd ≥ 2fc
The minimum permissible digitizing frequency (exactly twice fc) is called
the Nyquist frequency. In practice, it is better to sample at two or more
times the Nyquist frequency. Thus, fd = 5fc is commonly used. This means
that, if the MultiClamp 700B filter is set at 5 kHz, your interface should be
capable of digitizing at 25 kHz.
Risetime
The risetime is typically given as the time taken for a signal to increase
from 10% to 90% of its peak value. The more heavily a step response is
filtered, the greater the 10-90% risetime. For the 4-pole Bessel filter in the
MultiClamp 700B, the filtered 10-90% risetime (Tr, in ms) of a step input
depends on fc (in kHz) approximately as:
Tr ≈ 0.35/fc
(This can be measured by applying Seal Test to the model BATH in
V-Clamp mode and looking at “Primary Output: Membrane Current” while
changing the filter setting.) Suppose you are interested in measuring action
potentials, for which you expect the 10-90% risetime to be about 0.4 ms.
You would then choose the filter cutoff frequency to be high enough that
the filter risetime is about ten times faster than 0.4 ms so the action
potentials are minimally distorted by the filter. According to the above
equation, then, the appropriate filter setting would be 10 kHz. In practice,
you may need to make other compromises. For example, if the signal is
very noisy you may wish to filter more heavily and accept that the action
potential risetime is artifactually slowed.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 97
High-pass Filter
The Primary Output and Scope signals can be high-pass filtered by setting the AC
value in the Output Gains and Filters section of the main MultiClamp 700B
Commander panel. This is typically done in order to remove a DC component of
the signal. When the filter cutoff is set to DC this high-pass filter is bypassed.
Command Filter Frequency
Command stimuli applied in V-Clamp or I-Clamp can be filtered at different cutoff
frequencies, selectable in the General tab under the Options button. You might
wish to do this in order to smooth out sharp transitions in the command signal that,
if unfiltered, might produce very large capacitance transients that saturate the
headstage circuitry, even after capacitance compensation. (See Chapter 5,
EXTERNAL COMMAND INPUTS.)
Grounding and Hum
•
Methods for minimizing line-frequency noise.
•
See also Noise, Power Supply.
A perennial bane of electrophysiology is line-frequency pickup, often referred to as
hum. Hum can occur not only at the mains frequency but also at multiples of it.
In a well-shielded enclosure the MultiClamp 700B has insignificant hum levels
(less than 0.01 pAp-p). To take advantage of these low levels great care must be
taken when incorporating the MultiClamp 700B into a complete recording system.
The following precautions should be taken.
•
Ground the preparation bath only by directly connecting it to the gold
ground connector on the back of the headstage.
•
Place the MultiClamp 700B in the rack in a position where it will not absorb
radiation from adjacent equipment. A grounded, thick sheet of steel placed
between the MultiClamp 700B and the radiating equipment can effectively
reduce induced hum.
Chapter 5
98 • Reference Section
•
Initially make only one connection to the MultiClamp 700B, from the
PRIMARY OUTPUT BNC to the oscilloscope. After verifying that the hum
levels are low, start increasing the complexity of the connections one lead at a
time. Leads should not be draped near transformers located inside other
equipment. In desperate circumstances, the continuity of the shield on an
offending coaxial cable can be broken.
•
Try grounding auxiliary equipment from a ground distribution bus. This bus
should be connected to the MultiClamp 700B via the SIGNAL GROUND
(4 mm) socket on the rear panel. The Signal Ground in the MultiClamp 700B
is isolated from the chassis and power ground.
•
Experiment. While hum can be explained in theory (e.g. direct pickup, earth
loops), in practice empiricism prevails. Following the rules above is the best
start. The final hum level can often be kept to less than 0.1 pAp-p. One
technique that should not be used to reduce hum is the delicate placement of
cables so that a number of competing hum sources cancel out. Such a
procedure is too prone to accidental alteration.
Headstage
•
Principles and properties of the V-Clamp and I-Clamp circuits in the CV-7
headstage.
•
See also Feedback Resistor, Mode, Noise, Series Resistance Compensation.
The CV-7 headstage contains two distinct circuits, a current-to-voltage (I-V)
converter used in V-Clamp mode, and a voltage follower used in I-Clamp mode.
The I-V converter is similar to that found in an Axopatch-1D headstage, whereas
the voltage follower is like that in an Axoclamp 2B headstage.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 99
Voltage Clamp Circuit
In V-Clamp mode, the goal is to hold the interior of an electrode at a command
potential while measuring the currents that flow down the electrode. An I-V
converter achieves this by producing a voltage output that is proportional to the
current input. There are two types of I-V converters used in patch clamp
headstages: capacitive feedback (used in the Axopatch 200B), and resistive
feedback (used in the Axopatch-1D and in the MultiClamp 700B). The essential
parts of a resistive-feedback headstage are shown in Figure 5.11.
PROBE
If
Rf
BOOSTCIRCUIT
+
Vo
+
OFFSET
AND
SCALING
HIGHFREQUENCY
BOOST
I
Vp
Figure 5.11. Resistive-feedback headstage.
The heart of the circuit is an operational amplifier (op amp) in the PROBE. The
behavior of this circuit depends upon two characteristics of an ideal op amp.
•
An op amp has infinite input resistance. Therefore, the current flowing out of
the electrode (Ie) must equal the current (If) flowing through the feedback
resistor (Rf) because no current is allowed to flow into the ‘–’ input of the op
amp.
•
An op amp does all it can to keep the voltage at its two inputs equal. Thus,
because the voltage at the ‘+’ input is Vp (= the command voltage), the voltage
at its ‘–’ input is also Vp. The voltage across Rf must therefore be Vp – Vo = If
·Rf by Ohm’s Law.
Combining both of these pieces of information, the electrode current (which is what
we want) is given by Ie = If = (Vp – Vo)/Rf. In practice Rf is a very large resistor
Chapter 5
100 • Reference Section
(GΩ) so this circuit can measure very small currents (pA). The differential
amplifier in the BOOST CIRCUIT does this calculation of Ie. Subsequent
amplifiers are used to scale the gain and remove voltage offsets.
High Frequency Boost
A fundamental problem of this circuit when used for patch clamping is that the
output bandwidth of the probe is inherently low. To a first approximation, the
product of Rf and the stray capacitance sets the bandwidth across it. For
example, if Rf is 500 MΩ and the stray capacitance is 0.5 pF, the bandwidth is
about 600 Hz. To overcome this limitation, the probe output is passed through
a high-frequency boost circuit. The gain of this circuit is proportional to the
frequency.
The high-frequency boost is applied to the output of the I-V converter and
cannot influence the events at the electrode. Thus, one might conclude that the
voltage clamp of the electrode must also be slow. This is not the case, for the
following reason. The PROBE op amp does everything it can to keep the
voltage at its ‘–’ input equal to the command voltage at its ‘+’ input. If the
command is a rapid step, then the voltage at the ‘–’ input (i.e. at the back of the
electrode) is also a rapid step. This means the voltage clamp of the electrode is
fast. The RC filtering effect mentioned above applies only to the output of the
I-V converter, which can therefore be subjected to post hoc boosting.
What is Clamped During Voltage Clamping?
We were careful to state in the above discussion that it is only the back of the
electrode that is voltage clamped, not the cell membrane. The voltage at the
cell membrane may differ from that at the back of the electrode because of
bandwidth and voltage errors due to uncompensated series resistance (Rs). For
this reason, it is always important to consider using Rs compensation. (See
Chapter 5, SERIES RESISTANCE COMPENSATION.)
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 101
Intrinsic Headstage Noise
The intrinsic noise of a resistive-feedback I-V converter (i.e. with an opencircuit input) is determined, in theory, by the resistance of the feedback
resistor. The rms current noise is given approximately by
Irms ≈ √(4kTfc/Rf)
where fc is the filter cutoff frequency and k and T are constants. Thus, for
low noise, a high value of Rf is desirable. This was pointed out in Chapter
5, FEEDBACK RESISTOR.
Current Clamp Circuit
In I-Clamp mode a separate headstage circuit is used, called a voltage follower.
The essential features of a voltage follower are shown in Figure 5.12. A1 is an
(effectively) infinite input resistance, unity-gain op amp, the output of which is the
pipette voltage, Vp. A2 is a summing amplifier used for injecting current into the
cell. The voltage across the headstage resistor Rf is equal to Vcmd regardless of Vp.
Thus the current through Rf is given exactly by I = Vcmd/ Rf. If stray capacitances
are ignored, all of this current is injected into the cell.
Figure 5.12. Voltage follower headstage.
Chapter 5
102 • Reference Section
Note that Vcmd is limited to 10 V in the MultiClamp 700B, which in turn limits the
maximum amount of current that can be injected through the headstage resistor into
the electrode. For example, with Rf = 500 MΩ, the maximum current that can be
injected is 10 V/500 MΩ = 20 nA. These current limits are listed in the
Options/Gains panel of the MultiClamp 700B Commander.
Mounting the Headstage
For maximum mechanical rigidity, the CV-7 headstage should be mounted directly
to the head of the micromanipulator using the dovetailed mounting plate.
Bath Connection
The bath (or ground) electrode should be connected to the gold-plated 1 mm plug
on the rear of the headstage. The bath should not contact any other ground (e.g.
Signal Ground).
Cleaning
Wipe the headstage connector with a damp cloth to clean salt spills. Avoid spilling
liquids on the headstage. The Teflon input connector should be kept very clean.
Effective cleaning can be done by spraying with alcohol or swabbing carefully with
deionized water.
Static Precautions
The headstage can normally be safely handled. However, if you are in a laboratory
where static is high (i.e. you hear and feel crackles when you touch things) you
should touch a grounded metal object immediately before touching the headstage.
Acoustic Pick-up
Rare cases have been reported in which the headstage was susceptible to low
amplitude acoustic pick-up. For example, the audible hum of a nearby isolation
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 103
transformer can acoustically couple to the input of the headstage. This was traced
to the silver wire of the electrode and was solved by trimming off a fraction of the
wire, thus changing its resonant frequency.
Help
• On-line Help facility used to provide brief descriptions of the features of the
MultiClamp 700B Commander.
• Help is accessed via the
Commander window.
button at the top of the main MultiClamp 700B
In order for the On-line Help to work properly, the computer running the
MultiClamp 700B Commander must have a web browser (Internet Explorer v. 4 or
later, or equivalent). JavaScript is required. When the user clicks on the Help
button, the browser will open automatically (if it is not already running) and the
relevant page will appear.
Holders
•
Design, use and maintenance of the HL-U electrode holders supplied with the
MultiClamp 700B.
The HL-U series holder provides a universal fit for a very wide range of electrode
diameters and will fit any of the U-type headstages of Axon amplifiers.
Holder Design
The barrel of the holder is made of polycarbonate for lowest noise. There are two
different barrel lengths (16 mm and 28 mm). The shorter length contributes less to
instrument noise and is therefore suited to single-channel patch clamp recordings.
Although the longer barrel will contribute more to the noise, the greater length may
provide the needed clearance between the headstage case and other components in
the experimental setup. To further minimize the noise contributed by the holder in
Chapter 5
104 • Reference Section
single-channel recording, the holder uses a small (1 mm) pin for the electrical
connection and a large amount of insulating Teflon.
Mechanical stability of the electrode is assured in several ways. (See Figure 5.13.)
As the pipette cap is closed, the cone washer is compressed on the electrode from
the force applied to the front and back of the cone washer. The cap also forces the
blunt end of the electrode against the rear wall of the holder bore. (The electrode
should always be inserted as far as it will go in the holder.) The holder mates with
the threaded Teflon connector on U-type Axon headstages and is secured in place
with a threaded collar.
Figure 5.13. Exploded view of the HL-U holder.
The bore size of the HL-U accepts pipettes with an outer diameter (OD) of
1.0-1.7 mm. Pipettes are secured by a cone washer with an inner diameter (ID) that
accommodates the pipette OD. Color-coding aids identification of the four sizes of
cone washers:
1.0 mm (orange), 1.3 mm (clear), 1.5 mm (orange) and 1.7 mm (clear). When the
pipette OD falls between two sizes of cone washers, the larger size cone washer
should be used. For instance, if the pipette OD is 1.6 mm, then use a cone washer
with an ID of 1.7 mm.
An Ag/AgCl pellet offers no greater stability than properly chlorided silver wire.
Moreover, the diameter of the pellet (1 mm) restricts its use to pipettes with a large
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 105
ID (> 1.1 mm). Therefore, the HL-U is supplied with 0.25 mm silver wire, which
must be chlorided before use. (See below.)
Spare components included with each holder are: one 50 mm length of silver wire,
40 cone washers (10 of each size), and one 70 mm length of silicone tubing. Cut
into 2 mm lengths, the silicone tubing will yield approximately 30 replacement
silicone seals. Additional cone washers, silicone tubing, pins and silver wire can be
purchased from Axon Instruments, as well as optional Ag/AgCl pellet assemblies.
Optional Ag/AgCl Pellets
The HL-U holder will accommodate a 1 mm diameter Ag/AgCl pellet that should
provide many months of DC-stable recordings. The inner diameter (ID) of the
pipette must be > 1 mm. A wax-sealed Teflon tube surrounds the silver wire. This
ensures that the electrode solution only contacts the Ag/AgCl pellet. Three pellet
assemblies are sold as HLA-003.
Figure 5.14. Ag/AgCl pellet assembly.
Holder Use
Insertion of Electrode
Make sure the electrode cap is loosened so that pressure on the cone washer is
relieved, but do not remove the cap. Push the back end of the electrode through
the cap and cone washer until it presses against the end of the bore. Gently
tighten the cap so that the electrode is gripped firmly.
Chapter 5
106 • Reference Section
To minimize cutting of the cone washer by the sharp back end of the electrode,
you can slightly smooth the edges by rotating the ends of the electrode glass in
a Bunsen burner flame prior to pulling.
Filling Electrodes
Only the taper and a few millimeters of the shaft of the pipette should be filled
with solution. The chlorided tip of the wire should be inserted into this
solution. Avoid wetting the holder since this will increase the noise.
Silver Chloriding
It is up to you to chloride the end of this wire as required. Chloriding
procedures are contained in many electrophysiology texts1. Typically the
chlorided wire will need to be replaced or rechlorided every few weeks. A
simple yet effective chloriding procedure is to clean the silver wire down to the
bare metal using fine sand paper and immerse the cleaned wire in bleach for
about 20 minutes, until the wire is uniformly blackened. This provides a
sufficient coat of AgCl to work reliably for several weeks. Drifting or
otherwise unstable offsets during experiments is suggestive of the need for
rechloriding. The chlorided region should be long enough so that the electrode
solution does not come in contact with the bare silver wire.
Heat smoothing the back end of the electrode extends the life of the chloride
coating by minimizing the amount of scratch damage. Another way to protect
the AgCl coating is to slip a perforated Teflon tube over the chlorided region.
1
For easy-to-use recipes see Microelectrode Methods for Intracellular Recording and Ionophoresis, by R.D. Purves, London: Academic
Press, 1981, p. 51 or The Axon Guide. Foster City, CA: Axon Instruments, Inc., 1993, p. 83.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 107
Holder Maintenance
Cleaning
For lowest noise, keep the holder clean. Frequently rinse the holder with
distilled water. If more thorough cleaning is required, briefly wash in ethanol
or mild soapy water. Never use methanol or strong solvents.
Replacing the Silver Wire
To replace the silver wire, insert the nonchlorided end through the hole of the
silicone seal and bend the last 1 mm of wire over to an angle of 90°. Press the
wire into the back of the barrel making sure that the silicone seal is flush with
the back of the barrel. Slip the threaded collar over the back of the barrel.
Adapters
HLR-U right-angle adapters allow the HL-U series holder to emerge at 90° from the
headstage. Use the HLR-U with the HL-U holder.
HLB-U BNC-to-Axon adapter allows conventional BNC-type holders to be used
with Axon U-type headstages. Use the HLB-U with all U-type CV and HS
headstages. These headstages have a threaded white Teflon collet.
Input/Output Connections
•
Description of the different connectors on the front and rear panels of the
MultiClamp 700B main unit.
•
See also External Command Inputs, Oscilloscope Triggering, Mode.
Front Panel
Inputs
COMMAND: Voltage or current commands to the MultiClamp 700B are
accepted at this input. The External Command Sensitivity is set in the
Gains panel under the Options toolbar button.
Chapter 5
108 • Reference Section
MODE: This is enabled when the user has checked the Ext checkbox
under Channel 1 or 2 Mode in the MultiClamp 700B Commander. A
TTL Low input at MODE will select I-Clamp; a TTL High (3.5-5 V)
input will select V-Clamp. For example, these inputs can be a TTL
Digital Signal controlled by pCLAMP.
Scaled Outputs
PRIMARY: The output signal at this BNC is selected from the list in
the Primary Output section of the main window of the MultiClamp
700B Commander. The choices include:
•
Membrane Current
•
Membrane Potential
•
Pipette Potential (VC) or Command Current (IC)
•
100 x AC Membrane Potential (High-passed filtered at 1 Hz, this
special high-gain output is useful for viewing very small extracellular
signals.)
•
External Command Potential (VC) or Current (IC)
•
Auxiliary Potential (if HS-2 headstage attached) or Current (if VG-2
headstage attached).
SCOPE: The signal available here is the same as that at PRIMARY
OUTPUT, except that it can be independently low-pass filtered using the
Scope control in the Primary Output section of the main window of the
MultiClamp 700B Commander.
SECONDARY: The output signal at this BNC is selected from the list in the
Secondary Output section of the main window of the MultiClamp 700B
Commander. All signals are identical to the Primary outputs, except that
Membrane Potential also includes the commands applied by the Rs
Compensation circuitry.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 109
Headphone Jack: This will drive headphones or a remote powered speaker if
it is desired to monitor the audio output of the MultiClamp 700B. The
output is the same as that available at the rear panel AUDIO OUTPUT jack.
Rear Panel
HEADSTAGE #1 / #2: The CV-7 headstages are plugged into the corresponding
25-pin DB connectors. Note that Headstage #1 refers to Channel 1
inputs/outputs on the front panel, and Headstage #2 for Channel 2
inputs/outputs.
AUXILIARY HEADSTAGE #1 / #2: The optional voltage-following (HS-2) or
Bath (VG-2) is plugged into this 15-pin DB connector.
10 AUX1/ 10 AUX2: These BNC outputs provide x10 output signal for the
respective AUXILIARY HEADSTAGE channels.
USB:
The USB port of the host computer is connected via of a USB cable.
AUDIO INPUT: This connector is used if you wish to mix the audio output of the
MultiClamp 700B with the audio output of your PC. Connect the audio output
of your PC’s sound card to the AUDIO INPUT socket and the MultiClamp
AUDIO OUTPUT socket to the PC-powered speakers.
AUDIO OUTPUT: This output can be used in conjunction with AUDIO INPUT,
as described above. It can also drive headphones or a remote powered
speaker, like the front panel Headphones Socket.
SYNC OUTPUT: The signal available at this BNC connector is intended to be
used as an external trigger for an oscilloscope when internal commands (Seal
Test), Tuning) or Pulse are activated, or to indicate the Mode state of the
amplifier (commanded either externally by the Mode BNC or internally by the
Auto Mode switch feature). The Sync Output is a 0 to (approximately) 5 V
step aligned with the onset of the Seal Test, Tuning or Pulse step. Or, when
following Mode, 5 V corresponds to VC, while 0 V corresponds to IC. See the
Options / General tab to select the function of the SYNC output.
Chapter 5
110 • Reference Section
SIGNAL GROUND: This 4 mm socket is an alternative signal grounding point for
the MultiClamp 700B, and is isopotential with the CV-7 input signal. It can be
connected to a central grounding bus in order to combine other sources of noise
in your setup, such as the Faraday cage, perfusion system, etc.
Screw connector with nut (labeled with standard ground symbol): This provides an
alternative chassis or power supply ground.
Leak Subtraction
•
Leak Subtraction provides a quick method of subtracting linear leak currents
from the Membrane Current in V-Clamp mode.
•
Leak Subtraction is activated by checking the Checkbox and pressing the
button in the Leak Subtraction box in the V-Clamp pane.
•
See also Capacitance Compensation.
Leak Subtraction is typically used when you are trying to measure single-channel
currents that are sitting on top of a relatively large leak current. Imagine, for
example, a channel that opens during a 100 mV voltage step that is applied to a
patch with a 1 GΩ seal resistance. The seal (leak) current during the step will be
100 pA. Because of this relatively large leak current, the gain of the MultiClamp
700B cannot be turned up very far without saturating the amplifier, but at a low
gain setting the single-channel openings may not be resolved very well.
Leak Subtraction solves this problem by subtracting from the membrane current, in
this case, a 100 pA step of current before the Output Gain is applied. The Primary
Output signal will now be a flat line on which the single-channel activity is
superimposed. (This assumes that the capacitance transients at the start and end of
the step have already been canceled using Capacitance Compensation. Indeed,
Leak Subtraction can be thought of as a kind of capacitance compensation that
applies to leak currents.)
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 111
Leak Subtraction works by scaling the command potential waveform (Vc(t)) by the
seal resistance (Rseal) to obtain a time-varying estimate of the leak current (Ileak(t)),
which is then subtracted from the membrane current. It differs from Output Zero,
which simply subtracts a constant offset without regard to changes in the command
potential with time. In order to perform its correction, Leak Subtraction must be
provided with an estimate of Rseal. This is done by pressing the Auto Leak
Subtraction button, or by manually entering an estimate of Rseal to the right of the
button. When it is correctly adjusted, voltage steps that are known to elicit no
active currents (e.g. small hyperpolarizing steps) will produce a flat line in the
Membrane Current signal (ignoring the brief capacitance transients, if these are still
uncompensated).
We recommend that Leak Subtraction be used with caution, because it assumes that
Rseal is constant for all voltage steps. This may not be true if, for instance, the patch
contains small channels or electrogenic transporters that do not produce discernible
single-channel events. These will appear to be part of the seal current and may
impart apparent non-linear behavior to the seal.
For subtracting leak currents in whole-cell recordings, it is safer to use a computer
program like pCLAMP, which allows off-line leak correction.
Mode
•
Recording mode is switchable between voltage clamp (VC), normal current
clamp (IC) and current clamp in which all external inputs are disconnected
(I=0).
•
buttons, or remotely by checking the
Mode is selected using the
Ext check box and applying a voltage to the MODE input on the front panel of
the MultiClamp 700B (0 V for IC, 3.5-5 V for VC).
•
See also Headstage, Input/Output Connections.
Switching between V-Clamp and I-Clamp modes in the MultiClamp 700B activates
a switch between two distinct circuits in the CV-7 headstage. Voltage clamp is
achieved with a current-voltage converter, whereas current clamp is achieved with a
Chapter 5
112 • Reference Section
voltage follower. This contrasts with the design of other patch clamp amplifiers, in
which the same basic circuit is used for voltage clamp and current clamp, producing
a compromised performance.
The I=0 mode is a special case of I-Clamp in which all external inputs are
disconnected. This is convenient if you wish to quickly return to the resting
potential of the cell, or if you want to check the electrode offset at the end of the
experiment. (See Chapter 4, GENERAL ADVICE.)
Mode switching in the MultiClamp 700B can, under some circumstances, produce a
small transient at the input of the headstage, a transient that is seen by the cell. We
have extensively tested the headstage with many cell types and all recording
configurations, and have not encountered any problems with the transients causing
damage of the cell membrane.
Model Cell
•
PATCH-1U model cell is a standard accessory provided with the MultiClamp
700B. It is useful for setting up, testing and doing the tutorials described in
Chapter 3.
The model cell is a small metal box with three connectors labeled BATH, CELL
and PATCH, and an unlabeled 2 mm gold plug which connects to the 1 mm
grounding plug on the rear of the CV-7 headstage. The circuit is shown in Figure
5.15 (right). A 10 MΩ resistor models the electrode, the cell is modeled by
500 MΩ in parallel with 33 pF (the membrane time constant is 16.5 ms), and a
10 GΩ resistor models the patch. The pipette capacitance is about 4-6 pF. The
charging time constant is approximately 330 µs (10 MΩ x 33 pF).
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 113
The PATCH-1U model cell has been made without a switch to change the model
between the BATH, PATCH and CELL positions. This is because even the best
switches have an enormous amount of leakage resistance and capacitance that
increases the noise three to five times beyond what can be achieved with a good
seal. Instead of switches, three separate plug positions have been provided and you
can rotate the model cell into the position required. With this technique the noise
contribution of the model cell is still somewhat more than can be achieved with a
good seal, but the increase is less than 50%.
Figure 5.15. PATCH-1U model cell.
Chapter 5
114 • Reference Section
Noise
•
Sources of instrument noise in the MultiClamp 700B.
•
See also Feedback Resistor, Filters, Grounding and Hum, Headstage, Power
Supply, Series Resistance Compensation.
Measurement of Noise
Noise is reported in two different ways in this manual.
•
Peak-to-peak (p-p) noise. This measure finds favor because it is easily
estimated from an oscilloscope and its meaning is intuitively obvious. A
disadvantage is that it is very insensitive to structure in the noise (e.g. different
frequency components). For this reason, it is most commonly used for
quantifying “white” noise. (See Chapter 5, FILTERS.)
•
Root-mean-square (rms) noise. This is essentially the “standard deviation” of
the noise and can be calculated using a computer or an electronic circuit
designed for this purpose. For white noise, the rms noise is approximately onesixth the peak-to-peak noise. The MultiClamp 700B Commander displays the
rms noise on the Membrane Current signal in V-Clamp mode after checking the
Irms checkbox below the meters. The measurement is made with a bandwidth
of 30 Hz to 5 kHz (4-pole Butterworth filter). See the table on page 92 in the
Feedback Resistor section for noise measurements using the CV-7 headstage.
When reporting measured noise, the bandwidth (i.e. filter cutoff frequency) must
always be stated.
Sources of Noise
Cell and Seal
V-Clamp: The higher the resistance (R) and the smaller the capacitance (C)
between the interior of the electrode and ground, the smaller the current
noise. Thus, minimum noise is achieved for an isolated patch (large R,
small C) with a high seal resistance (large R). In whole-cell recordings
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 115
from larger cells (smaller R, larger C) the noise of the cell usually
dominates, meaning that subsequent noise sources (listed below) become
less important. (See Chapter 4, PATCH CLAMPING.)
I-Clamp: The voltage noise is dominated by the load resistance but is also
affected by the stray capacitance. For a purely resistive load the noise is
given approximately by 12√R µVrms (10 kHz bandwidth), where R is the
parallel combination of the feedback resistor (Rf) and the load resistance
(i.e. the electrode resistance plus input resistance of the cell). Thus, a low
resistance electrode/cell combination is preferred. A large stray
capacitance will reduce the noise by acting like an RC filter, but this will
also reduce the measurement bandwidth. Increasing the Capacitance
Neutralizaton setting will improve the bandwidth but increase the noise.
Electrode and Holder
V-Clamp: Current noise increases markedly with electrode capacitance.
This can be minimized by coating the electrode and other strategies. (See
Chapter 4, PATCH CLAMPING.) Increasing electrode resistance
apparently decreases the current noise, but this is due to the RC filtering
effect of the electrode resistance in parallel with the electrode capacitance.
In fact, it is desirable to decrease the electrode resistance in order to
maximize the bandwidth of the clamp, even if this apparently increases the
noise of the recording.
I-Clamp: Voltage noise increases markedly with electrode capacitance and
resistance. Thus, both should be minimized as much as possible. (See
Chapter 4, SHARP MICROELECTRODE RECORDING.)
Headstage Circuit
V-Clamp: Current noise decreases as the value of the feedback resistor (Rf)
is increased. Thus, for minimum noise the largest Rf should be chosen,
subject of course to range limitations. (See Chapter 5, FEEDBACK
RESISTOR.)
Chapter 5
116 • Reference Section
I-Clamp: Voltage noise decreases as the value of Rf is decreased, but Rf
should be chosen so that it matches the load resistance (i.e. sum of
electrode and cell resistance) within a 1:10 ratio (a 1:5 ratio is optimal).
Thus, Rf = 50 MΩ will work optimally for loads between 10 MΩ and
250 MΩ. This range limitation is determined by the effectiveness of the
Capacitance Neutralization circuit.
Compensation Circuits
V-Clamp: Adjusting Rs Compensation increases the current noise, because
the compensation circuit employs positive feedback that injects noise back
into the headstage. Further, the effect of Rs compensation is to reduce the
electrode series resistance, which reduces the effect of the RC filter
mentioned above (“Electrode and Holder”).
I-Clamp: Increasing Pipette Capacitance Neutralization increases the
voltage noise, for reasons similar to those just mentioned for Rs
Compensation.
Although both of these compensation circuits increase the noise in the
signal of interest, they are most likely to be required in whole-cell
recordings where the dominant noise source is the cell. In any case,
correction of Series Resistance and Pipette Capacitance errors must
normally take precedence over noise concerns in whole-cell experiments.
Power Supply
Noise can arise from earth loops, power supply glitches and radiation from
nearby instruments. (See Chapter 5, GROUNDING AND HUM, and POWER
SUPPLY.)
Oscilloscope Triggering
•
SYNC output on the rear panel of the MultiClamp 700B provides a signal for
triggering an oscilloscope (or for triggering in Clampex).
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 117
•
See also Input/Output Connections.
The signal available at this BNC connector is intended to be used as an external
trigger for an oscilloscope when internal commands (Seal Test), Tuning) or Pulse
are activated, or to indicate the Mode state of the amplifier (commanded either
externally by the Mode BNC or internally by the Auto Mode switch feature). The
Sync Output is a 0 to (approximately) 5 V step aligned with the onset of the Seal
Test, Tuning or Pulse step. Or, when following Mode, 5 V corresponds to VC,
while 0 V corresponds to IC. See the Options / General tab to select the function of
the SYNC output.
Output Zero
•
Subtracts the steady-state current offset (in VC mode) or voltage offset (in IC
mode).
•
Activated by pressing the
button in the Output Zero box, or by
checking the checkbox and manually adjusting the value to the left of the
button.
•
See also Leak Subtraction, Bridge Balance.
The purpose of this control is to zero the output, that is, to remove the DC voltage.
Output Zero works by sampling the current or voltage over a ~70 ms time window
immediately after pressing the button, and then subtracting this value from all
subsequent Primary Output signals. Unlike Leak Subtraction or Bridge Balance, it
does not account for currents or voltages that change as a result of time-varying
command pulses; it simply provides a constant offset adjustment.
The Auto Output Zero only affects the signal on the Primary Output. In other
words, the cell is not affected by the Output Zero command. No other input or
outputs are affected.
Output Zero is useful for recording small signals that are riding on a large, constant
offset current or voltage. However, in general we recommend that it not be used,
since potentially useful information about the biological signal is lost.
Chapter 5
118 • Reference Section
Overload
•
OVERLOAD light on the front panel of the MultiClamp 700B warns when the
signal presented at PRIMARY OUTPUT or SCOPE saturates (i.e. exceeds
±10.5 V longer than 10 µs) at any point in the internal circuitry of the amplifier.
•
See also Capacitance Compensation, Feedback Resistor.
Inadvertent overloading of the internal circuitry of the MultiClamp 700B is a
problem because it may cause distortion of the signal of interest. The OVERLOAD
light helps to avoid this problem in two ways.
•
By reporting saturation in internal circuits. The PRIMARY OUTPUT might
not appear to be saturated because it may be heavily filtered, reducing the size
of any saturating transients at the output. OVERLOAD reports any saturation
that occurs before the signal is conditioned.
•
By expanding transients. Very fast saturating spikes (e.g. uncompensated
capacitance transients) may be missed under visual inspection on an
oscilloscope, because they are too fast to be seen clearly. The overload sensing
circuitry in the MultiClamp 700B catches any signals that exceed saturation for
longer than 10 µs and illuminates the OVERLOAD light for at least 500 ms.
If saturation occurs, first try reducing the Output Gain. If the problem persists,
indicating that saturation occurs in the headstage, reduce the Feedback Resistor.
Polarity Conventions
•
Current and voltage sign conventions used in the MultiClamp 700B system.
Biological Polarity Conventions
Inward Current
Current (carried by positive ions) that flows across the cell membrane, from the
outside surface to the inside surface.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 119
Outward Current
Current that flows from the inside to the outside surface of the cell.
Membrane Potential
The potential inside the cell minus the potential outside the cell:
Vm = Vin – Vout.
Depolarization
A positive shift in Vm (e.g. from –60 mV to +80 mV) caused by a flow of
inward current.
Hyperpolarization
A negative shift in Vm.
MultiClamp Polarity Conventions
The conventions described here apply to all amplifiers manufactured by Axon
Instruments.
To prevent confusion, Axon always uses current and voltage conventions based on
the instrument's perspective. That is, the current is defined with respect to the
direction of flow into or out of the headstage. Axon amplifiers do not have
switches that reverse the current or the voltage command polarities. This prevents
forgetting to move the switch to the correct position. The data are recorded
unambiguously and the correct polarity can be determined during subsequent data
analysis.
Positive Current
Current that flows out of the headstage into the electrode and out of the
electrode tip into the cell.
Chapter 5
120 • Reference Section
Positive/Negative Potential
A positive/negative voltage at the headstage input with respect to the bath
ground.
With these definitions it is easy to work out the correct polarity for every
recording configuration. For example, in the whole-cell or outside-out patch
configuration the electrode tip is on the intracellular face of the cell. Thus, a
negative potential, Vp, at the headstage input (=electrode interior) is a negative
potential inside the cell. The cell’s membrane potential under voltage clamp is
therefore Vm = Vin – Vout = Vp – 0 = Vcmd. Positive current flowing out of the
electrode must then flow from the inside to the outside surface of the cell,
which means that it is outward current.
Polarity Summary for Different Recording Configurations
Whole Cell/Outside-out Patch
Positive current = outward membrane current
Membrane potential = Vp
Inside-out Patch
Positive current = inward membrane current
Membrane potential = – Vp
Cell-attached Patch
Positive current = inward membrane current
Membrane potential = Vrest – Vp
Power Supply
•
Behavior and maintenance of the power supply used in the MultiClamp 700B.
•
See also Grounding and Hum.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 121
Supply Voltage Selection
The MultiClamp 700B can be directly connected to all international supply
voltages. The input range is from 85 to 260 VAC. No range switching is required.
Alternatively, a DC voltage of 110 – 340 VDC can power the instrument.
Changing the Fuse
The MultiClamp 700B uses a 0.5 A, 250 V slow acting 5 x 20 mm fuse. Before
changing the fuse investigate the reason for its failure. To change the fuse:
1. Disconnect the power cord.
2. Use a screwdriver or a similar device to rotate the fuse holder
counterclockwise.
3. Replace the fuse with another fuse of the same rating.
4. Reconnect the power cord.
Glitches
The MultiClamp 700B has been designed to minimize the effects of power-supply
transients (glitches). Although normally inconsequential, glitches could cause
transients to appear on the voltage and current outputs that may corrupt highsensitivity recordings.
The most effective way to gain immunity from mains glitches is to eliminate them
at the source. Most glitches are due to the on/off switching of other equipment and
lights on the same power-supply circuit. Precautions to be taken include:
1. Avoid switching equipment and lights on or off while recordings are being
made.
2. Water baths, heaters, coolers, etc. should operate from zero-crossing relays.
3. Radio Frequency Interference filters should be installed in glitch-producing
equipment.
Chapter 5
122 • Reference Section
Select Device
•
Selection of Demo or Hardware modes and the Serial number.
•
Selection is made using the Select Device (
) button in the toolbar.
When the MultiClamp 700B Commander is run for the first time, the Select Device
window is displayed. (See Chapter 2, INSTALLATION AND BASIC
OPERATION.) When the MultiClamp 700B Commander is run subsequently, this
window is bypassed. The window can be accessed again by pressing the Select
Device toolbar button.
Select Device offers the following options.
•
Demo Mode. This allows the MultiClamp 700B Commander to be run without
a MultiClamp 700B amplifier being connected or switched on. Demo Mode is
useful for exploring the features of the MultiClamp 700B Commander. Note
that telegraphs are active during Demo mode, since they are communicated
through software messaging.
•
MultiClamp Hardware. This option only works when a functioning
MultiClamp 700B is connected to a USB port on the computer that is running
the MultiClamp 700B Commander. The unique hardware Serial Number is
identified by this operation.
Series Resistance Compensation
•
Theory and practice of compensating the series resistance in V-Clamp mode.
•
Adjusted using the
•
See also Capacitance Compensation, Headstage.
controls in the V-Clamp pane.
Introduction to Rs Compensation
Series resistance (Rs) is defined as the total resistance that is interposed between the
circuitry of the headstage and the membrane of the cell. Contributors to Rs include:
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 123
•
The resistance of the solution inside the electrode, dominated by that at the
narrow tip.
•
The resistance caused by intracellular organelles that partially clog the
electrode tip.
•
The resistance due to glial cells or connective tissue that cover the cell
membrane.
•
The resistance of the bath solution and the bath electrode (usually minor).
Series resistance causes three major problems in voltage clamp recordings.
1. Steady-state voltage errors. Suppose you are measuring a 1 nA membrane
current under V-Clamp. If Rs = 10 MΩ, there will be a voltage drop of
IRs = 1 nA x 10 MΩ = 10 mV across the series resistance. Since Rs is
interposed between the headstage and the cell membrane, the actual cell
membrane potential will be 10 mV different from the command potential at the
headstage. (The direction of the error will depend on the direction of current
flow.) Worse, the error will vary as the membrane current varies. In extreme
situations in the presence of voltage-gated channels, complete loss of control of
membrane potential can occur.
2. Dynamic voltage errors. Following a step change in command potential, the
actual cell membrane potential will respond with an exponential time course
with a time constant given by τs = RsCm, where Cm is the cell membrane
capacitance.
This time constant is 330 µs for the model cell provided with the MultiClamp
700B (Rs = 10 MΩ, Cm = 33 pF). This means that the actual membrane
potential response to a step voltage command will have a 10-90% risetime of
more than 0.7 ms and will not settle to within 1% of its final value until about
1.5 ms after the start of a step command. If you are interested in fast membrane
currents, like sodium currents, this slow relaxation of the voltage clamp is
unacceptable.
Chapter 5
124 • Reference Section
3. Bandwidth errors. The Rs appears in parallel with the membrane capacitance,
Cm, of the cell. Together they form a one-pole RC filter with a –3 dB cutoff
frequency given by 1/2πRsCm. This filter will distort currents regardless of
their amplitude. For the parameters of the model cell, this filter restricts true
measurement bandwidth to 480 Hz without Rs compensation.
Fortunately, electronic techniques have been developed to partially correct for the
errors caused by series resistance. In V-Clamp mode, the techniques are generally
referred to as Rs Compensation.
Series resistance errors can also occur in I-Clamp mode. These errors are generally
corrected using the techniques of Bridge Balance and Capacitance Neutralization.
(See these entries in Chapter 5.)
Is Rs Compensation Necessary?
Before embarking on Rs compensation, it is worth examining whether it is really
necessary in your application. The size of Rs can be estimated by selecting the
Whole Cell checkbox in the MultiClamp 700B Commander and pressing the Auto
button to compensate the whole-cell capacitance. (See Chapter 5, CAPACITANCE
COMPENSATION.) The estimated Rs is the MΩ value displayed to the right of the
manual adjust button under Whole Cell. If Rs = 10 MΩ and the maximum
membrane current you anticipate is 100 pA, the steady-state voltage error will be at
most 10 MΩ x 100 pA = 1 mV which is probably insignificant. In this case you
might think that Rs compensation is not necessary.
However, it should be remembered that dynamic voltage errors and bandwidth
errors can still occur in the above example, because these depend on Rs and Cm and
not on the size of the membrane current. Even if you are measuring only small
membrane currents in a whole-cell recording, application of Rs compensation can
greatly improve the fidelity of the voltage clamp.
As a general rule, it is best to try Rs compensation to see if it makes a difference.
This is certainly advisable in all whole-cell recordings. Compensation is rarely
useful with isolated membrane patches, which typically have small capacitance and
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 125
membrane currents. Indeed, the Whole Cell controls (which must be set before
using Rs compensation) are disabled with the 5 and 50 GΩ feedback resistors
typically used for isolated patch recordings. An exception is macropatches or
nucleated outside-out patches, in which the currents can be quite large and for
which Rs compensation may be necessary.
If Rs compensation is found not to be necessary, it is best to turn it off. This is
because Rs compensation increases noise.
Adjusting Rs Compensation
It is recommended that you practice adjusting Rs compensation with the
PATCH-1U model cell before using compensation in a real experiment. (See
Chapter 5, MODEL CELL.) Connect the CELL connector to the CV-7 headstage.
Set Primary Output to monitor Membrane Current Signal, and increase output Gain
to 10. Set the feedback resistor to 500 MΩ (for Voltage Clamp) and Seal Test to
100 mV at 50 Hz. Check the Seal Test checkbox and observe Membrane Current at
a fast sweep speed on an oscilloscope, triggering the oscilloscope so you can clearly
see the rising edge of the signal (Figure 5.15).
Chapter 5
126 • Reference Section
The first step is to fully compensate both the electrode capacitance (using the Cp
Fast/Slow controls) and the whole-cell capacitance (using the Whole Cell controls).
(See Chapter 5, CAPACITANCE COMPENSATION.) The estimated Rs – which is
the resistance we wish to compensate – is the MΩ value displayed under the Whole
Cell checkbox. After compensation the trace will look like Figure 5.16.
Figure 5.16. Uncompensated response (with saturating transients).
Figure 5.17. After compensating transients.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 127
Figure 5.18. After setting Prediction = 90%, Correction = 0%.
Figure 5.19. After setting Prediction = 90%, Correction = 90%.
Figure 5.20. After optimizing Cf, Rs and Cm to minimize transients.
Chapter 5
128 • Reference Section
Check the Rs Compensation checkbox. If the Prediction and Correction controls
are locked together ( ), press the Lock button to unlock them ( ). Set Rs
Compensation Bandwidth to 15 kHz.
Bandwidth vs. Lag
The MultiClamp 700B Rs Compensation Bandwidth control replaces the “Lag”
control on the Axopatch-1D and 200 series amplifiers. The relationship of
Bandwidth (BW) to Lag is defined as:
BW = 1 / (2 * π * Lag)
The default MultiClamp Rs Correction Bandwidth value is 1 kHz, which
equates to a Lag value of 160 µs. (2 kHz BW = 80 µs Lag, 10 kHz BW = 16 µs
Lag, etc.)
Increase Prediction to 90% (Figure 5.18). Note that Prediction is an open loop
process, i.e. it does not involve feedback, and instability is only possible if the
internal circuitry that develops the prediction signals is pushed too far. Generally,
the circuit is stable up to values of about 98%, but it can become non-linear,
depending on the magnitude of Vcmd. This may only become noticeable after
increasing the Primary Output Filter to 50 kHz bandwidth. Reduce Prediction
slightly if severe oscillations are observed.
Carefully increase the Correction value to equal that under Prediction. A rather large
transient should appear in the current at the beginning and end of the command step.
Its peak-to-peak amplitude should be 2-4 nA and it should undergo several distinct
“rings” requiring 1 ms to disappear into the noise (Figure 5.19). To eliminate this
transient, begin by reducing by a few percent the value of Rs (MΩ) displayed under
Whole Cell. As you reduce this setting, the amplitude of the transient first decreases
and then begins to increase. A distinct minimum exists and the desired value of Rs is
at this minimum.
Next, slightly adjust the Cp Fast settings, trying to further minimize any fast
leading-edge transients. When this has been done, small adjustments in the Whole
Cell capacitance (pF) value should completely eliminate any remaining transients
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 129
(Figure 5.20). If this is not possible in the real experiment, iterative fine
adjustments of Cp Fast and Whole Cell Rs may achieve the desired cancellation. If
all of this fails and the oscillations are too severe, you may need to go back to the
beginning and set the Prediction and Correction controls to lower values.
By reducing the Bandwidth control under Rs Compensation you can usually
increase the percent compensation without instability. However, this is likely to be
a false improvement if it is pushed too far. Reducing the Bandwidth slows down
the feedback circuit used in Rs compensation, reducing its dynamic response. For
example, if you limit the Bandwidth to 1 kHz, the Rs Compensation will be reduced
substantially for conductance changes faster than 160 us. Bottom line: if you
increase the Bandwidth value, you can measure faster conductance changes, but
you sacrifice Rs compensation stability. One tremendous advantage of the
MultiClamp 700B is that you can choose to automatically disable or reduce Rs
Compensation if oscillations should occur due to changes in membrane or pipette
properties during an experiment (see Tutorial 5 in Chapter 2).
In order to see the improvement brought about by Rs compensation, check and
uncheck the Rs Compensation checkbox. A dramatic speeding-up of the Membrane
Current should be apparent with the compensation correctly adjusted.
Theory of Rs Compensation
The MultiClamp 700B uses a dual approach for Rs compensation, like the Axopatch
200 series of amplifiers. This provides superior correction and stability.
Chapter 5
130 • Reference Section
For Rs compensation to function properly, whole cell compensation must have been
adjusted and the Whole Cell checkbox must be checked. Whole cell compensation
provides estimates of Rs and Cm, which together determine the shape of the
correction current that is injected through capacitor C2 (Figure 5.20). Note that this
C2 correction current does not improve the speed of clamping of the cell; rather, it
charges the membrane capacitance as slowly as before but in a way that is invisible
to the user, because it bypasses the feedback resistor in the headstage.
Figure 5.21. Schematic whole-cell compensation circuit.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 131
The ‘Prediction’ Control
After switching on Rs Compensation in the MultiClamp 700B Commander, the
Prediction control adds a transient signal to the command potential, speeding the
rate at which the true membrane potential will change in response to a step voltage
command. It is similar to the idea of “Supercharging” introduced by Armstrong
and Chow (1987). The signal added to the command is derived from the command
input and from the setting of the Whole Cell compensation parameters. It enables
the actual membrane potential to be a faithful replica of the command potential; i.e.
the effects of series resistance in distorting the command potential at the cell
membrane are removed up to the percentage setting of the control (e.g. a 98%
setting means that, in effect, only 2% of the original series resistance remains in
terms of command potential). The signal added by Prediction is injected through
the C2 capacitor used by whole cell capacitance compensation (See Figure 5.7).
The magnitude and time constant of this signal are determined by the pF and MΩ
settings under Whole Cell and by the Prediction setting.
For example, consider a whole-cell voltage clamp situation where Rs = 10 MΩ and
Cm = 50 pF and the resting membrane resistance Rm is very large with respect to Rs.
Assume that Whole Cell pF and MΩ are set at 10 MΩ and 50 pF, respectively, so
that the whole-cell capacity transient is perfectly canceled. If the Prediction control
is 0%, the signal applied to the headstage capacitor C2 (5 pF for 500M range and
53 pF for 50M range) in response to a step voltage command will have a time
constant of 500 µs and an amplitude that is appropriate to cancel a whole-cell
capacitance transient arising from these parameters (about 10 Vc). With 0%
Prediction nothing is added to the command potential waveform. In response to a
step voltage command the cell membrane potential will change to its new value
with a time constant of 500 µs (RsCm). If the % Prediction control is advanced to
50%, a transient will be added to the command potential step, Vc, with a time
constant of 250 µs and an amplitude equal to that of the command step itself. This
will have the effect of changing the cell membrane potential in response to a step
command with a time constant given by RsCm (1 - % Prediction /100); here this is
250 µs.
Chapter 5
132 • Reference Section
More formally, the command potential with the Prediction signal included, Vcp, can
be expressed in terms of the command input, Vc, by:
Vcp = Vc (1+sτs)/(1+sτsrp)
where τs = RsCm, τsrp = RsrpCm, where Rsrp is the residual (uncompensated) series
resistance in terms of Prediction, given by Rsrp = Rs (1 - % Prediction /100), and, in
the frequency domain s = jw (w is the natural frequency, w = 2πf), or in the time
domain s is the operator d/dt. Thus, Vcp = Vc·(1 + (Rs / Rsrp - 1)e-t/τsrp).
Moreover, the membrane potential, Vm, is given by Vm = Vcp/(1+sτs) = Vc/(1+sτsrp),
or Vm = Vc (1 - e-t/τsrp). Therefore, advancing the Prediction setting to 80% gives
Rsrp of 2 MΩ andτsrp of 100 µs. That is, the speed with which the membrane
potential responds to a voltage command is improved 5-fold over that which is
achieved with 0% Prediction. Prediction of 98% gives Rsrp of 200 kΩ and τsrp of
10 µs. The membrane potential will now respond to a step voltage command with a
10-90% risetime of about 22 µs and will settle to within 1% of its final value in less
than 50 µs.
Saturation Effects
Note that the equation presented above for Vcp (i.e. the command potential plus
Prediction signal) can be used to define the maximum allowable % Prediction for a
given size step voltage command. (This limit should not be confused with
limitations imposed by the stability of the Prediction circuit itself.) The command
plus Prediction signal is attenuated at the headstage by a 10:1 voltage divider.
Since the circuitry in the MultiClamp 700B main unit will saturate at about
±11-12 V, Vcp is limited in absolute value to about 1.1 to 1.2 V. To be conservative, we will use 1.1 V in the following calculations.
The peak amplitude of Vcp for a step voltage command, Vc, is given by Vc (Rs /Rsrp )
that can be rewritten as Vc / (1 - % Prediction /100). So we may state the limitation
on Vc as a function of % Prediction as:
Vc ≤ 1.1(1 - % Prediction /100)
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 133
or the limitation on % Prediction as a function of Vc as:
% Prediction ≤ 100(1 - Vc /1.1)
Thus, for example, if it is known that the maximum command step to be used in a
particular experiment is 100 mV, Prediction may be set at 91% without fear of
saturation of Vcp; this is true regardless of the value of Rs or Cm. In fact, this is a
rather conservative estimate since it is derived on the assumption that the signal Vcp
will instantly jump to its maximum value following a step voltage command. In
fact, due to limitations in the speed of the Prediction circuitry, this over-estimates
the maximum value of Vcp, particularly when % Prediction is large. In actual
practice, Prediction can typically be set to about 94% for a 100 mV command step.
Readjustment of Whole Cell Compensation with ‘Prediction’
As the Prediction potentiometer is advanced, the signal applied to the headstage
capacitor C2 is modified appropriately so that it will continue to cancel the wholecell capacity transient despite the fact that the speed of this transient has increased.
This is simply accomplished by reducing the time constant of this signal as %
Prediction is increased. If the circuitry worked perfectly, and if the whole-cell
capacity transient had been perfectly canceled with 0% Prediction, no transient
would appear as % Prediction is increased up to the maximum allowable values.
However, due to the complexity of this circuitry and a variety of non-ideal
characteristics, cancellation of whole-cell capacity transients does not remain
perfect as % Prediction is increased. The small residual transient that emerges can,
however, be completely removed by small readjustments of the setting of the Cp
Fast and Whole Cell controls. (See “Adjusting Rs Compensation”, above.)
It should be noted that Prediction would work for any command waveform, not just
steps. This may be useful for capacitance measurements using phase sensitive
techniques or lock-in amplifiers.
Chapter 5
134 • Reference Section
The ‘Correction’ Control
Although Prediction can greatly speed the response time of the true membrane
potential with respect to the command potential and, thus, overcome one important
effect of series resistance, it does not correct for the effects of series resistance
associated with the flow of membrane ionic current (i.e. IR drops and filtering
effects described above). This is the role of the % Correction value. Correction
feeds back a portion of the measured membrane current; this signal is added to the
command potential. The percentage set by the Correction potentiometer refers to
the Rs (MΩ) value under Whole Cell. For example, if this value is 10 MΩ, a 90%
setting of the Correction control means that 9 MΩ of series resistance is
compensated; the residual (uncompensated) series resistance in terms of Correction,
Rsrc, is 1 MΩ.
The Bandwidth setting under Rs Compensation gives the –3 dB cutoff frequency of
a one-pole RC filter through which the Correction signal is passed prior to being
summed with Vc. The Bandwidth is used to ensure stability when large amounts of
Correction are used. It is generally good practice to begin using Correction with the
Bandwidth set at 10 kHz or less. However, once the desired level of Correction has
been achieved, it is usually possible (if desired) to significantly increase the
Bandwidth setting; 30 kHz is usually quite achievable for 90% Correction.
Continuing with the example considered above (Rs = 10 MΩ, Cm = 50 pF), a 90%
Correction setting will reduce voltage errors in the true membrane potential
resulting from the flow of ionic current to 10% of the error present with 0%
Correction. For example, a 2 nA ionic current would produce a 20 mV error in Vm
with 0% Correction, whereas 90% Correction will reduce this error to only 2 mV.
At the same time, the use of Correction will reduce the filtering effect of Rs and Cm
on the measured current. With 0% Correction the actual bandwidth of current
measurement prior to any output filtering is limited to 1/2πRsCm, which will be
about 320 Hz in this example. As % Correction is increased this “filter” changes to
1/2πRsrcCm, so that for 90% Correction the possible bandwidth for current
measurement is increased to 3.2 kHz in this example. With 95% Correction the
possible bandwidth is increased to 6.4 kHz and with 98% it is further increased to
16 kHz (although the effects of the Bandwidth value should not be forgotten).
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 135
Readjustment of Whole Cell Compensation with ‘Correction’
If the capacity transient has been canceled prior to the use of Correction (and for
now assume that Prediction has already been set at 95%) then, in principle, there is
no capacity current to feed back when Correction is utilized. Note that the
discussion here of capacity current should be distinguished from the discussions of
the ionic current. Therefore, no transient should develop as Correction is advanced.
In practice, however, a small transient will emerge as % Correction is increased.
Again, this is due to non-ideal characteristics of the circuitry. As in the case of
‘Prediction’, the small residual transient that emerges can be completely removed
by small readjustments of the setting of the Cp Fast and Whole Cell controls. (See
“Adjusting Rs Compensation”, above.)
Setting ‘Prediction’ and ‘Correction’ Values
There are many situations in which it will be desirable to have the % Prediction and
the % Correction controls set at different values. For example, for a 200 mV step
command Prediction should be limited to about 80% to avoid saturation. (See
“Saturation Effects”, above.) However, it is usually possible to compensate series
resistance up to 90 to 95% or more by use of the Correction control. In other patch
clamps the issue of saturation would limit the amount of compensation used for
ionic currents; this is not true in the MultiClamp 700B. On the other hand, in some
cases it might be impossible to advance the Correction percentage beyond about
70% without causing instability. Nevertheless, Prediction, which is inherently
stable up to 98% or more, can be set to a value substantially higher than 70% (about
95%), thereby ensuring that the true transmembrane potential changes rapidly in
response to the command potential even though a substantial series resistance
remains uncompensated in terms of ionic currents.
Oscillations
One of the practical problems when using the % Correction function of Rs
Compensation is that there is a great risk of oscillations because the Correction
circuitry is a form of positive feedback. The main cause of oscillations is the
Chapter 5
136 • Reference Section
inability of the circuitry to distinguish between current that flows down the
electrode and into the cell from current that flows through the stray capacitance of
the electrode into the bath. The current that flows through the electrode resistance
into the cell is the current that is intended to be compensated. The Correction
circuitry also tries to compensate for the current into the electrode capacitance.
However, in this case there is no significant series resistance component to
compensate, and the Correction circuit will oscillate as soon as the Correction
control is advanced.
The tendency to oscillate therefore depends on the relative magnitude of the
electrode resistance to the electrode capacitance and the degree of compensation of
the electrode capacitance. Thus, one should take care that Cm is well compensated
as one advances correction. In addition, the tendency to oscillate can be reduced by
limiting the bandwidth of the positive-feedback circuit. This is the function of the
Bandwidth control.
Limitations of Rs Compensation
Series-resistance compensation is an attempt to electronically reduce the effect of
the electrode resistance. Because of practical limitations, it is never perfect. Even
if 100% compensation could be used with stability, this would only apply to DC
and medium-speed currents. Very fast currents cannot be fully corrected.
For best results, the cell membrane resistance should be many-fold higher than the
electrode resistance. This is normally the case for cells at rest containing small
drug-activated or synaptic currents. However, during voltage activation the cell
membrane resistance could fall a hundredfold or more to values similar to or less
than the series resistance. In these cases it is probable that:
1. There will be a significant error due to the voltage drop across the
electrode. This error is not obvious to the user because the patch clamp
controls the combined voltage drop across the electrode and the cell.
2. The setting of the Whole Cell compensation controls will become
erroneous because it is based on the time constant to charge the membrane
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Reference Section • 137
capacitance before the change in membrane resistance occurred. Since this
time constant depends on the parallel value of membrane resistance and the
electrode series resistance, this error could become substantial.
If the cell input resistance becomes comparable to, or less than, the electrode
resistance, the whole-cell patch clamp technique will probably not work. In this
situation it would be preferable to use a discontinuous (chopped) single-electrode
voltage clamp, such as the Axoclamp.
SoftPanel Configuration
The SoftPanel is an optional instrument that provides knob and button control in
place of mouse gliders and clicks in the MultiClamp 700B Commander software.
The SoftPanel is merely a hardware extension of the Commander, and replicates the
many Commander control functions of the MultiClamp 700B.
The SoftPanel comes with a magnetic overlay with pre-defined functions assigned
to the various knobs and buttons. However, the SoftPanel can easily be reconfigured in the MultiClamp 700B Commander software. Click on the Configure
SoftPanel toolbar icon ( ) to access the menus for re-configuring each knob or
button.
After assigning the desired functions to each knob or button, remove the predefined magnetic overlay to reveal the erasable surfaces at each knob or button.
Re-label the position with the appropriate function using a marking pen. (Sharpie®
pens are appropriate on this special surface.)
Figure 5.22
Chapter 5
138 • Reference Section
Status
•
STATUS light on the front panel of the MultiClamp 700B indicates traffic on
the USB cable.
•
See also Chapter 6, TROUBLESHOOTING.
The STATUS light illuminates whenever data is being transmitted on the USB
cable that connects the MultiClamp 700B to the host computer. Under quiescent
conditions the STATUS light flashes at about 2 Hz, indicating that the MultiClamp
700B Commander is interrogating the MultiClamp 700B in order to update its
meter displays.
The STATUS light is useful for troubleshooting. If it does not flash continuously, a
communication problem is indicated. (See TROUBLESHOOTING.)
Zap
•
Zap applies a large, brief voltage pulse to the electrode when in V-Clamp mode,
to facilitate breaking into a cell for whole-cell recording.
•
Zap is triggered by pressing the
button in the V-Clamp pane.
The conventional method for rupturing a membrane patch to go to whole-cell
recording is to apply a pulse of suction. Sometimes this method damages the cell.
Zap provides an alternative method. It applies a large (1 V) voltage pulse that
ruptures the patch, presumably by causing dielectric breakdown of the membrane.
The Zap duration can be varied; it is best to use the minimum duration that is likely
to achieve the desired result, because too long a Zap could cause the seal resistance
to deteriorate. A duration of 0.5 or 1 ms is suggested for initial attempts.
Apply a repetitive test pulse (e.g. Seal Test) and press the Zap button while
carefully monitoring Membrane Current. Sometimes it helps to apply steady
suction while Zapping. Successful break-through is signaled by an increase in the
current noise and by large whole-cell capacitance transients.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Troubleshooting • 139
Chapter 6
Troubleshooting
It has been our experience at Axon Instruments that the majority of troubles
reported to us have been caused by faulty equipment connected to our instruments.
If you have a problem, please physically disconnect all instruments connected to
the MultiClamp 700B except for the oscilloscope. Ideally, remove the MultiClamp
700B from the rack. Work through Chapter 2, INSTALLATION AND BASIC
OPERATION. This can often uncover a problem that is in your setup. In order to
force the MultiClamp 700B Commander to recheck the hardware configuration,
press the Select Device button in the toolbar. (See Chapter 5, SELECT DEVICE.)
Some common problems are listed below.
Symptom: The MultiClamp 700B is not responding to commands. The Status
light is not flashing.
Possible causes: The USB cable is not plugged in properly or is defective.
The PC’s USB port is defective. Select Device has been set to Demo rather
than MultiClamp Hardware, or the correct Device Number has not been set.
Suggestions: Check the USB cable. Check that the PC’s USB port works
with other serial instruments, or try a different port. Press the Scan button in
the Select Device window to ensure that the MultiClamp 700B Commander
can find the correct device.
Chapter 6
140 • Troubleshooting
Symptom: Unable to adjust the Pipette Offset to zero.
Possible causes: There may be a break in the connection between the
headstage input and ground, causing the input to float. The bath may be
leaking, producing a short circuit to the microscope. In I-Clamp mode, the
capacitance neutralization circuit may be oscillating.
Suggestions: Check the electrical continuity and DC stability of the electrode
holder and bath electrode. Check for bubbles in the microelectrode. Check
that the outside of the chamber is dry. Set Pipette Capacitance Neutralization
to zero.
Symptom: Extraneous noise is present in the Primary Output signal. Pipette
Offset is drifting rapidly.
Possible cause: The Ag/AgCl pellet or Ag wire in the electrode holder may
be defective. Dirt or corrosion may have built up in the holder or headstage
connector socket.
Suggestions: Check the DC stability of the pellet and replace if necessary.
Rechloride the Ag wire. Clean the holder and headstage connectors.
If the problem cannot be resolved, please contact Axon Instruments for technical
support (1-800-635-5577 or [email protected]).
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Specifications • 141
Chapter 7
Specifications
Unless otherwise specified, TA = 20oC, 1 hr warm-up time.
Main Unit
Line Voltage 85 - 260V
Line frequency 50 - 60 Hz
Fuse 5 mm x 20 mm 2A slow
Case 8.89 cm high x 48.26 cm x 30.48 cm deep (3.5˝ x 19˝ x 12˝ deep) rack
mountable
CV-7 Headstage
Dimensions 4.06 x 8.38 x 2.03 cm (1.6˝ x 3.3˝ x 0.8˝)
Voltage Clamp
Gain: Rf = 50 GΩ , 5 GΩ, 500 MΩ, 50 MΩ
10 kHz Noise (8-pole Bessel filter):
50 G
5G
500 M
50 M
0.28 pArms
0.9 pArms
1.4 pArms
3.0 pArms
Specifications
142 • Specifications
5 kHz Noise (4-pole Butterworth filter):
50 G
5G
500 M
50 M
0.15 pArms
0.5 pArms
0.8 pArms
2.0 pArms
Fast capacitance compensation magnitude:
0 - 12 pF for 50 G range.
0 - 36 pF on all other ranges.
Fast capacitance compensation tau:
0.5 µs to 1.8 µs.
Slow capacitance compensation magnitude:
0 - 1 pF for 50 G range.
0 - 3 pF on all other ranges.
Slow capacitance compensation tau:
10 µs to 10 ms in two ranges (10 – 200 µs and 200 – 4000 µs).
Whole cell capacitance compensation:
Cm from 1 pF to 100 pF and Rs from 400 k to 1000 M on 500 M range.
Cm from 2.5 pF to 1000 pF and Rs from 100 k to 100 M on 50 M range.
Series Resistance compensation:
Bandwidth is adjustable from 0.32 to 16 kHz.
Series resistances corrected varies from 0.4 to 1000 M on 500 M range and
0.1 to 100 M on 50 M range.
Current Clamp
Rise time < 10 µs for load of 10 M on 50 M range (Output Filter bypassed).
Rise time < 30 µs for load of 100 M on 500 M range.
Rise time < 150 µs for load of 1 G on 5 G range.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Specifications • 143
Test Signals
Voltage Clamp
The available test signals are Seal Test, Pulse and Zap.
Seal Test and Pulse amplitudes are selectable from 0 to ±1 V at the electrode.
Seal Test frequency is selectable from 2 to 1000 Hz.
Pulse duration is selectable from 0.1 to 500 ms.
Zap is fixed at +1V at the electrode but with selectable 0.1 to 50 ms duration.
Current Clamp
The available test signals are Tune, Pulse, Buzz and Clear (+/-).
Tune and Pulse amplitudes are selectable from 0 to ±10 V/Rf amps at the
electrode.
Tune frequency is selectable from 2 to 1000 Hz.
Pulse duration is selectable from 0.1 to 500 ms.
Buzz amplitude is fixed at ±15 V signal to the headstage capacitor but with
selectable 0.05 to 500 ms duration.
Clear (+/-) amplitude is fixed at ±15 V signal to the headstage capacitor.
DC Holding Commands
Voltage Clamp
±1000 mV range in 30 µV steps
Auto Pipette Offset adjusts DC holding potential to zero Membrane Current.
Current Clamp
±20 nA range in 0.7 pA steps (50 MΩ range)
±2 nA range in 0.07 pA steps (500 MΩ range)
±0.2 nA range in 0.007 pA steps (5 GΩ range)
Note: External command can provide up to 10 times the above holding
currents.
Auto Pipette Offset adjusts DC holding current to zero Membrane Potential.
Specifications
144 • Specifications
Output Gain and Filters
Output Gain
Primary: Post-filter gain of 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000.
Secondary: Post-filter gain of 1, 2, 5, 10, 20, 50, 100.
Primary Output Filters
Lowpass four-pole Bessel frequencies (Hz): 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280,
300, 400, 600, 800, 1k, 1k2, 1k4, 1k6, 1k8, 2k, 2k2, 2k4, 2k6, 2k8, 3k, 4k, 6k,
8k, 10k, 12k, 14k, 16k, 18k, 20k, 22k, 24k, 26k, 28k, 30k, Bypass.
Lowpass four-pole Butterworth frequencies (Hz): 3, 6, 9, 12, 15, 18, 21, 24, 27,
30, 33, 36, 39, 42, 45, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390,
420, 450, 600, 900, 1k2, 1k5, 1k8, 2k1, 2k4, 2k7, 3k, 3k3, 3k6, 3k9, 4k2, 4k5,
6k, 9k, 12k, 15k, 18k, 21k, 24k, 27k, 30k, 33k, 36k, 39k, 42k, 45k, Bypass.
Highpass single-pole Bessel frequencies (Hz): DC, 0.1, 1, 3, 10, 30, 100, 300.
Secondary Output Filters
Lowpass single-pole Bessel filter fixed at 10 kHz frequency, or Bypass.
Scope Filter
Lowpass two-pole Bessel filter with four –3 dB cutoff frequencies (Hz): 1k, 3k,
10k, Bypass.
Command Inputs
20 mV/V or 100 mV/V sensitivity for V-Clamp;
400 pA/V or 2 nA/V sensitivity for I-Clamp.
Input impedance is 10 kΩ.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Specifications • 145
Mode Switching
External
When enabled in MultiClamp 700B Commander software, 0 V input to MODE
BNC selects I-Clamp mode and 5 V input selects V-Clamp mode. This mode
can be used in conjunction with Internal Auto Mode switching to return mode
to I-Clamp (see Internal Mode Switching).
Internal
When enabled in MultiClamp 700B Commander Options / Auto menu, switch
from I-Clamp to V-Clamp is automated when Vm threshold crossing is
detected.
Positive to Negative or Negative to Positive crossing
Vm threshold: ±1000 mV
Delay to switch: 0-500 ms, in 2 ms steps
Delay to return from V-Clamp: 20ms – 500 seconds, in 10 ms steps
(this can also be done manually or with External Mode BNC)
Switching Speeds
Auto, from I-Clamp to V-Clamp: < 0.5 ms
Auto, from V-Clamp to I-Clamp: ≈ 22 ms
Mode switching performed manually with the mouse, keyboard or SoftPanel
interface will always be slower than automatic switching, due to delays in
computer operating system communication. Add approximately 30 msec to the
above speeds to estimate typical manual switching speeds.
Audio Monitor
The Audio Monitor output can select Current, Voltage or Voltage x 100 for either
Channel 1 or Channel 2. The selected signal is available for direct monitoring or
via a voltage-to-frequency converter (VCO). The VCO ranges from ~4000 Hz @
+100 mV to ~300 Hz at –100 mV.
Specifications
References • 147
References
Armstrong, C.M. and Chow, R.H. Supercharging: a new method for improving
patch-clamp performance. Biophys. J. 52:133-136, 1987.
Ebihara, S., Shirato, K., Harata, N. and Akaike, N. Gramicidin-perforated patch
recording: GABA response in mammalian neurones with intact intracellular
chloride. J. Physiol. 484:77-86, 1995.
Cota, G. and Armstrong, C.M. Potassium channel “inactivation” induced by softglass patch pipettes. Biophys. J. 53:107-109, 1988.
Finkel, A.S. and Redman, S.J. Optimal voltage clamping with a single
microelectrode. In: Voltage and Patch Clamping with Microelectrodes, Smith,
T.G., Lecar, H., Redman, S.J., Gage, P.W. (Eds), Williams & Wilkins: Baltimore,
1985.
Furman, R.E. and Tanaka, J.C. Patch electrode glass composition affects ion
channel currents. Biophys. J. 53: 287-292, 1988.
Hamill, O.P., Marty, A., Sakmann, B. and Sigworth, F.J. Improved patch-clamp
techniques for high-resolution current recording from cells and cell-free membranes
patches. Pflügers Arch. 391: 85-100, 1981.
Johnston, D. and Brown, T.H. Interpretation of voltage-clamp measurements in
hippocampal neurons. J. Neurophysiol. 50:464-486, 1983.
Purves, R.D. Microelectrode Methods for Intracellular Recording and
Ionophoresis. Academic Press: London, 1981.
References
148 • References
Rae, J., Cooper, K., Gates, P. and Watsky, M. Low access resistance perforated
patch recordings using amphotericin B. J. Neurosci. Meth. 37:15-26, 1991.
Sakmann, B. and Neher, E. Single-Channel Recording. (Second Edition) Plenum
Press: New York, 1995.
Sherman-Gold, R. The Axon Guide for Electrophysiology & Biophysics
Laboratory Techniques. Axon Instruments, Foster City, CA. 1993. Available at
http://www.axon.com
Yawo, H. and Chuhma, N. An improved method for perforated patch recordings
using nystatin-fluorescein mixture. Jap. J. Physiol. 43:267-273, 1993.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Technical Assistance • 149
Technical Assistance
If you need help to resolve a problem, there are several ways to contact Axon
Instruments / Molecular Devices:
World Wide Web
www.axon.com
Phone
1 (800) 635-5577
Fax
+1 (510) 675-6300
E-mail
[email protected]
Questions?
See Axon's Knowledge Base: http://support.axon.com
Technical Assistance
Warranty and Repair Service • 151
Warranty and Repair Service
Warranty
Axon Instruments / Molecular Devices Corp. warrants its non-consumable
hardware products to be free from defects in materials and workmanship for 12
months from date of invoice. The warranty covers the cost of parts and labor to
repair the product. Products returned to our factory for repair must be properly
packaged with transportation charges prepaid and the shipment fully insured. Axon
Instruments / Molecular Devices will pay for the return shipping of the product to
the customer. If the shipment is to a location outside the United States, the
customer will be responsible for paying all duties, taxes and freight clearance
charges if applicable.
The warranty is valid when the product is used for its intended purpose and does
not cover products which have been modified without approval from Axon
Instruments, or which have been damaged by abuse, accident or connection to
incompatible equipment.
To obtain warranty service, follow the procedure described in the Repair Service
section. Failure to do so will cause long delays and additional expense to the
customer.
This warranty is in lieu of all other warranties, expressed or implied.
Warranty and Repair Service
152 • Warranty and Repair Service
Repair Service
The company reserves the right to cease providing repair maintenance, parts and
technical support for its non-consumable hardware products five years after a
product is discontinued. Technical support for old versions of software products
will cease 12 months after they are upgraded or discontinued.
If you purchased your instrument from a Distributor or OEM Supplier, contact them
for repair service.
If you purchased your instrument from Axon Instruments / Molecular Devices,
contact our Technical Support Department. If it is determined your instrument
must return to the factory for repair, the Technical Support Representative will
issue a Service Request (SR) number. Our Logistic Coordinator will contact you
with specific instructions.
Shipping
The MultiClamp 700B is a solidly built instrument designed to survive shipping
around the world. However, in order to avoid damage during shipping, the
MultiClamp 700B must be properly packaged.
In general, the best way to package the MultiClamp 700B is in the original factory
carton. If this is no longer available, we recommend that you carefully wrap the
MultiClamp 700B in at least three inches (75 mm) of foam or "bubble-pack"
sheeting. The wrapped instrument should then be placed in a sturdy cardboard
carton. Mark the outside of the box with the word FRAGILE and an arrow
showing which way is up.
We do NOT recommend using loose foam pellets to protect the MultiClamp 700B.
During shipping, there is good chance that the instrument will shift within the loose
pellet packing and be damaged.
If you need to ship the MultiClamp 700B to another location, or back to the factory,
and you do not have a means to adequately package it, Axon Instruments /
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices
Warranty and Repair Service • 153
Molecular Devices can ship the proper packaging material to you for a small fee.
This may seem an expense you would like to avoid, but it is inexpensive compared
to the cost of repairing an instrument that has sustained shipping damage.
It is your responsibility to package the instrument properly before shipping. If the
packaging is inadequate, and the instrument is damaged during shipping, the
shipper will not honor your claim for compensation.
Warranty and Repair Service
Circuit Diagrams Request Form • 155
Circuit Diagrams Request Form
All the information that you require for operation of the MultiClamp 700B is included in the operator's manual. In
the normal course of events, the MultiClamp 700B does not require any routine maintenance. If, for some reason,
the headstage is changed, the MultiClamp 700B must be recalibrated. In anticipation of this, the recalibration
procedures are described in the operator's manual, and circuit diagrams are not required.
Should you need the circuit diagrams for the MultiClamp 700B, Axon Instruments / Molecular Devices will be
pleased to supply them to you. However, we caution you that the MultiClamp 700B is a sophisticated instrument
and that service should only be undertaken by talented electronics experts. Diagrams for the CV-7 or B headstages
are not available.
To request a copy of the circuit diagrams and the parts lists, please complete the form at the bottom of this page and
mail it to:
Axon Instruments / Molecular Devices Corp
Sales Department
3280 Whipple Road
Union City, CA 94587
USA
This form must be completed in full and signed. Telephone orders will not be accepted.
Name of registered owner: ________________________________________________________________________
Department: ___________________________________________________________________________________
University/Institute: _____________________________________________________________________________
Street address: __________________________________________________________________________________
City: ________________________________ State: ________ Zip Code: _____________ Country: ______________
Telephone: ______________________________________________ Fax: _________________________________
Model: MultiClamp 700B
Serial number: ____________________
Declaration
Please send me the circuit diagrams and parts lists for the MultiClamp 700B. I agree that I will only use the circuit
diagrams and parts lists for service of the MultiClamp 700B. I will not use them to create equivalent or competing
products. If I transfer the circuit diagrams or copies thereof to someone who is assisting in the service of the
MultiClamp 700B, I will ask them to make the same undertaking that I am declaring herein.
Signature:______________________________________________________
Date: _____________________
Name:_________________________________________________________
Title: _____________________
Circuit Diagrams Request Form
Declaration of Conformity • 157
Declaration of Conformity
Manufacturer:
Axon Instruments / Molecular Devices
3280 Whipple Road
Union City, CA 94587
USA
Type of Equipment:
Computer-Controlled Microelectrode Amplifier
Model Number:
MultiClamp 700B
Year of Manufacture:
2003
Application of Council Directives:
EC EMC Directive 89/336/EEC as amended
EC Low Voltage Directive 73/23/EEC as amended
Harmonized Standards to which Conformity is Declared:
EMC:
EN 61326-1: 1997 (A1: 1998 A2: 2001)
EN 55011/CISPR11: 1998 AS/NZS 2064: 1997
Safety: EN 61010-1: 2001
I, the undersigned, hereby declare that the equipment specified above conforms to
the above Directives and Standards.
Authorized Signature and Date:
(Signature on file)
Declaration of Conformity
Important Safety Information • 159
Important Safety Information
DISCLAIMER
Safe Environmental Conditions
THIS EQUIPMENT IS NOT INTENDED TO BE USED AND
SHOULD NOT BE USED IN HUMAN EXPERIMENTATION OR
APPLIED TO HUMANS IN ANY WAY.
1. Indoor use.
2. Mains supply fluctuations: not to exceed ±10% of the
nominal voltage.
3. Temperature: between 5 ºC and 40 ºC.
4. Altitude: up to 2000 m.
5. This instrument is designed to be used under laboratory
conditions. Operate in a clean, dry environment only. Do
not operate in a wet or damp environment.
WARNING
IF THIS EQUIPMENT IS USED IN A MANNER NOT SPECIFIED
BY THE MANUFACTURER, THE PROTECTION PROVIDED BY
THE EQUIPMENT MAY BE IMPAIRED.
Power-Supply Voltage Selection and Fuse Changing
Supply Voltage
The MultiClamp 700B can be directly connected to all
international supply voltages. The input range is from 100 to
240 V~. No range switching is required. Alternatively, the
instrument can be powered by a DC voltage of 120 to 310 V.
Changing the Fuse
The MultiClamp 700B uses a 250 V~, T2A, 5 x 20 mm fuse.
In the event of fuse failure, disconnect the power cord.
Before changing the fuse investigate the reason for its failure.
To change the fuse:
1. Disconnect the power cord.
2. Use a screwdriver or a similar device to rotate the fuse
holder counterclock-wise.
3. Replace the fuse with another fuse of the same rating.
4. Reconnect the power cord.
Basic Equipment Setup and Safety
1. Connections: Use the included IEC power cord to connect
the instrument to a GROUNDED power receptacle.
2. Mounting: Table or rack.
3. Assembly: The headstage connects to the instrument
through the rear panel, 25 pin D-sub connector marked
"Headstage". Power should always be turned OFF when
connecting headstages to the main unit.
4. Use: Do not operate this equipment with covers or panels
removed.
5. Cleaning: Wipe the headstage connector with a damp cloth
to clean salt spills. Avoid spilling liquids on the headstage.
The Teflon input connector should be kept very clean.
Effective cleaning can be done by swabbing carefully with
denatured alcohol or deionized water. If possible, avoid the
use of Freon since it is thought to be detrimental to the
environment.
Static Precautions
If you are in a laboratory where static is high (i.e., you hear
and feel crackles when you touch things), you should touch a
grounded metal object immediately before touching the
headstage.
Shipping the MultiClamp 700B
The MultiClamp 700B is a solidly built instrument designed to
survive shipping around the world. However, in order to avoid
damage during shipping, the MultiClamp 700B must be
properly packaged.
In general, the best way to package the MultiClamp 700B is in
the original factory carton. If this is no longer available, we
recommend that you carefully wrap the MultiClamp 700B in at
least three inches (75 mm) of foam or "bubble-pack" sheeting.
The wrapped MultiClamp 700B should then be placed in a
sturdy cardboard carton. Mark the outside of the box with the
word FRAGILE and an arrow showing which way is up.
We do not recommend using loose foam pellets to protect the
MultiClamp 700B. If the carton is dropped by the shipper,
there is a good chance that the MultiClamp 700B will shift
within the loose pellet packaging and be damaged.
If you need to ship your MultiClamp 700B to another location,
or back to the factory, and you do not have a means to
adequately package it, Axon Instruments can ship the proper
packaging material to you for a small fee. This may seem like
an expense you would like to avoid, but it is inexpensive
compared to the cost of repairing an instrument that has
sustained shipping damage.
It is your responsibility to package the instrument properly
before shipping. If it is not, and it is damaged, the shipper will
not honor your claim for compensation.
Important Safety Information
160 • Renseignments Importants
Renseignments Importants
LIMITE DE RESPONSABILITE
CE MATERIEL N'A PAS ETE CONCU POUR DES EXPERIENCES SUR
LES ETRES HUMAINS; ET NE DOIT DONC PAS ETRE UTILISE A
CETTE FIN.
ATTENTION
L'EMPLOI DE CE MATERIEL D'UNE MANIERE DIFFERENTE A CELLE
SPECIFIEE PAR LE FABRICANT AFFECTERA LE NIVEAU DE
PROTECTION FOURNIT PAR L'APPAREIL.
Sélection du voltage et changement du fusible
Voltage d'alimentation
Le MultiClamp 700B peut être directement branché sur toutes
alimentations comprises entre 100 et 240 V~. Aucun changement
n'est nécessaire afin de sélectioner le voltage de l’appareil. En outre,
l'appareil peut être aussi alimenté en courant continu (DC) de 120 à
310 V.
Changement du fusible
Le MultiClamp 700B emploie un fusible de 250 V~, T2A, 5 × 20
mm.
En cas de rupture du fusible, débrancher la prise de courant.
Avant de changer le fusible, chercher la raison de la panne.
Pour changer le fusible:
1. Débrancher la prise de courant.
2. A l'aide d'un tournevis ou autre outil de ce genre, faire tourner le
support du fusible dans le sens opposé des aiguilles d'une
montre.
3. Remplacer le fusible par un fusible de même valeur.
4. Rebrancher la prise de courant.
Installation du matériel et sécurité
1. Branchement: Employer le fil electrique IEC fourni pour brancher
l'appareil a une prise de courant comprenant UNE TERRE.
2. Pose: Table ou rack.
3. Montage: La tête de l'amplificateur (“headstage”) est connectée
à l'appareil sur le panneau arrière, par l'intermediere d'une prise
D-sub à 25 fiches portant l'indication “Headstage”.
4. Emploi: Ne pas utiliser ce matériel sans son couvercle et ne pas
le couvrir lors de son utilisation.
5. Nettoyage: Essuyer la prise du “headstage” avec un linge
humide pour nettoyer les traces de sel. Eviter de renverser des
liquides sur le “headstage”.
La prise d'entrée en Téflon doit être maintenue trés propre. Un
nettoyage efficace consiste à vaporiser de l'alcool ou á essuyer
soigneusement avec de l'eau désionisée Si possible, éviter
l'emploi de Fréon, ce produit étant considéré comme nuisible
pour l'environnement.
2. Fluctuations du réseaux d'alimentation: ne doivent pas dépasser
±10% de la tension nominale.
3. Température: entre 5 °C et 40 °C.
4. Altitude: jusqu'à 2000 m.
5. Cet appareil a été étudié pour l'emploi en laboratoire et il doit être
situé dans un environnement sec et propre. Ne pas l'utiliser
dans un environnement mouillé ou humide.
Précautions statiques
Le “headstage” peut être maniée sans danger. Cependant, dans un
laboratoire avec un niveau élevé d'electricité statique (c'est-à-dire
lorsque vous sentez et voyez des décharges électriques), touchez
un objet métallique pour une mise à la terre avant de toucher le
“headstage”.
Ne pas d'ébrancher le MultiClamp 700B lors de la manipulation de
l'entrée du “headstage”, ceci risque de déranger son équilibre
thermique.
Expédition du MultiClamp 700B
Le MultiClamp 700B est un appareil de construction robuste, étudié
en vue d'expéditions dans le monde entier. Cependant, l'appareil
doit être correctement emballé pour éviter tout domage pendant son
transport.
En général, la meilleure façon d'emballer le MultiClamp 700B est de
le mettre dans son carton d'origine. Si celui-ci n'est plus disponible,
il est recommandé d'envelopper soigneusement le MultiClamp 700B
dans au moins trois inches (75 mm) de mousse ou de feuilles
d'emballage à bulles. Le MultiClamp 700B ainsi protégé devra alors
être placé dans un carton solide. Indiquer la mention FRAGILE sur
l'extérieur de la boîte ainsi qu'une flèche vers le haut montrant la
position verticale.
Il n'est pas recommandé d'employer des boulettes de mousse pour
protéger le MultiClamp 700B. En cas de chute de la boîte durant
son transport, le MultiClamp 700B pourrait se déplacer à l'intérieur
et être endommagé.
Si vous devez expédier le MultiClamp 700B à un autre endroit, ou le
renvoyer au fabricant, et si les matériaux d'emballage nécessaires
corrects ne sont pas disponibles, ces derniers peuvent être obtenus
chez Axon Instruments pour un prix minime. Bien que ceci puisse
sembler être une dépense que vous pourriez éviter, elle est
cependant insignificante en comparaison à celle que coûterait la
réparation d'un appareil endommagé pendant le transport.
La responsabilité vous incombe de bien emballer l'appareil avant
son expédition. Si ceci n'est pas fait, le transporteur ne pourra pas
satisfaire vos réclamation de compensation en cas d'avaries.
Conditions à respecter pour un emploi sans danger
1. Emploi à l'intérieur.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
Wichtige Informationen • 161
Wichtige Informationen
UNZULÄSSIGE VERWENDUNG
DIESER APPARAT IST NICHT VORGESEHEN, BEI MENSCHLICHEN
VERSUCHEN VERWENDET ZU WERDEN UND AUCH NICHT AN
MENSCHEN IN IRGENDEINERWEISE ANWENDBAR.
WARNUNG
WEN DIESER APPARAT IN EINER ART UND WEISE ANGEWENDET
WIRD, DIE NICHT VOM HERSTELLER SPEZIFISCH ERWÄHNT
WIRD, KANN DIE SCHUTZVORRICHTUNG DES APPARATES
BEEINTRÄCHTIGT WERDEN.
Spannungswahl für die Stromversorgung und Auswechseln der
Sicherung
Netzspannung
Der MultiClamp 700B kann direkt an alle internationalen
Netzspannungen angeschlossen werden. Die Eingangsspannung
reicht von 100 bis 240 V~. Ein Umschalten des
Spannungsbereichs ist nicht erforderlich. Das Instrument kann auch
mit Gleichstromspannungen von 120 bis 310 V betrieben werden.
Auswechseln der Sicherung
Der MultiClamp 700B verwendet eine 250V~, T2A, 5 x 20 mm
Sicherung.
Im Falle des Ausfalls der Sicherung das Netzkabel ausschalten.
Vor dem Auswechseln der Sicherung den Grund für ihren Ausfall
untersuchen.
Schritte zum Auswechseln der Sicherung:
1. Das Netzkabel ausschalten.
2. Die Fassung der Sicherung mit einem Schraubenzieher oder
einem ähnlichen Werkzeug entgegen dem Uhrzeiger drehen.
3. Die Sicherung mit einer anderen Sicherung mit gleicher
Nennleistung ersetzen.
4. Das Netzkabel wieder anschließen.
Grundlegende Hinweise zu Installation und Sicherheit der Ausrüstung
1. Netz- und Erdungsanschlüsse: Das Instrument mit dem
beigefügten IEC Netzkabel an einen Erdungsschalter anschließen.
2. Anbringung: Tisch oder Rahmengestell.
3. Montage: Der Vorverstärker (“headstage”) wird über einen mit
der Aufschrift “Headstage gekennzeichneten 25 Pin DUnterstecker an der Rückwand des Instrumentes verbunden.
4. Gebrauch: Dieser Apparat darf nicht mit abgenommenen
Abdeckungen oder Platten in Betrieb gesetzt werden.
5. Reinigung: Zur Reinigung von verschüttetem Salz den
Vorverstärkeranschluß mit einem feuchten Tuch abwischen. Das
Verschütten von Flüssigkeiten auf den “headstage” ist zu
vermeiden.
Der Teflon-Eingangsstecker sollte in sehr sauberem Zustand
gehalten werden. Durch Besprühen mit Alkohol oder
vorsichtigem Abtupfen mit entionisiertem Wasser ist eine
wirksame Reinigung möglich. Die Benutzung von Freon ist nach
Möglichkeit zu vermeiden, da diese Substanz als
umweltschädigend angesehen wird.
Umweltsichere Betriebsbedingungen
1. Verwendung in Innenräumen.
2. Netzschwankungen: darf nicht ±10% der Nennspannung
überschreiten.
3. Temperatur: zwischen 5 °C und 40 °C.
4. Höhe: bis zu 2000 m.
5. Dieses Instrument ist für den Gebrauch unter Laborbedingungen
vorgesehen. Nur in sauberer, trockener Umgebung in Betrieb
setzen. Nicht in nasser oder feuchter Umgebung in Betrieb setzen.
Schutzmaßnahmen gegen statische Aufladung
Der “headstage” kann normalerweise sicher gehandhabt werden.
Falls Sie sich jedoch in einem Labor mit höher statischer Aufladung
befinden (D.h. Sie hören und fühlen beim Berühren von Objekten ein
Knacken), sollten Sie unmittelbar vor dem Berühren der “headstage”
ein geerdetes Objekt aus Metall anfassen.
Bei Handhabung des Vorverstärkereingangs sollten Sie die Stromzufuhr
zum MultiClamp 700B nicht abschalten, um das
Temperaturgleichgewicht nicht zu stören.
Versand des MultiClamp 700B
Bei dem MultiClamp 700B handelt es sich um ein solide gebautes
Instrument, das beim weltweiten Versand keinen Schaden nehmen
sollte. Um jedoch Versandschäden zu verhindern, muß der
MultiClamp 700B ordnungsgemäß verpackt werden.
Im allgemeinen läßt sich der MultiClamp 700B am besten im
Originalkarton des Werks verpacken. Ist dieser nicht mehr
vorhanden, empfehlen wir, den MultiClamp 700B vorsichtig in
mindestens 75 mm starkem Schaumstoff oder Bubblepackungen
einzuwickeln. Der so eingewickelte MultiClamp 700B sollte dann in
einen festen Pappkarton gesetzt werden. Die Außenseite des
Kartons ist mit dem Worten ZERBRECHLICH (FRAGILE) und einem
Pfeil, der auf die Oberseite des Kartons weist, zu kennzeichnen.
Sollte der Karton vom Spediteur fallengelassen werden, besteht eine gute
Möglichkeit, daß der MultiClamp 700B innerhalt der losen
Schaumstoffkugelverpackung bewegt wird und dadurch beschädigt
werden kann.
Wenn Sie den MultiClamp 700B an einen anderen Ort oder zurück
ans Werk senden müssen und Ihnen kein angemessenes
Verpackungsmaterial zur Verfügung stehen, kann Axon Instruments
Ihnen das geeignete Verpackungsmaterial gegen eine kleine Gebühr
zustellen. Sie mögen dies zwar als unnötige Zusatzkosten
betrachten, doch ist dieser Aufwand im Vergleich zu den
Reparaturkosten fur ein während des Transports beschädigtes
Instrument gering.Sie sind selbst für das richtige Verpacken des
Instruments vor dem Versand verantwortlich. Bei einer nicht
ordnungsgemäßen Verpackung, die eine Beschädigung zur Folge
hat, wird der Spediteur ihren Schadensersatzanspruch nicht
anerkennen.
Wichtige Informationen
162 • Importante Informacion sobre la Seguridad
Importante Informacion sobre la Seguridad
LÍMITE DE RESPONSABILIDADES
Condiciones de seguridad ambiental
ESTE EQUIPO NO ESTÁ DISEÑADO PARA USO EN HUMANOS
Y NO DEBE USARSE PARA EXPERIMENTACIÓN O
APLICACIÓN EN SERES HUMANOS BAJO NINGUNA
CIRCUNSTANCIA.
1. Para uso interior.
2. Fluctuaciones eléctricas en la fuente de suministro: no
deben exceder ±10% del voltaje nominal.
3. Temperatura: entre 5 °C y 40 °C.
4. Altitud: hasta 2.000 m
5. Este instrumento está diseñado para ser usado en
condiciones de laboratorio. Debe operarse únicamente en
un ambiente limpio y seco. No lo use en un ambiente
húmedo ni mojado.
ADVERTENCIA
SI ESTE EQUIPO SE USA DE MANERA NO ESPECIFICADA POR
EL FABRICANTE SE PODRÍA PERDER LA PROTECCIÓN
PROVISTA POR EL EQUIPO.
Selección del suministro de corriente y cambio de fusibles
Voltaje de entrada
El MultiClamp 700B puede conectarse directamente a todos
los suministros de energía. El límite de voltaje va entre 100 y
240 V~. No es necesario efectuar cambios en el selector.
Además, el instrumento puede ser alimentado a partir de una
fuente de corriente continua con voltajes entre 120 y 310 V.
Cambio de fusible
El MultiClamp 700B utiliza un fusible de 250 V~, T2A, 5 × 20
mm.
En el caso de que un fusible falle, desconecte el cordón
eléctrico.
Antes de cambiar el fusible investigue la causa de la falla.
Para cambiar el fusible:
1. Desconecte el cordón eléctrico.
2. Use un destornillador o un dispositivo similar para girar el
portafusibles en sentido contrario al de las manecillas del
reloj.
3. Reemplace el fusible existente con otro de la misma
capacidad.
4. Conecte nuevamente el cordón eléctrico.
Instalación básica y seguridad del equipo
1. Suministro de corriente y conexión a tierra: Use el cordón
eléctrico IEC incluido para conectar el instrumento a una
toma de corriente CON CONEXIÓN A TIERRA.
2. Montaje: Sobre una mesa o en un estante.
3. Ensamblaje: El cabezal (“headstage”) se conecta al
instrumento en el tablero posterior con el conector de 25
clavijas D-sub, marcado “Headstage”.
4. Uso: No utilice este equipo sin las cubiertas o paneles.
5. Limpieza: Limpie el conector del “headstage” con un paño
húmedo a fin de quitar los derrames de sales. Evite
derramar líquidos sobre el “headstage”.
El conector de entrada fabricado de Teflon debe
mantenerse muy limpio. Puede hacerse una limpieza
efectiva rociando con alcohol o con un algodón
humedecido con agua desionizada. En la medida de lo
posible evite el uso del gas freón, puesto que es dañino
para el medio ambiente.
Precauciones contra la estática
El “headstage” puede manejarse con seguridad, bajo
condiciones normales. Sinembargo, si usted se encuentra en
un laboratorio donde la estática es alta (por ejemplo, si
escucha y percibe chispas cuando toca los objetos), usted
debería tocar inmediatamente un objeto metálico que esté en
contacto con tierra, antes de tocar el “headstage”.
No apague el interruptor principal del MultiClamp 700B cuando
manipule la entrada del “headstage” ya que esto afectará el
equilibrio térmico.
Envío del MultiClamp 700B
El MultiClamp 700B es un instrumento de construcción sólida,
diseñado para soportar el transporte a cualquier parte del
mundo. Sinembargo, para evitar los daños que pudieran
ocurrir durante su envío, el MultiClamp 700B debe empacarse
adecuadamente.
En general, la mejor manera de empacar el MultiClamp 700B
es en la caja original de fábrica. Si ésta ya no se encuentra
disponible, le recomendamos que envuelva cuidadosamente el
MultiClamp 700B en una funda o sábana de espuma o de
“empaque de burbujas” con un espesor mínimo de 3 pulgadas
(75 mm). El MultiClamp 700B, envuelto así, deberá colocarse
en una caja de cartón resistente. Marque el exterior de la caja
con la palabra FRÁGIL y una flecha que indique la posición
hacia arriba.
No recomendamos el uso de bolitas de espuma sueltas para
proteger el MultiClamp 700B. Si la caja se cae
accidentalmente durante el transporte, es muy probable que el
MultiClamp 700B se desplace dentro del contenedor con las
bolitas de espuma sueltas y se dañe.
Si necesita enviar su MultiClamp 700B a otra localidad, o de
regreso a la fábrica, y no posee el empaque adecuado, Axon
Instruments puede enviarle el material necesario por un cargo
mínimo. Esto podría parecerle un gasto superfluo que
preferiría evitar, pero es económico comparado con lo que
costaría la reparación de un instrumento que ha sufrido daños
durante el envío.
Es su responsabilidad empacar el instrumento adecuadamente
antes de enviarlo. Si no lo hace así y resulta dañado, el
transportista no será responsable ni aceptará su reclamo de
indemnización.
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
Index • 163
Index
Audio Monitor, 68, 69, 70, 71, 145
Auto
Bridge Balance, 32, 33, 65, 75, 76, 77, 84, 86,
117, 124
Leak Subtraction, 28, 29, 51, 54, 110, 111, 117
Output Zero, 29, 33, 111, 117
Whole Cell, 130, 142
Bandwidth, Rs Compensation, 128, 129, 134
BATH HEADSTAGE, 45, 72, 74
VG, 74
Bridge Balance, 32, 33, 65, 75, 76, 77, 84, 86, 117,
124
Buzz, 65, 78, 87, 143
Calibration, 11
Capacitance Compensation
Current Clamp, 6, 19, 31, 90, 101, 142
Electrode, 17, 19, 45, 58, 60, 61, 78, 79, 105,
115, 116
Fast, 7, 12, 13, 24, 25, 28, 29, 51, 53, 80, 83,
126, 128, 133, 135, 142
Slow, 24, 25, 51, 80, 83, 126, 142
Whole-cell, 51, 81, 130, 142
Whole-Cell, 12
Capacitance Neutralization, 33, 62, 65, 75, 77, 84, 86,
116, 124, 140
Cleaning
Headstage, 47, 58, 72, 91, 93, 98, 101, 102, 111,
114, 115, 122, 141
Holders, 58, 103
Clear, 65, 78, 87, 143
Correction, 30, 127, 128, 129, 134, 135
Current Clamp
Capacitance Compensation, 78, 79, 80, 83, 89,
90, 110, 118, 122, 124, 126
Model Cell, 112, 125
Whole-cell, 51, 81
Electrochemistry, 87
External Command, 78, 84, 89, 90, 91, 93, 97, 107
External Command Inputs, 78, 84, 89, 91, 93, 97, 107
Filter, 89, 90, 95, 97, 128, 142, 144
Functional Checkout, 6
Gain, 23, 24, 110, 118, 141, 144
Glass
Dimensions, 141
Types, 95
Grounding, 97, 114, 116, 120
Headstage
Adapters, 107
Cleaning, 102, 107, 159
Gain, 23, 110, 118, 141, 144
Mounting, 102, 159
Help
On-line, 67, 103
Holder, Pipette
Cleaning, 102, 107
Use, 74, 83, 84, 86, 105, 107, 121, 159, 162
Holding, 7, 27, 29, 31, 37, 51, 63, 65, 90, 143
Hum, 97, 114, 116, 120
Input/Output Connections, 107, 111, 117
Installation, 3, 122, 160, 161
Lag, 128
Leak Subtraction, 28, 29, 51, 54, 110, 111, 117
Mode, 6, 31, 89, 91, 93, 98, 107, 108, 111, 112, 122
Index
164 • Importante Informacion sobre la Seguridad
Automatic Switch, 37
I=0, 6, 37, 45, 59, 111, 112
I-Clamp, 20, 31, 46, 47, 59, 64, 75, 78, 84, 87,
90, 91, 93, 97, 98, 101, 108, 109, 111, 112,
115, 116, 117, 124, 140
V-Clamp, 7, 17, 46, 47, 59, 78, 89, 90, 91, 96, 97,
98, 99, 108, 109, 110, 111, 114, 115, 116,
117, 122, 123, 124, 138
Model Cell, 16, 112, 125
MultiClamp 700B Commander, 3, 4, 5, 6, 7, 9, 10, 11,
12, 13
Low Noise Techniques, 48, 55
Perforated-patch Recording, 54
Sharp Microelectrode Recording, 47, 59, 85, 86,
115
Whole-cell Voltage Clamp Recording, 51
Prediction, 30, 127, 128, 129, 131, 132, 133, 134,
135
Primary Output, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27,
28, 29, 30, 31, 32, 33, 37, 49, 64, 65, 76, 86, 93,
96, 97, 110, 117, 128, 140, 144
Pulse, 30, 37, 89, 90, 109, 117, 143
Noise, 11, 47, 91, 92, 93, 97, 98, 101, 114, 116, 141,
142
Quick Select, 9, 10
Options, 10, 12, 21, 24, 51, 83, 89, 90, 91, 93, 97,
107
Oscillations, 135
Reduce Compensation, 33, 34
Oscilloscope Triggering, 107, 116
Output
Filter, 89, 90, 95, 97, 128, 142, 144
Gain, 23, 110, 118, 141, 144
Overload, 91, 118
Pipette, 16, 18, 49, 56, 62, 63, 64, 65, 77, 86, 116,
140, 143
Chloriding, 106
Cleaning, 102, 107
Filling, 64, 106
Glass, 56, 61
Insertion, 105
Pipette Offset, 18, 20, 49, 63, 64, 140, 143
Polarity Conventions, 67, 118, 119
Power Supply
Glitches, 121
Voltage Selection, 121, 159
Practical Guide
Forming a Gigaseal, 49
Interfacing a Computer, 46
Reset to Program Defaults, 17, 26
Seal Test, 8, 12, 18, 19, 24, 26, 27, 29, 49, 90, 96,
109, 117, 125, 138, 143
Secondary Output, 144
Select Device, 122, 139
Series Resistance
Compensation, 30, 54, 78, 82, 98, 100, 114, 116,
122, 124, 125, 128, 129, 131, 133, 134, 135,
136
Correction, 30, 127, 128, 129, 134, 135
Fast, 7, 25, 29, 51, 53, 80, 83, 126, 128, 133,
135, 142
Lag, 128
Oscillations, 135
Prediction, 30, 127, 128, 129, 131, 132, 133, 134,
135
Slow, 51, 80, 83, 126, 142
Status, 138, 139
Tutorials
Giga Seal Configuration, 21
VG-2, 72
Zap, 52, 138, 143
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
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