HEKA EPC 9 Manual de usuario
8.52
EPC 9 Manual
HEKA Elektronik Dr. Schulze GmbH
Wiesenstraße 71 • D-67466 Lambrecht • Germany
Tel: +49 (0) 6325 9553 0 • Fax: +49 (0) 6325 9553 50
Web Site:
http://www.heka.com
E-mail:
•
Table of Contents EPC9 Manual 2
Table of Contents
1. Introduction 5
Introducing the EPC 9 _____________________________________________ 5
References ____________________________________________________ 6
Naming Conventions______________________________________________ 8
EPC 9, EPC 9 Double, and EPC 9 Triple...................................................................................8
Windows versions ................................................................................................................8
Support Hotline _________________________________________________ 9
2. Description of the Hardware 10
Probe _______________________________________________________ 10
Main Unit_____________________________________________________ 11
EPC 9 Double and Triple __________________________________________ 15
3. Installation 16
Installation Procedure ____________________________________________ 16
Calibrating the EPC 9 ____________________________________________ 16
Creating the C-fast Lookup Table_____________________________________ 18
4. Verifying and Testing the EPC 9 19
Testing the EPC9 with the Model Circuit ________________________________ 19
The Model Circuit MC 9.......................................................................................................19
Step 1: Applying the Test Pulse.............................................................................................20
Step 2: ”On-Cell” Voltage-Clamp Recording.............................................................................23
Step 3: ”Whole-Cell” Voltage-Clamp Recording ........................................................................24
Step 4: ”Whole-Cell” Current-Clamp Recording.........................................................................26
Step 5: Measuring the Noise of the Amplifier............................................................................29
Making a ”Full Test” _____________________________________________ 31
Measuring the Frequency Response __________________________________ 34
Table of Contents EPC9 Manual 3
5. E9SCREEN Software 35
EPC9 Window _________________________________________________ 35
Main Controls....................................................................................................................35
Hidden Controls.................................................................................................................46
Notebook Window_______________________________________________ 48
Drop-Down Menus ______________________________________________ 48
File Menu .........................................................................................................................48
Edit Menu.........................................................................................................................49
EPC9 Menu ......................................................................................................................50
Notebook Menu .................................................................................................................52
Calibrate Menu ..................................................................................................................53
6. Operating Modes 54
Voltage-Clamp Mode _____________________________________________ 54
Current-Clamp Mode _____________________________________________ 54
Test Mode ____________________________________________________ 57
Search Mode __________________________________________________ 58
7. Compensation Procedures 59
Series Resistance Compensation ____________________________________ 59
Capacitance Compensation ________________________________________ 62
Offset Compensation_____________________________________________ 63
8. Patch-Clamp Setup 67
Mounting the Probe ............................................................................................................67
Ground Wires....................................................................................................................67
Grounding the Microscope ...................................................................................................67
External Shielding ..............................................................................................................68
Connections to other Instruments ..........................................................................................68
Pipette Holder and Electrode ................................................................................................ 68
Bath Electrode...................................................................................................................69
9. Patch-Pipettes 71
Glass Capillaries................................................................................................................71
Pulling..............................................................................................................................72
Table of Contents EPC9 Manual 4
Coating ............................................................................................................................72
Heat Polishing ...................................................................................................................73
Use of Pipettes .................................................................................................................. 73
10. Using the Patch Clamp 75
Forming a Seal_________________________________________________ 75
Initial Setup.......................................................................................................................75
Entering the Bath ...............................................................................................................75
Forming a Gigaseal ............................................................................................................76
Cell-Attached Recording __________________________________________ 76
Whole-Cell Recording ____________________________________________ 77
Breaking the Patch ............................................................................................................. 77
Capacitive Transient Cancellation..........................................................................................78
Series Resistance Compensation .......................................................................................... 78
Current-Clamp Recording__________________________________________ 79
11. Low-Noise Recording 80
Appendix I: Controlling E9SCREEN 83
Communication between E9Screen and other Programs _____________________ 83
Reserving the AD/DA-board for exclusive use .......................................................................... 83
The “EPC9out.EPC” file.......................................................................................................83
Controlling the EPC9 from another Program _____________________________ 85
Sending Commands to E9Screen..........................................................................................85
Error Messages..................................................................................................................87
Implemented Commands and Messages................................................................................. 87
Notes to Programmers.........................................................................................................91
Sample program ................................................................................................................93
Appendix II: Technical Data 98
Digital I/O Connector_____________________________________________ 98
Standard EPC9..................................................................................................................98
EPC9 Double and Triple ...................................................................................................... 99
Index 101
Introduction EPC9 Manual 5
1. Introduction
The patch-clamp technique was introduced by
Neher and Sakmann (1976) for recording the
currents in a small patch of membrane under
voltage-clamp conditions. In the intervening
years a number of changes have occurred, most
notably the development of the “gigaseal” by E.
Neher (1981). Various recording configurations
allow intracellular recordings to be made with
the same type of recording setup as used for
patch recording from the cell surface or cell-free
membrane patches (Hamill et al., 1981).
Introducing the EPC 9
The EPC9 represents roughly the ninth in the
series of patch-clamp designs in use in the
Göttingen laboratories. It is a logical successor to
the EPC7, retaining all of its features but adding
a number of capabilities, the most important
being implementation of full digital control of
the various functions. Thus, the new digitally
controlled EPC9 patch-clamp amplifier has no knobs, switches or dials. The Pulse
software replaces the analog controls of conventional amplifiers by using Macintosh
computers and a built-in ITC-16 interface. The convenient graphics display and
mouse operations provide unsurpassed versatility and ease of operation.
In addition to the controls for the amplifier and the built-in filters, the Pulse software
contains a powerful data acquisition system (sampling and storage in pulse, ramp
and continuous modes), a fully programmable pulse generator, and a digital
oscilloscope. Thus, the EPC9 offers all the features of a complete workstation for
controlling experiments and acquiring data. Furthermore, there is the PulseFit
software package as well as TAC (Threshold Analysis for single Channels) available,
which allow data analysis, data export, and graphics output.
The EPC9 also accepts a stimulus input and provides current monitor outputs just
like conventional amplifiers to operate in combination with a host computer running
custom and commercial software from other sources. The versatility of the EPC9 can
Introduction EPC9 Manual 6
best be appreciated by the variety of experiments that can be carried out with it.
Besides high-resolution recordings of single channels, it can be used in studies of
whole-cell voltage and current clamp, exocytosis (by monitoring changes in cell
membrane capacitance), and recordings from artificial membranes or loose-patch
experiments. Technically, the EPC9 is noteworthy for three special features, the
range-changing capability of the head stage, the extremely wide bandwidth available
from the current monitor circuitry, and the integrated transient cancellation
(automatically if desired) and series-resistance compensation functions. Together
these features mean that a single head stage suffices for both single-channel and
whole-cell recordings, and that both kinds of recordings can be made with high time
resolution and low noise.
This manual is designed to provide a general guide for setting up and using the
EPC9 for experiments. It covers general information about the hardware, the
E9Screen program, and basic principles o f the EPC9's functions and patch-clamp
techniques.
It is assumed that the reader has some familiarity with patch-clamp techniques;
should you be a newcomer to the field perhaps the best place to start would be the
paper by Hamill et al., where the basic gigaseal techniques are described and the first
three chapters of Single Channel Recording (B. Sakmann & E. Neher, eds., Plenum
Press, New York, 1995). Certainly, it will be worthwhile to read this manual
carefully. Many users will want to read some of the more advanced and complete
discussions of individual topics which can be be found in original articles and in the
books Single Channel Recording (B. Sakmann & E. Neher, eds., Plenum Press, New
York, 1995) and Methods in Enzymology, vol. 207 (Academic Press, New York, 1992).
References
Original Articles
Hamill, O. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. (1981) Improved
patch clamp techniques for high-resolution current recording from cells and cell-free
membrane patches. Pflügers Arch. 391, 85-100.
Neher, E. (1981) Unit conductance studies in biological membranes. In: Techniques in
Cellular Physiology (P. F. Baker, ed.) Elsevier/North Holland.
Neher, E. & Sakmann, B. (1976) Single-channel currents recorded from membrane of
denervated frog muscle fibres. Nature 260, 779-802.
Introduction EPC9 Manual 7
Rae, J. & Levis, R. (1984) Patch clamp recordings from the epithelium of the lens obtained
using glasses selected for low noise and improved sealing properties. Biophys. J. 45, 144-
146.
Barry, P. H. & Lynch, J. W. (1991) Liquid junction potentials and small cell effects in
patch-clamp analysis. J. Memb. Biol. 121, 101-117.
Sigworth, F. J., Affolter, H. & Neher, E. (1995) Design of the EPC-9, a computer-
controlled patch-clamp amplifier. 2. Software. J. Neurosci. Methods 56, 203-221.
Book Chapters
Penner, R. (1995) Chapter 1: A practical guide to patch clamping. In: Single-Channel
Recording (B. Sakmann & E. Neher, eds.) Plenum Press, New York.
Marty, A. & Neher, E. (1995) Chapter 2: Tight-seal whole-cell recording. In: Single-
Channel Recording (B. Sakmann & E. Neher, eds.) Plenum Press, New York.
Heinemann, S. H. (1995) Chapter 3: Guide to data acquisition and analysis. In: Single-
Channel Recording (B. Sakmann & E. Neher, eds.) Plenum Press, New York.
Sigworth, F. J. (1995) Chapter 4: Electronic design of the patch clamp. In: Single Channel-
Recording (B. Sakmann & E. Neher, eds.) Plenum Press, New York.
Neher, E. (1995) Chapter 6: Voltage offsets in patch-clamp experiments. In: Single
Channel-Recording (B. Sakmann & E. Neher, eds.) Plenum Press, New York.
Colquhoun, D. & Sigworth, F. J. (1995) Chapter 19: Fitting and statistical analysis of
single-channel records. In: Single-Channel Recording (B. Sakmann & E. Neher, eds.)
Plenum Press, New York.
Neher, E. (1992) Correction for liquid junction potentials in patch clamp experiments. In:
Methods in Enzymology 207, 123-131, Academic Press, New York.
Introduction EPC9 Manual 8
Naming Conventions
EPC 9, EPC 9 Double, and EPC 9 Triple
Throughout the present manual we will address all three amplifier types as “EPC9”.
We will explicitly mention the particular amplifiers, where it is required.
Windows versions
The EPC9 is supported on Windows 3.1, Windows 95, Windows 98,
Windows NT 3.51, Windows NT 4.0, and Windows 2000.
Throughout the present manual we will address all the above Windows versions as
“Windows”. We will explicitly mention the particular Windows versions, whenever
it is required.
Introduction EPC9 Manual 9
Support Hotline
If you have any question, suggestion, or improvement, please contact HEKA’s
support team. The best way is to send us an e-mail or fax specifying:
•Your postal and e-mail address (or fax number)
•The program name:
E9SCREEN, PULSE, PULSEFIT, etc.
•The program version number:
v8.31, v8.50
•Your operating system and its version:
MacOS 7.6.1, MacOS 8.5,
Windows 98, Windows NT 4.0, etc.
•Your type of computer:
Mac PPC 8500, Pentium II 300 MHz, etc.
•Your acquisition hardware, if applicable:
EPC9, ITC–16, ITC–18
•Your amplifier, if applicable:
EPC9, EPC9 Double, Axon 200B, etc.
•The serial number and version of your EPC9, if applicable:
EPC9 single, version “920552 D”.
•The questions, problems, or suggestions you have
•Under which conditions and how often the problem occurs
We will address the problem as soon as possible.
HEKA Elektronik GmbH phone: +49 (0) 6325 9553 0
Wiesenstrasse 71 fax: +49 (0) 6325 9553 50
D-67466 Lambrecht/Pfalz e-mail:
Germany web:
http://www.heka.com
Description of the Hardware EPC9 Manual 10
2. Description of the Hardware
The hardware components of the EPC9 patch-clamp system consist of the head stage
(or probe), the amplifier main unit with the integrated ITC-16 interface board, and
the computer system. Specific information about the hardware installation is given
below in Chapter 3. Installation on page 16.
Probe
The probe, or “head stage” of the EPC9 is contained in a small enclosure designed to
be mounted on a micromanipulator and directly attached to the recording
micropipette. It contains the sensitive amplifier that constitutes the current-to-
voltage converter, as well as components for injecting test signals into that amplifier.
On the probe are the following connectors:
Input Connector: This is a Teflon-insulated BNC connector. The standard pipette
holder plugs directly into this connector; the center pin is the amplifier input, and
the shield is driven with the command potential Vp.
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