NPI SEC-05X Operating instructions

OPERATING INSTRUCTIONS AND
SYSTEM DESCRIPTION OF THE
SEC-05X
SINGLE-ELECTRODE CLAMP
AMPLIFIER
VERSION 2.0
npi 2015
npi electronic GmbH, Bauhofring 16, D-71732 Tamm, Germany
Phone [phone]; Fax: [phone]
[email protected]; http://www.npielectronic.com

SEC-05X User Manual
version 2.0 page 2
Table of Contents
About this Manual................................................................................................................... 4
1. Safety Regulations.............................................................................................................. 5
2. Introduction......................................................................................................................... 6
2.1. Why a Single-Electrode Clamp? ................................................................................. 6
2.2. Principle of Operation ................................................................................................. 8
Major advantages of the npi SEC System................................................................... 10
3. SEC-05X System................................................................................................................ 10
3.1. SEC-05X Components ................................................................................................ 10
3.2. Description of the Front Panel..................................................................................... 12
3.3. Description of the Rear Panel...................................................................................... 20
4. Headstages .......................................................................................................................... 21
4.1. Standard Headstages.................................................................................................... 21
4.2. Low-noise Headstage (SEC-HSP)............................................................................... 23
5. Setting up the SEC-05X System......................................................................................... 24
6. Passive Cell Model ............................................................................................................. 24
6.1. Cell Model Description ............................................................................................... 25
6.2. Connections and Operation ......................................................................................... 26
7. Test and Tuning Procedures ............................................................................................... 28
7.1. Headstage Bias Current Adjustment ........................................................................... 28
7.2. Electrode Selection...................................................................................................... 29
7.3. Offset Compensation................................................................................................... 29
7.4. Bridge Balance (in BR mode) ..................................................................................... 30
7.5. Switching Frequency and Capacitance Compensation (in switched modes).............. 32
Criteria for the selection of the switching frequency .................................................. 32
7.6. Capacity Compensation - Tuning Procedure............................................................... 34
First part: basic setting................................................................................................. 34
Second part: fine tuning............................................................................................... 40
7.7. Testing Operation Modes ............................................................................................ 41
Current Clamp (in BR- or discontinuous CC mode)................................................... 41
Voltage Clamp............................................................................................................. 41
8. Special Modes of Operation ............................................................................................... 43
8.1. Dynamic Hybrid Clamp (DHC) Mode (optional) ....................................................... 43
General Description..................................................................................................... 43
Operation..................................................................................................................... 43
8.2. Linear Mode (optional)................................................................................................ 43
General Description..................................................................................................... 43
Operation..................................................................................................................... 43
8.3. VCcCC mode (optional).............................................................................................. 44
General Description..................................................................................................... 44
Operation..................................................................................................................... 44
Current Clamp Input.................................................................................................... 45
9. Sample Experiments........................................................................................................... 46
9.1. Sample Experiment using a Sharp Microelectrode ..................................................... 46
9.2. Sample Experiment using a Patch Electrode............................................................... 49
10. Tuning VC Performance.............................................................................................. 51
General Considerations................................................................................................ 51
Tuning Procedure ........................................................................................................ 52
11. Trouble Shooting......................................................................................................... 53
12. Appendix ..................................................................................................................... 54
12.1. Theory of Operation .................................................................................................... 54

SEC-05X User Manual
version 2.0 page 3
12.2. Speed of Response of SEC Single-Electrode Clamps................................................. 55
12.3. Tuning Procedures for VC Controllers........................................................................ 56
Practical Implications.................................................................................................. 56
13. Literature ..................................................................................................................... 58
13.1. Papers in Journals and Book Chapters about npi Single-electrode Clamp Amplifiers58
13.2. Books........................................................................................................................... 70
14. SEC-05X Specifications – Technical Data.................................................................. 71
15. Index............................................................................................................................ 74

SEC-05X User Manual
version 2.0 page 4
About this Manual
This manual should help the user to setup and use SEC systems correctly and to perform
reliable experiments.
If you are not familiar with the use of instruments for intracellular recording of electrical
signals please read the manual completely. The experienced user should read at least chapters
1, 3, 7 and 10.
Important: Please read chapter 1 carefully! It contains general information about the safety
regulations and how to handle highly sensitive electronic instruments.
Signs and conventions
In this manual all elements of the front panel are written in CAPITAL LETTERS as they
appear on the front panel.
System components that are shipped in the standard configuration are marked with ✓,
optional components with ➪. In some chapters the user is guided step by step through a
certain procedure. These steps are marked with ❏.
Important information and special precautions are highlighted in gray.
Abbreviations
Cm: cell membrane capacitance
Cstray: electrode stray capacitance
GND: ground
Imax: maximal current
Ra: access resistance
Rm: cell membrane resistance
REL: electrode resistance
SwF: switching frequency
τCm: time constant of the cell membrane
VREL: potential drop at REL

SEC-05X User Manual
version 2.0 page 5
1. Safety Regulations
VERY IMPORTANT:Instruments and components supplied by npi electronic are NOT
intended for clinical use or medical purposes (e.g. for diagnosis or treatment of humans),
or for any other life-supporting system. npi electronic disclaims any warranties for such
purpose. Equipment supplied by npi electronic must be operated only by selected,
trained and adequately instructed personnel. For details please consult the GENERAL
TERMS OF DELIVERY AND CONDITIONS OF BUSINESS of npi electronic, D-71732
Tamm, Germany.
1) GENERAL: This system is designed for use in scientific laboratories and must be
operated only by trained staff. General safety regulations for operating electrical devices
should be followed.
2) AC MAINS CONNECTION: While working with the npi systems, always adhere to the
appropriate safety measures for handling electronic devices. Before using any device
please read manuals and instructions carefully.
The device is to be operated only at 115/230 Volt 60/50 Hz AC. Please check for
appropriate line voltage before connecting any system to mains.
Always use a three-wire line cord and a mains power-plug with a protection contact
connected to ground (protective earth).
Before opening the cabinet, unplug the instrument.
Unplug the instrument when replacing the fuse or changing line voltage. Replace fuse
only with an appropriate specified type.
3) STATIC ELECTRICITY: Electronic equipment is sensitive to static discharges. Some
devices such as sensor inputs are equipped with very sensitive FET amplifiers, which can
be damaged by electrostatic charge and must therefore be handled with care. Electrostatic
discharge can be avoided by touching a grounded metal surface when changing or
adjusting sensors. Always turn power off when adding or removing modules,
connecting or disconnecting sensors, headstages or other components from the
instrument or 19” cabinet.
4) TEMPERATURE DRIFT / WARM-UP TIME: All analog electronic systems are
sensitive to temperature changes. Therefore, all electronic instruments containing analog
circuits should be used only in a warmed-up condition (i.e. after internal temperature has
reached steady-state values). In most cases a warm-up period of 20-30 minutes is
sufficient.
5) HANDLING: Please protect the device from moisture, heat, radiation and corrosive
chemicals.

SEC-05X User Manual
version 2.0 page 6
2. Introduction
Npi electronic’s SEC (Single-Electrode Clamp) systems are based on the newest
developments in the field of modern electronics and control theory (see also chapter 8). These
versatile current/voltage clamp amplifiers permit extremely rapid switching between current
injection and current-free recording of true intracellular potentials.
The use of modern operational amplifiers and an innovative method of capacity compensation
makes it possible to inject very short current pulses through high resistance microelectrodes
(up to 200 MΩand more) and to record membrane potentials accurately, i.e. without series
resistance error, within the same cycle.
Although the system has been designed primarily to overcome the limitations related to the
use of high resistance microelectrodes in intracellular recordings, it can also be used to do
conventional whole-cell patch-clamp recordings or perforated patch recordings. The whole-
cell configuration allows to investigate even small dissociated or cultured cells as well as cells
in slice preparations in both current and voltage clamp mode, while the intracellular medium
is being controlled by the pipette solution.
2.1. Why a Single-Electrode Clamp?
Voltage clamp techniques permit the analysis of ionic currents flowing through biological
membranes at preset membrane potentials. Under ideal conditions the recorded current is
directly related to the conductance changes in the membrane and thus gives an accurate
measure of the activity of ion channels and electrogenic pumps.
The membrane potential is generally kept at a preselected value (command or holding
potential). Ionic currents are then activated by sudden changes in potential (e.g. voltage-gated
ion channels), by transmitter release at synapses (e.g. electrical stimulation of fiber tracts in
brain slices) or by external application of an appropriate agonist. Sudden command potential
changes used to activate voltage-gated currents are especially challenging, because the
membrane will adopt the new potential value only after its capacitance (Cmin Figure 1 and
Figure 2) has been charged. Therefore, the initial transient current following the voltage step
should be as large as possible to achieve rapid membrane charging. In conventional patch-
clamp amplifiers, this requires a minimal resistance between the amplifier and the cell interior
– a simple consequence of Ohm’s law (∆U = R*I), i.e. for a given voltage difference (∆U),
the current (I) is inversely proportional to the resistance (R). In this context, R is the access
resistance (Rain Figure 1 and Figure 2) between the electrode and the cell interior.
Rais largely determined by certain electrode properties (mainly electrode resistance) and the
connection between the electrode and the cell. Sharp microelectrodes usually have much
larger resistances (30 to 150 MΩor even more) than patch-clamp electrodes. This makes
rapid charging of the cell membrane to attain a new voltage level more difficult than in patch-
clamp experiments.

SEC-05X User Manual
version 2.0 page 7
Figure 1: Model circuit for whole-cell patch-clamp recording.
Cm: membrane capacitance, Cstray: electrode stray capacitance, Ra: access
resistance, Rm: membrane resistance
Figure 2: Model circuit for intracellular recording using a sharp electrode
Cm: membrane capacitance, Cstray: electrode stray capacitance, Ra: access
resistance, Rm: membrane resistance
Besides slowing down the voltage response of the cell, Racan also cause additional adverse
effects, such as error in potential measurement. Ra, together with the membrane resistance
(Rm) forms a voltage divider (see Figure 1 and Figure 2). Current flowing from the amplifier
to the grounded bath of a cell preparation will cause a voltage drop at both, Raand Rm. If Ra
<< Rm, the majority of the voltage drop will develop at Rmand thus reflect a true membrane

SEC-05X User Manual
version 2.0 page 8
potential. If, in an extreme case, Ra= Rm, the membrane potential will follow only one half of
the voltage command. In order to achieve a voltage error of less than 1% Ramust be more
than 100 times smaller than Rm. This condition is not always easy to accomplish, especially if
recordings are performed from small cells. If sharp intracellular microelectrodes are used, it is
virtually impossible. If Rais not negligible, precise determination of the membrane potential
can be achieved only if no current flows across Raduring potential measurement. This is the
strategy employed in npi electronic’s SEC amplifier systems.
The SEC amplifiers inject current and record the potential in an alternating mode (switched
mode). Therefore, this technique is called discontinuous SEVC (dSEVC). This ensures that no
current passes through Raduring potential measurement and completely eliminates access
resistance artefacts.
After each injection of current, the potential gradient at the electrode tip decays much faster
than the potential added at the cell membrane during the same injection. The membrane
potential is measured after the potential difference across Rahas completely dropped (see
chapter 2.2). The discontinuous current and voltage signals are then smoothed and read at the
CURRENT OUTPUT and POTENTIAL OUTPUT connectors.
2.2. Principle of Operation
Figure 3: Model circuit of SEC systems

SEC-05X User Manual
version 2.0 page 9
Figure 4: Principle of dSEVC operation
Figure 3 and Figure 4 illustrate the basic circuitry and operation of npi SEC voltage clamp
amplifiers.
A single microelectrode penetrates the cell or is connected to the cell interior in the whole-cell
configuration of the patch-clamp technique. The recorded voltage is buffered by an x1
operational amplifier (A1 in Figure 3). At this point, the potential (V[A1] in Figure 4) is the
sum of the cell’s membrane potential and the voltage gradient which develops when current is
injected at the access resistance. Due to npi’s unique compensation circuitry, the voltage at
the tip of the electrode decays extremely fast after each injection of current and therefore
allows for a correct measurement of Vcell after a few microseconds. At the end of the current-
free interval, when the electrode potential has dropped to zero, the sample-and-hold circuit
(SH1 in Figure 3) samples Vcell and holds the value for the remainder of the cycle (VSH1 in
Figure 4).
The differential amplifier (A2 in Figure 3) compares the sampled potential with the command
potential (Vcom in Figure 3). The output of this amplifier becomes the input of a controlled
current source (CCS in Figure 3), if the switch S1 (Figure 3) is in the current-passing position.
The gain of this current source increases as much as 100 µA/V due to a PI (proportional-
integral) controller and improved electrode capacity compensation. In Figure 3 S1 is shown in
the current-passing position, when a square current is applied to the electrode. When the
current passes the electrode a steep voltage gradient develops at the electrode resistance. Vcell
(Figure 4) is only slightly changed due to the slow charging of the membrane capacitance.
The amplitude of injected current is sampled in the sample-and-hold amplifier SH2 (Figure
3), multiplied by the fractional time of current injection within each duty cycle (1/8 to 1/2 in
SEC-05 and SEC-10, 1/4 in SEC-03 systems) and read out as current output (ISH2 in Figure 4).
S1 then switches to the voltage-recording position (input to CCS is zero). The potential at A1
decays rapidly due to the fast relaxation at the (compensated) electrode capacity. Exact
capacity compensation is essential to yield an optimally flat voltage trace at the end of the

SEC-05X User Manual
version 2.0 page 10
current free interval when Vcell is measured (see also Figure 13). The cellular membrane
potential, however, will drop much slower due to the large (uncompensated) membrane
capacitance. The interval between two current injections must be long enough to allow for
complete (≤1%) settling of the electrode potential, but short enough to minimize loss of
charges at the cell membrane level, i.e. minimal relaxation of Vcell. At the end of the current-
free period a new Vcell sample is taken and a new cycle begins. Thus, both current and
potential output are based on discontinuous signals that are stored during each cycle in the
sample-and-hold amplifiers SH1 and SH2. The signals will be optimal smooth at maximal
switching frequencies.
Major advantages of the npi SEC System
Npi electronic’s SEC amplifiers are the only systems that use a PI controller to avoid
recordings artefacts known to occur in other single-electrode clamp systems (“clamping of the
electrode”). The PI controller design increases gain to as much as 100 µA/V in frequencies
less than one-fourth the switching frequency. The result is very sensitive control of the
membrane potential with a steady-state error of less than 1% and a fast response of the clamp
to command steps or conductance changes.
The use of discontinuous current and voltage clamp in combination with high switching
frequencies yields five major advantages:
1. The large recording bandwidth allows accurate recordings even of fast signals.
2. High clamp gains (up to 100 µA/V) can be used in voltage clamp mode.
3. Very small cells with relatively short membrane time constants can be voltage-
clamped.
4. Series resistance effects are completely eliminated for a correct membrane potential
control even with high resistance microelectrodes.
5. The true membrane potential is recorded also in the voltage clamp mode (whereas
continuous feedback VC amplifiers only reflect the command potential).
3. SEC-05X System
3.1. SEC-05X Components
The following items are shipped with the SEC-05X system:
✓SEC-05X amplifier
✓Headstage
✓GND- and DRIVEN SHIELD (2.6 mm banana plug) connectors
Please open the box and inspect contents upon receipt. If any components appear damaged or
missing, please contact npi electronic or your local distributor immediately
([email protected]).