pCLAMP™ 10 Data Acquisition & Analysis Software User Guide 1-2500-0180 G January 2016 pCLAMP 10 Data Acquisition and Analysis Software User Guide This document is provided to customers who have purchased Molecular Devices equipment, software, reagents, and consumables to use in the operation of such Molecular Devices equipment, software, reagents, and consumables. This document is copyright protected and any reproduction of this document, in whole or any part, is strictly prohibited, except as Molecular Devices may authorize in writing. Software that may be described in this document is furnished under a non-transferrable license. It is against the law to copy, modify, or distribute the software on any medium, except as specifically allowed in the license agreement. Furthermore, the license agreement may prohibit the software from being disassembled, reverse engineered, or decompiled for any purpose. 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All rights reserved. 2 1-2500-0180 G Contents Chapter 1: Introduction 9 New Features 10 AxoScope Software 10 MiniDigi Digitizer 11 pCLAMP Software Software Documentation 12 Overview of User Guide 14 Utility Programs 15 History of pCLAMP 15 Chapter 2: Description 17 Definitions 17 Data Aquisition Modes 20 Terms and Conventions in Electrophysiology 21 The Sampling Theorem in pCLAMP Software 25 Optimal Data Acquisition 26 File Formats 29 pCLAMP Software Quantitative Limits 31 Chapter 3: Setup 35 Computer System 35 Software Setup and Installation 39 Digitizer Configuration in Clampex Software 41 Chapter 4: Clampex Software Features 45 Clampex Software Windows 45 Telegraphs 49 Lab Bench 50 Overrides 51 Handling Data 51 Edit Protocol 52 Data Acquisition 60 Real Time Controls 60 Seal and Cell Quality: Membrane Test 65 Time, Comment, and Voice Tags 69 1-2500-0180 G 3 pCLAMP 10 Data Acquisition and Analysis Software User Guide Junction Potential Calculator 70 Calibration Wizard 70 Sequencing Keys 71 LTP Assistant 71 Chapter 5: Clampex Software Tutorials 87 I-V Tutorial 87 Membrane Test Tutorial 102 Scenarios 103 Chapter 6: Clampfit Software Features 111 Clampfit Software Windows 111 File Import 116 Data Conditioning 117 Event Detection 119 Single-Channel Analysis in pCLAMP Software 121 Fitting and Statistical Analysis 131 Creating Figures in the Layout Window 132 Chapter 7: Clampfit Software Tutorials 133 Creating Quick Graphs 133 Preconditioning Noisy Single-Channel Recordings 136 Evaluation of Multicomponent Signals: Sensillar Potentials with Superimposed Action Potentials 140 Separating Action Potentials by Their Shape 147 Chapter 8: Digital Filters 155 Finite vs. Infinite Impulse Response Filters 155 Digital Filter Characteristics 157 End Effects 157 Bessel Lowpass Filter (8 Pole) Specifications 158 Boxcar Smoothing Filter Specifications 160 Butterworth Lowpass Filter (8 Pole) Specifications 161 Chebyshev Lowpass Filter (8 Pole) Specifications 163 Gaussian Lowpass Filter Specifications 165 Notch Filter (2 Pole) Specifications 168 RC Lowpass Filter (Single Pole) Specifications 169 RC Lowpass Filter (8 Pole) Specifications 171 4 1-2500-0180 G RC Highpass Filter (Single Pole) Specifications 173 Bessel Highpass Filter (8-Pole Analog) Specifications 174 The Electrical Interference Filter 175 Chapter 9: Clampfit Software Analysis 185 The Fourier Series 185 The Fourier Transform 186 The Fast Fourier Transform 187 The Power Spectrum 187 Limitations 188 Windowing 188 Segment Overlapping 189 Transform Length vs. Display Resolution 189 Chapter 10: pCLAMP Analysis 191 Membrane Test 191 Template Matching 194 Single-Channel Event Amplitudes 194 Level Updating in Single-Channel Searches 195 Kolmogorov-Smirnov Test 196 Normalization Functions 197 Variance-Mean (V-M) Analysis 199 Burst Analysis 201 Peri-Event Analysis 203 P(Open) 203 Chapter 11: Curve Fitting 205 Fitting Model 205 Fitting to Split-Clock Data 205 Function Parameters 206 Parameter Errors 206 Fitting Failure 207 Numerical Limitations 207 Units 208 The Levenberg-Marquardt Method 208 The Simplex Method 210 The Variable Metric Method 212 1-2500-0180 G 5 pCLAMP 10 Data Acquisition and Analysis Software User Guide The Chebyshev Transform 213 Maximum Likelihood Estimation 227 Model Comparison 229 Defining a Custom Function 230 Multiple-Term Fitting Models 231 Minimization Functions 232 Weighting 233 Normalized Proportions 234 Zero-shifting 235 Chapter 12: Fitting Functions 237 BETA Function 237 Binomial 238 Boltzmann, Charge-voltage 239 Boltzmann, Shifted 240 Boltzmann, Standard 240 Boltzmann, Z-Delta 241 Current-Time Course (Hodgkin-Huxley) 241 Exponential, Alpha 241 Exponential, Cumulative Probability 242 Exponential, Log Probability 242 Exponential, Power 242 Exponential, Probability 243 Exponential, Product 243 Exponential, Sloping Baseline 243 Exponential, Standard 244 Exponential, Weighted 244 Exponential, Weighted/Constrained 244 Gaussian 245 Goldman-Hodgkin-Katz 245 Goldman-Hodgkin-Katz, Extended 246 Hill (4-parameter Logistic) 246 Hill, Langmuir 247 Hill, Steady State 247 Lineweaver-Burk 247 6 1-2500-0180 G Logistic Growth 248 Lorentzian Distribution 248 Lorentzian Power 1 248 Lorentzian Power 2 249 Michaelis-Menten 249 Nernst 249 Parabola, Standard 250 Parabola, Variance-Mean 250 Poisson 250 Polynomial 251 Straight Line, Origin at Zero 251 Straight Line, Standard 251 Voltage-Dependent Relaxation 251 Constants 251 Appendix A: References 253 Primary Sources 253 Further Reading 256 Appendix B: Troubleshooting 257 Software Problems 257 Hardware Problems 257 Obtaining Support 258 Appendix C: Resources 259 Programs and Sources 259 Index 261 1-2500-0180 G 7 pCLAMP 10 Data Acquisition and Analysis Software User Guide 8 1-2500-0180 G Chapter 1: Introduction 1 pCLAMP™ Data Acquisition & Analysis Software is the data acquisition and analysis suite from the Axon Instruments Conventional Electrophysiology product line of Molecular Devices. Designed for a variety of experiments, pCLAMP Software Version 10.6 is the latest version that has become the standard for electrophysiological experimentation and analysis. The flexibility that pCLAMP software offers allows researchers to adapt it to many uses outside its traditional applications in electrophysiology. The pCLAMP Software Version 10.6 suite contains the following: Clampex Software, for data acquisition and production of stimulus waveforms. Clampfit Software, for data analysis. AxoScope Software , for background chart recording. The optional accessory MiniDigi 2-Channel Digitizer can be ordered separately. Clampex Software is a versatile and powerful software tool for acquiring digitized data of all types. While excellent for the acquisition of patch-clamp data, it is not limited to measuring voltage-clamp or current-clamp responses. Clampex Software can be used to measure any physical parameter that can be linearly converted into a voltage. For example, you can monitor and acquire end-plate currents, measure the fluorescence signal from a photomultiplier tube, measure pressure from a strain gauge, or acquire any other combination of analog signals. Clampfit Software is powerful data analysis software with a wide variety of statistics, analyses, transforms, and layout tools for electrophysiological data. Together, AxoScope Software and the optional MiniDigi digitizer provide the functionality traditionally performed by a separate chart recorder, for example, for concurrent background recording. 1-2500-0180 G 9 pCLAMP 10 Data Acquisition and Analysis Software User Guide New Features AxoScope Software and Clampex Software See Enabling the HumSilencer System Controls on page 55, for further description of the following new feature. There are three models of the Digidata 1550B digitizer, one of which excludes the HumSilencer Adaptive Noise Cancellation System (ANC). The other