Axon AutoMate Scientific MultiClamp 700B User manual

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. .comaxon
800.998.MATE | www.autom8.com | 650 University Ave # 5, Berkeley, CA 94710 USAt e l 5 1 0 . 8 4 5 . 6 2 8 3 | f a x 5 1 0 . 6 6 5 . 3 9 7 5 | e - m a i l i n f o @ a u t o m 8 . c o m

i
P LEASE 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
R
b
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 R
s
Compensation .................................................................. 122
Is R
s
Compensation Necessary? .................................................................... 124
Adjusting R
s
Compensation .......................................................................... 125
MultiClamp 700B Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices

Table of Contents •vii
Theory of R
s
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 R
s
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, high-
speed 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-by-
step 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 double-
headed 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 (τ
s
).
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 root-
mean-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