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WINEDR User Guide - Spider

1. m Curve fitting Fit Curve x Set Zero Level None S C 1 pA SavetoLog Current To plot the histogram 1 Select All Points In State from the Histogram Type list 2 If more than one signal channel is available select the channel to be used from the Channel list 3 Select the All option to use all detected channel transition events in the recording or select Range and enter the sub range of events to be included Select the channel state s Close Open X1 or All to be included in the histogram from the State list D 5 A numter of zero or more samples at the beginning and end of each state can be excluded from the histogram to eliminate the parts of the signal associated with the transition between states Enter the number samples to be excluded in the Margin Points box Enter the width of the histogram bin in the Bin Width box Ne g Click the New Histogram button to compile and display the histogram 115 Analysing Single channel Currents gt Current Amplitudes gt Amplitude Histograms gt Mean State Amplitude Histogram This histogram displays the mean amplitude for each detected channel state computed from the average of the sample points contained within the selected set of channels states closed open and all Note This option can only be used after transition detection has been completed Single channel C
2. Computing Averages To compute the event average waveform 1 Select the All Events option to average all detected events or select Range and enter a selected range of events 2 Select the Count Matched option if you wish to compute a count matched average and enter the number of events to be averaged in the No Events box The count matched average is an average of a defined number of the largest amplitude events within the selected event range It provides a method for comparing average event amplitude before and after an experimentally induced reduction in amplitude which compensates for the failure to detect small events which have fallen below the detection threshold See Stell amp Mody J Neuroscience 2002 Vol 22 RC223 3 Set the of the averaged waveform to be before the event detection point in the Pre detection samples box 4 Set the Event Analysis Polarity of the average waveform measurements Select Positive for positive going waveforms and Negative for negative going 5 Define the peak detection region using the pair of a cursors This option can be used to exclude stimulus artefacts or other signals feature from the waveform analysis 6 Click the Compute Average button to calculate and display the average waveform Fitting curves to averages To fit one or more exponential functions to the average 1 Define the segment of the average waveform be fitted using the p
3. Analog Outputs A0 M No Channels h gt L 5500 ms Protocol Waveforms A00 one ne gt Protocol A0 0 Stimulus type AO Waveforms ne Voltage mv Digital Outputs DO No Channels RE Up to 4 voltage waveform output channels AOO AO3 are available and 8 TTL digital pulse channels DOO DO7 A diagram of the output waveforms appears in the Waveform display box To create a stimulus protocol click the New Protocol button to create a blank protocol Next Recording Experimental Signals gt Creating Stimulus Protocols gt Analog amp Digital Output Channels Analog Outputs AO Select the number of analog output channels to be used in the analog outputs No Channels list For each AO channel defined select the type of stimulus Voltage or Current and the stimulus units in the Stimulus Type list Analog Outputs 40 No Channels 1 A00 Stimulus type voltage im Digital Outputs DO Select the number of digital output channels to be used in the digital outputs No Channels list M Digital Outputs DO No Channels RE X Note during execution of a stimulus protocol the analog and digital holding levels override the default holding levels set in the Seal Test or Default Settings windows Next Recording Experimental Signals gt Creating Stimulus Protocols gt P
4. The height of each histogram bin indicates the number of samples falling within the limits of the bin The number of events within each bin and the dwell time at its mid point can be read out using the green readout cursor The number of events contained in the bins falling within a selected range of dwell times and the mean dwell time of that region can be displayed by placing the pair of grey f f cursors at the limits of the region of interest 0 0265 6 02 0 0 Mean 0 0206 s Events 230 Seven basic types of histogram can be produced from the dwell time data in the channel state list Open times distribution of channel open times Closed times distribution of channel close times Burst Length distribution of the duration of bursts of openings a burst being defined as a series of opening interspersed by short closures terminated by a closure longer than a predefined period Single open times distribution of open times of channel openings which do not form part of a burst Opens times within bursts distribution of open times of channel openings which form part of a burst Openings per burst distribution of the number of channel openings within a burst External file distribution of times loaded from an external file Analysing Single channel Currents gt Channel Dwell Time Analysis gt Plotting a Dwell Time Histogram To plot a dwell time histogram 1 Set the type of histogram to be plotted using the
5. Parameter Initialisation _oK Automatic Manual Initialise Click the OK button to begin fitting The best fitting gaussian function s are superimposed in red on the histogram The values of the best fitting function parameters along with their estimated standard errors are displayed in curve fitting results area at the bottom of the display 90 905 16 0 6 0 8 Peak nA Fit Results vod Peak exp Oep 2i 20 u 0 8804 0 003153 sd nA o 0 02931 0 003225 sd nA Peak 15 51 1 345 sd Residual D 2 611 Degrees of freedom 18 104 Detecting amp Analysing Events gt Averaging Events The averaged waveform of a series of events can be computed and displayed on the Average page Averages can be computed from a specified range of events or using the count matched averaging process to average the n largest events EO lx Detect Events ReviewEdit Events XY Plot Histogram Average Compute Average 15 Abort Events All events Range Im 1 128 n M Count Matched No Events mo 05 m Event Analysis Avg of 1 128 128 Peak a aj 0 79714 nA Area a aj 2 0438 nA ms Ttrisej 0 2 ms Cure fitting T 90 5 6 ms Fit Curve Tau decayj 2 3197 ms Duration 6 1 ms None 8o0G8 ta t 0 011 0 055086 n al 0 0 002 0 004 0 006 0 008 0 01 0 012 0 014 0 0160 018 s
6. Get C 1 Ce H i yi Width alf128ms pl The simplest approach to the classification of the single channel current signal into open and closed states is to place a transition threshold at the mid point between the zero and single channel current levels If the signal lies above the threshold the channel is deemed to be open if it lies below the channel is deemed to be closed An idealised sequence of channel open and closed periods is thus obtained by scanning through the digitised recording point by point counting the time between threshold crossings Analysing Single channel Currents gt Detecting Channel Transitions gt Detecting Channel Open Close Transitions Single channel Current Analysis Amplitude Histograms Transition Detection Euit Channel States Dwell Time Histograms SummaryStability 4 gt Detect Transitions Abort m Range Whole file Range 0 10 035 s Use C0 C1 m Detection parameters Channelfcn o Im S C current 7 pA Threshold 50 0 level Trend Removal Linear AddBlock_ o Clear Blocks Subtract trend Restore Original Cursors Get C 0 Get C 1 m 0 342 0 344 0 346 0 348 0 35 0 352 xj width alf128ms gt l To detect the channel open close transitions in a single channel current recording 1 Define the section of the recording to scanned Select the Whole file
7. HoldingVoltage R W Property Reads sets the selected DAC channel holding voltage V applied to the cell E g W HoldingV oltage 0 06 DAChannel R W Property Reads sets the selected D A channel to which HoldingVoltage is applied TriggerMode R W Property Read sets the recording sweep trigger mode F Free run E External trigger NumTriggerSweeps R W Property Read set number of triggered sweeps to be acquired when recording with TriggerMode E StimulusProtocol R W Property Read sets the selected stimulus pulse protocol e g W StimulusProtocol prot01 StartStimulus Method Start stimulus selected by StimulusProtocol Status Read Only Reads the current operational status of Property WinEDR 0 idle 1 seal test running 2 recording to disk Seal test functions WinWCP s seal test function can be initiated via the command interface and used to apply test pulses to cells and calculate the cell membrane conductance capacity access conductance and pipette seal resistance These measurements can be read via the command interface while the seal test is running The seal test commands are listed below Seal Test Methods and Properties StartSealTest Method Displays the seal test window and applies the seal test pulse SealTestPulseAmplitude R W Property Reads sets the amplitude Volts of the seal test pulse e g W SealTestPulse A mplitude 0 01 SealTestPulseDuration R W Property Rea
8. Plenum Press 483 587 Cull Candy S G Howe J R amp Ogden D C 1988 Noise and single channels activated by excitatory amino acids in rat cerebral granule neurones J Physiol 400 189 222 Dempster J 1993 Computer Analysis of Electrophysiogical Signals Academic Press Dempster J 2001 The Laboratory Computer A practical guide for neuroscientists and physiologists Academic Press Defelice L J 1981 Introduction to membrane noise Plenum Press Eisenberg R S Frank M amp Stevens C F 1984 Membrane channels and noise Plenum Press Ogden D ed 1994 Microelectrode Techniques The Plymouth Workshop Handbook 2 ed The Company of Biologists Ltd Ruff R 1977 A quantitative analysis of local anaesthetic alteration of miniature end plate currents and end plate current fluctuations J Physiol 264 89 124 Sakmann B amp Neher E 1995 Single Channel Recording 2 ed Plenum Press
9. 4 Run WinEDR and select from the main menu Setup gt Laboratory Interface to open the Laboratory Interface Setup dialog box _ Laboratory Int 5 x Jaxon Instruments Digidata 1200 Unknown AD Converter Voltage Range 5 Y OK Cancel then select Axon Instruments Digidata 1200 from the list of laboratory interface options Signal input output connections Signal input and output connections are made via the BNC sockets on the front and rear of the Digidata 1200 I O box Digidata 1200 Analog In 5 Analog In 7 Analog Output Analog Out 1 O Digital Out 1 sd Cd Digital Out 3 Note 1 An active high TTL pulse on this input triggers the start a stimulus program which has been set up with the External Stimulus Trigger Y option Note 2 WinEDR only supports digital output lines 0 3 of the Digidata 1200 Troubleshooting There are two known problems which will prevent WinEDR from recording from a Digidata 1200 s analog input channels T O port conflict The Digidata 1200 default I O port addresses span the range 320H 33AH These settings conflict with the default MIDI port setting 330H of Creative Labs Sound Blaster 16 and similar sound cards There are a number of solutions to this problem 1 Change the Sound Blaster MIDI port setting to a value higher than 33AH 2 Remove the Sound Blaster card or disable it using the BIOS setup if it is built in to the computer motherboard DMA ch
10. 4xS D button can be used to set the amplitude threshold to 4x the standard deviation computed from the displayed signal Dead Time To avoid repeated detection of the same signal set the time period before another signal can be detected in the Dead Time box Usually set equal to the expected duration of the longest signals being detected Rising Edge Window Click Detect Events to begin the event detection process Detecting amp Analysing Events gt Reviewing amp Editing Events After a list of detected events has been created each event can be inspected on the Revie w Edit Events page Event Detection Detect Events ReviewEdit Events x y Plot Histogram Average Event 57 128 1 5 M Event Analysis Detected at 5 10555 Peak a aj 0 10758 nA Area a aj 1 9317 nA ms T rise 0 05 ms T 90 0 1 ms Tau decayj 0 ms Duration 0 2 ms gt Event polarity C Positive Negative Zero level Atstart At event Subt Baseline Trend Pts Avod 20 Gap jo gt TEX decay time Decay From g0 Peak 0 5 al al 5 104 5 106 5 108 5 11 5 112 5 114 5 116 5 118 Det 1 M Edit Events Insert F1 Delete F2 m mena GAGRE T a aa a T a rT 0 0 002 0 004 0 006 0 008 0 01 0 012 0 014 0 0 JV Init cursor to detection point Export Events KI E ___Export Events _Abort Width Pre detect
11. 5 0 cm Top 50cm Line width 2 pts Botomf50cm Marker size 5ps Use colour a cence Click the OK button to print the graph Analysing Single channel Currents gt Current Amplitudes gt Copying Histograms to the Clipboard Copying the histogram data points to the Windows clipboard The numerical values of the X Y data points which generate the histogram can be copied to the clipboard by selecting Edit Copy Data The data is placed on the clipboard as a table of data values in tab text format defining the histogram There are 4 values per row and one row for every bin in the histogram Each row has the format lt Bin Lower Limit gt lt tab gt lt Bin Mid point gt lt Bin Upper Limit gt lt tab gt lt Bin Count gt lt cr gt lt lf gt Copying an image of the histogram to the Windows clipboard An image of the histogram plot can be copied to the clipboard by selecting Edit Copy Image to open the dialog box Copy Image x Typeface Image Size arial width J 600 pixels Size 12 pixels Height 500 ixels Line width 2 pixels Marker size 5 pixels Use colour E The dimensions pixels of the bit map which will hold the image can be set using the Width and Height image size boxes The size and style of the typeface can be set using the Typeface and Size boxes When the image parameters have been set click the OK button to copy the image
12. Amplifier 3 Amplifier 4 Manual gain entry v Input Channels Primary channel Im Scale factor 0 001 VipA Units pA On analog input al Ch 0 Secondary channel Vm Units mv Scale factor 9 001 VimV On analog input aich 1 Voltage clamp command channel Scale factor 1VN Output ao Ch 0 Current clamp command channel Scale factor 4 av Output ao Ch 0 Load Settings Save Settings Default Settings Enter the current clamp command potential scaling factor membrane current command voltage into the Scale factor box The current clamp command scaling factor can usually be found in the amplifier user manual Typical values are between 10 A V and 10 A V Usually the current command signal is applied to the same input on the amplifier as the voltage command AO Ch 0 for Amplifier 1 however on some there may be a separate current command input Saving Loading Settings Amplifier and input channel settings can be saved to a settings file by clicking the Save Settings button and saving the settings to an XML settings file Settings can be reloaded from a settings file by clicking Load Settings and selecting the file The default scaling and channel settings for an amplifier can be reset by clicking the Restore Default Settings button Default settings are also loaded whenever the amplifier type is changed Next Getting Started gt Amplifiers gt Channel Calibration Table E Input Channels amp Amplif
13. AxDD1550 DLL and AxDD1550A DLL and device drivers for the Digidata 1550 and 1550A Series 1 Install the AxoScope or PCLAMP software supplied with the Digidata 1550 1550A 2 Reboot the computer 3 Attach the Digidata 1550 or 1550A to a USB port and turn it on 4 Run WinWCP and select from the main menu Setup gt Laboratory Interface to open the Laboratory Interface Setup dialog box Then select Molecular Devices Digidata 1550 if you have a Digidata 1550 or Molecular Devices Digidata 1550A if you have a Digidata 1550A from the laboratory interface list nterface lol xi DD1550A vd100 3 4 firmware 00 AID Converter Voltage Range 10v oK Notes a The HumSilencer feature of the Digidata 1550A is not currently supported by WinWCP b When a recording is sweep manually terminated by the user clicking the Stop button in the Record to Disk window while WinEDR is waiting for an external START trigger pulse in the Ext Triggered recording mode an additional trigger pulse must be applied to the Digidata 1550 1550A START input to terminate recording c Support for analog input channel re mapping in the AI Ch column of the Input Channels table in the Input Channels amp Amplifier Setup dialog box is limited Recording channels can only be remapped to HIGHER analog input numbers Signal input output connections Signal input and output connections are made via the BNC sockets on the front of the Digidata 1440A digit
14. By combining eqns 3 and 4 the single channel conductance can be calculated from the Lorentzian curve fitted to the ionic current power spectrum fac So u 2 1 Oo I 5 Note In these circumstances no information can be derived concerning the total number of channels Miniature endplate current noise It is also possible to analyse the current fluctuations associated with the miniature endplate currents under conditions of the high frequency release evoked by increasing the K ion concentration of the bathing solution The MEPC noise spectrum can be modelled with the three parameter F F A function A IF F f f F F SUA Eqn 6 can be fitted to a spectrum by selecting MEPC Noise from the fitting function list Parameters F and F are associated with the MEPC rise and decay time course while A is associated with the overall noise amplitude The time constants for MEPC rise and decay and a can be computed from these After the and a time constants have been established by fitting eqn 6 to the MEPC noise power spectrum the frequency of MEPC release can be computed by clicking the MEPC Frequency button on the Variance Analysis page Noise Analysis gt Printing Plots or Spectra To print the displayed plot select File Print To open the dialog box Click the OK button to print the graph 174 Noise Analysis gt Copying Plots to the Clipbboard Copying the plot data
15. Editing tie Channel Event WISE sce cteact tle otS lol eee ee Nila taal a eran o ieee seca 135 Exporting the Channel State List cuccnio ate 8 ern aoe adel aa ae eeineliondniy 136 CUPSOR M asurem ents sienen spud ic ange tat aesvemncas a uaqssadcocaeanta e a aA 137 Channel Dwell Time Analysis Analysis of Channel Dwell Times cssscscecsccececesesssecssseesceesecscsceecsceeaceesecsceassceeceseeeaeeseeeaes 139 Plotting a Dwell Time Histogram ccccccececcssescsseseseseeseseescseseesesesecsesesesesenseseeecaeseeseseeesseeeseseeeeaes 141 Fitting exponential Functions to dwell time histOgraMs 0 0 0 ee eset csc etetsenececeteteneeenees 145 COSTOMMISING HIStOGAIIP OUS swsscicnsserdiracit dies ain cseee reek areata etn een AO 151 Prining Histograms sce costes ness niena anata A E EEE E cuntlas tees TAO ate 152 Copying Histograms to the Clipboard ee ceeceeccseeeeseeseeecseeseeecseeseeecseescseeseeeeeeeseeaeeaeeecaeeaeeetseeens 153 Stability Plots Stability PIOUS nannan a ene hast Geta agaes ae Ran eee Oe 155 Printing PlOtS cic 06 e325 oR Sathana ane Re la Bah i AE OS 157 Copying Plots totne Clipboard 4 224 ese rasta ee lar ale cal aS Ok es 158 Noise Analysis INGA Bea SIS ascetic waar cane cata wads thse cace ons ace ganas ates nok E 159 Signal GondivoninG 4223 26 vasin aE Senseo dee cea RG inden ee 161 Selection amp Inspection Of Variance RECOIS cccececsssesesssseseseeseseseeseseescseseescsesenseseescsesenseeesecseeee
16. Inst Frequency The average event frequency computed over a defined period of time Rate The peak amplitude positive or negative depending upon the setting of the Polarity option of the event peak The integral of the event waveform area The 10 90 rise time of the event T rise The X decay time after peak T X The time constant of exponential decay Tau decay Signal baseline level determined just before the onset of the signal Baseline Detecting amp Analysing Events gt Plotting Histograms of Event Analysis Measurements The Histogram page allows histograms of the distribution of event analysis measurements to be plotted Lx Detect Events Review Edit Events xY Plot Histogram average New Histogram 30 Peak 25 20 No Bins 100 15 Bin width 0 02 nA 10 Lower Limit 0 n 5 Upper Limit 2 na 0 31 28 i 0 91 F cumulative 0 02 04 06 08 1 12 14 16 1 P Percentage Peak nA Events All events Curve fitting Range Fit Curve 1 ii 128 None Set Axes Mean 0 90252 nA Min 0 80096 nA Max 1 6556 nA n 127 Plotting a Histogram To plot a histogram 1 Select the variable to be plotted from the Histogram variable list 2 Select the All Events option to plot the results from all events in the list or select Range and enter a selected range of events 3 Define the upper and l
17. Laboratory Interface to open the Laboratory Interface Setup dialog box Laboratory Interface Setup F iol xj National Instruments NIDAQ Mx juse 6211 8 ch 16 bit 10V ADC 2 ch 16 bit 10 DAC Device AJD Input Dey1 7 Differential AD Converter Voltage Range i 140 OK Cancel then select National Instruments NIDAQ MX from the list of laboratory interface options except for Lab PC or 1200 series cards in which case use National Instruments NIDAQ Trad 7 Select the device number of the card listed in the NI Devices amp Interfaces list from the Device list usually Dev1 if only one card is installed 8 Set the A D Input mode If you are using a BNC 2110 or BNC 2090 input output box select Differential Note The SE DI switches on a BNC 2090 panel must be set to DI If you are using a Lab PC or 1200 series card set the A D Input mode to Single Ended RSE Signal input output connections Signal input and output from National Instruments PCI cards are made via 68 pin sockets on the rear of the card attached to BNC socket input output panels BNC 2110 or BNC 2090 or screw terminal panels by appropriate shielded cables available from National Instruments i 21210 10 10 01010 10 10 0 2o f National Instruments X E amp M Series cards National Instruments X E amp M Series cards Analog Input 1 O Panel Screw terminal panel Ch 0 68 67 62 signal ground Ch 1 33 67
18. Points Amplitude Histogram The All Points histogram represents the distribution of current amplitude within the digitised record irrespective of the open close state of the channel It is used for initial analysis of the current amplitude distribution determination of single channel current amplitude number of channels in the patch and the steady state open channel probability popen The current amplitude range is divided into a series of equally spaced bins up to 512 Each sample point within the digitised signal is allocated to a bin according to the formula 7 i L Lond Bin no 1 lo where Ihi and Ilo are the upper and lower limits of the current range nb is the number of bins and i is the current for A D sample i The distribution is displayed as a histogram of the percentage of the total number of sample points contained within each histogram bin Single channel Current Analysis iol x Amplitude Histograms Transition Detection Edit Channel States Dwell Time Histograms SurmmaryiStabi 4 gt New Histogram Set Axes Abort Histogram Type All Points x Data Channel cho Im Whole file C Range 0 10 0352 Use C0 C1 No bins 200 Bin width 0 015 pA Lower Lim 1 453 pA Upper Lim 1 547 pA 0 4895 0 0 369 0 0 369 0 738 1 11 All Paints pA Mean 0 0956 pA Area 100 Curve fitting Fit Curv
19. _ S500 ms No Channels fr Protocol Waveforms A00 m Toolbox Protocol iE A00 pee peee pempem oe ae a pee PPE AO Waveforms Y voltage mv M Digital Outputs DO No Channels F Waveforms are constructed by dragging waveform step and ramp elements from the Toolbox and dropping them into the selected voltage channel AO 0 AO 4 or digital DO 0 DO 7 output list A plot of the resulting stimulus protocol for each output channel is shown in the protocol display panel A stimulus waveform on each output channel can consist of up 10 separate elements The amplitude and duration for each element is defined in its parameters table which can be made to appear by clicking on the element Eight analog and 4 digital waveform elements are available in the toolbox as detailed below Rectangular voltage pulse of fixed size A simple pulse which does not vary in amplitude and duration between records This element can be used to provide series of stimuli of fixed size or in combination with other elements to provide fixed pre conditioning pulses pf Parameters Initial Delay Delay at the holding level before the pulse begins Amplitude Pulse amplitude Family of rectangular pulses varying in amplitude H A rectangular voltage pulse whose amplitude is automatically incremented between recording sweeps This element is typically used to explore the voltage sensitiv
20. and save the file Standard CED 1401 The performance of the original CED 1401 interface unit is very limited compared to later models The number of samples record is limited to a total of 8192 and the achievable sampling rate when stimulus pulses are being generated is limited to a maximum of around 20 kHz divided by the number of channels In some circumstances the sampling rate set by WinEDR can exceed the capabilities of the standard 1401 resulting in samples between mixed up between channels This problem can be resolved by reducing the number of samples per record or by increasing the duration of the recording sweep The CED 1401 ISA card default I O port addresses are at 300H Check that these do not conflict with other cards within the computer The CED 1401 also makes use of DMA channel and an IRQ channel IRQ2 These may also conflict with other cards Some standard 1401 appear to fail the DMA direct memory access test in TRY 1401W and this also causes problems when running WinEDR If this error occurs disable the DMA channel by clicking on the CED 1401 icon within the Windows Control Panel and un checking the Enable DMA transfers check box Getting Started gt Laboratory Interfaces gt Axon Instruments Digidata 1320 Axon Instruments Inc now owned by Molecular Devices www moleculardevices com The Digidata 1320 Series 1320A 1322 interfaces consist of self contained mains powered digitiser units with BNC I O sockets
21. in the file size of record number and scaling of channels This is followed by a data block containing the A D converter samples stored as 16 bit integers If more than one input channel has been digitised samples are interleaved within the data block e g Ch 0 Ch 1 Ch 2 Ch 0 Ch 1 Ch 2 Given information on the size of each of the file header block and the number of channels the import module can extract the signals from the file These details of the data file structure can often be obtained from the user manuals associated with the software which created the data files Note that the sampling interval and other scaling information is discarded by the binary import module Raw Binary Import loj x File Description Channels File header size bytes 0 Ch Name Unitsibit Units No of signal channels 7 0 vm 0 001 Read in blocks of 512 Sample Format C Float Integer No of bytes sample 2 Max Value 2047 Sampling interval ims msecs secs mins OK Cancel The Import settings must be set up to match the characteristics of the file and data being imported 1 Enter the size of the file header in the File header box If no file header exists set this value to zero 2 Enter the number of analog data channels in the file in the No of signal channels box 3 Provide names scaling factors and units for each data channel in the Channels table 4 Set the nu
22. membrane current and voltage and computer generated voltage pulses are applied to the patch clamp command voltage input to stimulate the cell Patch Clamp AO Ch 0 Al Ch 0 Al Ch 1 Gain Telegraph Al Ch Mode Telegraph Al Ch 6 VO Panel WinEDR supports up to 4 patch voltage clamp amplifiers and for each amplifier in use up to 5 analog signal connections must be made between the amplifier and laboratory interface A pair of laboratory interface analog input channels designated the primary and secondary input channels are required to record the membrane current Im and membrane voltage Vm outputs from the amplifier An analog output must be connected to the amplifier command voltage input Vcom to provide current voltage stimulus waveforms Two additional analog inputs may be required to receive the amplifier gain GAIN and voltage current clamp mode MODE telegraph signals Note Some patch clamps do not support gain and or mode telegraphs others communicate gain mode information via USB or other communications lines WinEDR supports many of the commonly used models of patch and voltage clamp amplifiers and is able to read gain and mode telegraph signals allowing the current and voltage signal channels factors to be scaled correctly The required primary and secondary input channel command voltage output gain and mode telegraph signals connections for the amplifiers currently supported by WinEDR are shown in the table on
23. 0 cm Line width 2 pts Bottom 50m Marker size 5 pts Use colour E comet Click the OK button to print the graph Analysing Single channel Currents gt Stability Plots gt Copying Plots to the Clipboard Copying the plot data points to the Windows clipboard The numerical values of the X Y data points which generate the plot can be copied to the clipboard by selecting Edit Copy Data The data is placed on the clipboard as a table of data values in tab text format defining the histogram There are 4 values per row and one row for every bin in the histogram Each row has the format lt X gt lt tab gt lt Y gt lt cr gt lt lf gt Copying an image of the plot to the Windows clipboard An image of the histogram plot can be copied to the clipboard by selecting Edit Copy Image to open the dialog box Copy Image xj Typeface Image Size Arial Width 600 pixels Size 12 pixels Height 500 ixels Line width 2 pixels i Marker size 5 pixels Use colour E The dimensions pixels of the bit map which will hold the image can be set using the Width and Height image size boxes The size and style of the typeface can be set using the Typeface and Size boxes When the image parameters have been set click the OK button to copy the image to the clipboard Noise Analysis gt Noise Analysis To analyse ionic current noise select Analysis Noise Analysis
24. 62 65 67 62 30 67 62 Ch 5 60 67 62 Ch 6 25 67 62 Ch 7 57 67 62 Analog Output 22 55 Trigger Inputs Ext Sweep Trigger PFIO TRIG1 11 44 Ext Stimulus Trigger PFIO TRIG1 NIDAQ MX 11 44 See Note 1 PFI1 TRIG2 NIDAQ 10 44 Digital Output Output Ch 1 PO 1 17 53 Note 1 An active high TTL pulse on this input triggers the start a stimulus program which has been set up with the External Stimulus Trigger Y option The trigger signal is applied to PFI0 when the NIDAQ MxX interface library is in use Laboratory Interface Card National Instruments NIDAQ MX and PFI1 when the Traditional NIDAQ interface library for Lab PC 1200 cards only is in use Laboratory Interface Card National Instruments NIDAQ USB 6000 to USB 6005 Devices The input output connections for the low cost USB 6000 6005 devices are Ch 2 Al 2 2 signal ground Ch 3 Al 3 3 Signal ground Ch 0 AO 0 Gnd signal ground PFIO PFI1 and P0 0 MUST be connected together for the test pulse to generated in the Seal Test window Lab PC 1200 Series Cards The input output connections for 50 pin Lab PC and 1200 series boards are tabulated below ee fae som Se E meow ee ja jar oe us Analog Outputs Co jar ee fan C NOTE 2 Analog output channel 1 DAC1 is used to synchronise the start of the A D conversion and D A waveform generation and must be connected to EXTTRIG for WinEDR waveform generation functio
25. D A converter update interval to be used to produce the analog stimulus waveforms within the protocol D74 Update Interval 0 2734 ms Fix Interval The interval is normally set to Recording Duration No Samples see Recording Settings but may be greater than this if the laboratory interface cannot support this D A update rate or less if short duration pulses exist within the protocol If digitised analog waveform elements exist within the protocol the update interval is set to to the D A update interval for these waveforms To keep the D A update interval fixed at a specified value enter the update interval into the D A Update Interval box and tick the Fix Interval option Note If the laboratory interface cannot support this D A update rate it will be adjusted to the minimum possible update interval for the interface Next Recording Experimental Signals gt Creating Stimulus Protocols gt Saving and Loading Stimulus Protocols When you have created a stimulus protocol you can save it to a protocol file by clicking the Save Protocol As button to open the Save Stimulus Protocol dialog box Save stimulus rotcal Te Save in yprat c EJ T2 2 step 30mv Jan 10m A 03a Rab AP rate dep BCL 800 40m Jan 20mY My Recent 03b Rab AP rate dep BCL 500 S 50mv Jan 10m ee 03c Rab AP rate dep BCL400 2 cedtest S gt Jan 20m E 03d Rab P rate dep BCL 300 DigOPulseTrain Jan 3
26. Display Duration C ms s C min al 10s gt The display area of the screen acts like a digital oscilloscope displaying live traces of the incoming analogue signals The Signal Level box provides a readout of the signal level on each input channel The duration of the live display window can be adjusted using the Display slider bar at the bottom right of the screen or by entering a duration in the Dis play box The vertical magnification for each channel can be expanded to a selected region by moving the mouse to the upper limit of the region pressing the left mouse button drawing a rectangle to indicate the region and releasing the mouse button The vertical magnification can also be adjusted using the buttons at the right edge of each plot Individual channels can be added removed from the display by clicking the button at the left edge of each channel The vertical area of the display devoted to each channel can be adjusted by dragging the top left edge of each channel Y axis up or down Recording Experimental Signals gt Recording Modes gt Trigger Modes There are two trigger modes Free Run and External Trigger Triggger Mode Special Free Run External Trigger Trigger Level 5v OV When the trigger mode is set to Free Run recording starts immediately after the Record button is pressed and continues until the required recording duration has been acquired Choose the free run mode
27. E Sunil Logantha Testi Desktop Trevor Smart TSeries 06242009 1457 065 e E 04 02 2009 R txt B binarised 514Etm 1250 2250 txt My Documents E test cursor measurements txt BL My Computer K File name a EE Files of type Places J Open as read only Select the disk drive and folder from the Look In list Then select ASCII Text txt asc from the Files of Type list A list of available files in that type are displayed 2 Select one of the file names then click the OK button to open the ASCII Import dialog box ASCII Import xj 0 002 0 0611 0 00195 0 06115 0 0019 0 0611 0 00185 0 06115 0 0018 0 06115 0 00175 0 06115 0 0017 0 0611 m Column Separator Channels Tab Comma Space Ch Name Ym Units my No of title lines to be skipped 0 Time Time Data in Col 0 User Defined Sampling Interval Os Units C msecs secs mins oon one WH oO OK Cancel which allows you to view the format of the data to be imported and to specify how it should be imported Column Separator Select the character used to separate data columns in the file lt tab gt comma or single space character No of title lines to be skipped The first data row s in the table often contain labels or identification information which should not be treated as samples To skip one or more of these lines enter th
28. Ext Sweep Trigger input triggers the start of a recording sweep when Ext Trigger sweep trigger mode has been selected Note 3 An active high TTL pulse on the Ext Stimulus Trigger input triggers the start a stimulus program which has been set up with the External Stimulus Trigger Y option Troubleshooting tips Verify that the CED 1401 is working correctly before investigating problems using WinEDR Use the TRY 1401W program to test the CED 1401 WinEDR uses the commands ADCMEMI CMD MEMDACI CMD and DIGTIM CMD with the CED 1401 ADCMEM GXC MEMDAC GXC and DIGTIM GXC with the CED 1401 plus and ADCMEM ARM MEMDAC ARM and DIGTIM ARM with the CED Micro 1401 All three commands must be available within the 1401 directory Power 1401 The digital pattern output command DIGTIM appears to behave differently on different versions of the Power 1401 resulting in the the digital output waveforms in a WinEDR stimulus protocol being produced incorrectly On Off levels are inverted and pulse durations are incorrect This problem can be corrected by setting the CEDPOWER 1401DIGTIMCOUNTSHIFT entry in the WinEDR lab interface xml file in c WinEDR to 0 Stop the WinEDR program if it is running and open the file c WinEDR lab interface xml with the Notepad text editor and search for the entry CEDPOWER1401DIGTIMCOUNTS HIFT gt 1 lt CEDPO WER1401 DIGTIMCOUNTSHIFT Change it to CEDPOWER1401DIGTIMCOUNTS HIFT gt 0 lt CEDPO WER 1401 DIGTIMCOUNTSHIFT
29. Files EDR 7 Cancel Detecting amp Analysing Events gt Plotting X Y Graphs of Event Analysis Measurements The X Y Plot page allows the event analysis measurements for the series of detected events to plotted against each other Event Detection 5 x Detect Events ReviewEdit Events Y Plot Histogram Average New Plot Set Axes Abort m Plot X AXIS Event No Y Axis Peak T Events __ All events C Range 1 128 D To create an X Y plot 110 1 6443 60 80 100 120 Event No 1 Select the variable to be plotted on the horizontal axis from the X Axis variable list 2 Select the variable to be plotted on the vertical axis from the Y Axis variable list If Rate has been selected enter the interval over which the event frequency is to be calculated 3 Select the All Events option to plot the results from all events in the list or select Range and enter a selected range of events 4 Click the New Plot button to display the plot 5 If you want to customise the axes ranges click the Set Axes button The values of the x y points in the plot can be read out using the blue readout cursor Variables that can be plotted include e The time at which the event was detected Detected at e The time interval between successive events Interval e The instantaneous event frequency i e the reciprocal of Interval
30. Garena ae endeared es 28 Teel Tea PS PTH gH LT Ae 8 tcc lose ai dg Ss IR ONG sec Be OS 29 Heka Patch Clamps amp Interfaces ates ae el eh a eh eee Pala ed tak oF it al seta 30 Amplifiers Patch Voltage claMp Amplifiers cc ccsescescssesesseseescsssscsscssesscsseseescseescsesseescsessesscsessesaseaesseaeees 32 Sighal Connections Tableseressreris oie aes ae ake rene need neni 33 Configuring Amplifier Support in WINEDR eee eceeceeecseeseeecseeseeeeeeesceeeseeseeaesecaeeaeeecaeeaeeetseseees 35 C annel Calibration Tables Genesia nen cach winters E A E a 38 CED TOO 2 AiG Ob eeii nna erates a a eia e nie 39 Tecella Patch Clamp Amplifier Control Panel ccccccccssesesseseseseeseseseeseseeeceesenseeeeecseeeseseenaes 41 Heka EPC9 01 Patch Clamp Control Panel ccccceesseseseseeseeeseseseeseseseeseseeecaesenseseeecseeeseseeeaes 43 Molecular Devices Multiclamp 7OOA B cccccccssecescssesesseseseseeseseescseseesesesenseeeeecsesenseseeecseseeseseneeas 45 DCLAMP Dynamic Clam P rscene sensan a a een canna andes oes 46 Recording Experimental Signals Monitoring Input Signals amp Patch Pipette Seal TeSt ee eeecseeseeecneescneesesseseesecseeaeecseeaeeetaeeeens 48 Recording SIG Mall S corset cass seteceutentsncosessants eeen aa EE E ear a aai tenet 51 Recording Modes aae Modes a eS Rit ge A a a a A RAR nO a A er 53 roLO CAB Fe U0 E eer eee iets EEE nites E E E E 00 E E 54 Creating Stimulus Protocols Openin
31. at fixed intervals or random time intervals governed by an exponential distribution Gaussian background noise and sine wave interference can be added to the signals to test the abilities of the event detection system To create a data file containing simulated mPSCs 1 Create a new data file to hold the records by selecting File New and entering the name of a new data file Select Simulations Post synaptic currents to open the window _Postsynaptic Current Simulation Start Simulation Abort Duration 10s PSC Parameters Peak tpA St Dev oopa Tau rise ooms Tau decay 2 0 ms Units pa v r Event Frequency At Random Intervals At Fixed Intervals Frequency ioon Delay 2s St dev onz 0 P release 01 Rel Pool 100 Depression 0 Time Const 1s Noise amp Interference Sine Amplitude 0 00 pA Sine Frequency 50 00 Hz Random Noise 010 p 7 94 7 945 7 95 7 955 7 96s 3 Enter the time period over which PSCs are to be generated in the Duration box SO S08 Sa A Oh Ee 11 12 13 14 15 Enter the PSC peak amplitude in the Peak box Enter the standard deviation of the inter event variation in PSC peak amplitude in the St Dev box Enter the time constant of the PSC rising phase in the Tau rise box Enter the time constant of the PSC decay phase in the Tau decay box Select the current units from the Uni
32. can be to the dwell time histograms using either an iterative least squares method Fitting Exponential Probability Density Functions To fit an exponential mixture to a histogram 1 Define the range of dwell times containing the histogram bins exponential distributions to be fitted using the pair of 0 7 0029427 44 10 4 103 10 2 10 1 Closed times s Mean 0 00581 s Events 3857 grey I region of interest cursors l 2 Select the number of exponential components in the mixture to be fitted from the Curve Fitting list Curve fitting Fit Curve 2 Exponentials 3 Click the Fit Curve button and enter an appropriate set of initial guesses for the exponential function parameters Ai i A set of initial guesses are computed automatically but it is often necessary to adjust these to better match the location and size of the observed distribution E g set the mean dwell times to the peaks in a logarithmic histogram Individual function parameters can also be fixed at their initial values by ticking its associated Fixed box Set Fitting Parameters x Parameters Fixed Fixed A 75 7 u oos O a Ae 25 C 2 oos a E M Parameter Initialisation Automatic Cancel Manual Jnitialise Click the OK button to begin fitting The mixture and the best fitting individual exponential components are superimposed in red on the histogram
33. current xml A two step pulse protocol for recording tail currents A 500 msec pre pulse followed by a 60 mV 50 msec duration test pulse The pre pulse steps from 10mV to 120 mV Ramp 100 100mV 1s xml A voltage ramp slewing from 100 mV to 100 mV over a period of 1 sec A digitised 1572 sample point sine waveform Digpulse xml A digital stimulus program controlling digital outputs 0 and 1 Dig O is OFF initially and pulses ON for a period of 50 msec after a delay of 100 msec Dig 0 is ON initially and pulses OFF for 50 msec Next Recording Experimental Signals gt Creating Stimulus Protocols gt Default Output Settings The default output settings panel sets the default analog and digital output holding levels when a stimulus protocol is NOT in progress Select Setup Default Output Settings To open the Default Output Settings control panel Default Output Settings x m Digital Outputs 0 1 3 4 5 6 7 On C a pi 2 C 2 2 2 Off E iC iC o o O o g Analog Output Holding Levels gt Apply A00 A014 Oom Analog Output Holding Levels Sets the default holding level for the analog outputs Note When an amplifier is defined the default holding level for the command output of that amplifier can also be set by changing the holding level in the Pipette Seal Test window Digital Outputs Selecting the appropriate On Off switch se
34. for details Io ican be estimated either The Ga estimate from option determines how Io is estimated Peak Io estimated from the peak current of the capacity transient Exp Amp Io estimated from amplitude of the fitted exponential at the start of the voltage step Note If Ga Gm and Cm are to be estimated correctly the patch clamp s pipette series resistance compensation and capacity current cancellation features must be turned off Sweeps Averaged The Sweeps Averaged setting determines the number of test pulse sweeps averaged to calculate the displayed pipette and cell parameters Rpipette Rm Gm Cm Ra Ga Values can range from 1 no averaging to 10 averaging of the 10 most recent test pulses Recording Experimental Signals gt Recording Signals To make a recording of signals from your experiment select Record Record to disk to open the recording window ox Record Record E stop Duration 120 0s No sweeps No channels Sampling Interval 0 1 ms Stimulator ramp 5s Start Stop Analog Outputs A0 aoo aon Holding Level 400 mv Triggger Mode Special F Capacity Config Huorescence Event Frequency Resistance Amplifier Gain Mode 1 Gain 0 0005 VipA VClamp IClamp Update Gain Signal Level Im 73 24 pA Ym 73 58 mY Fixed zero levels Ident p 800 Vin m Mark Chart
35. form of keywords one word per line as follows KEY lt value gt lt cr gt lt l1f gt where lt value gt is a number or text depending on the parameter and lt cr gt lt If gt are the carriage return and line feed characters A typical header block from a file with 2 channels contains the following keywords VER 6 4 lt cr gt lt lf gt EDR file version number NC 2 lt cr gt lt 1f gt No of analogue input channels NP 102400 lt cr gt lt lf gt No of A D samples in data block NBH 2048 lt cr gt lt lf gt No of bytes in file header block AD 5 0000 lt cr gt lt lf gt A D converter upper limit of voltage range V ADCMAX 4095 lt cr gt lt lf gt Maximum A D sample value DT 1600 lt cr gt lt 1f gt A D sampling interval s YNO Im lt cr gt lt l1f gt Channel 0 name n 0 NC 1 YUO nA lt cr gt lt lf gt Channel 0 units YCFO 0 0001 lt cr gt lt 1l gt Channel 0 calibration factor V units YAGO 10 0 lt cr gt lt lf gt Channel 0 gain factor ZO 1024 lt cr gt lt lf gt Channel 0 zero level A D bits YOO 0 lt cr gt lt 1f gt Channel 0 offset into sample group in data block YNl Im lt cr gt lt lf gt Channel 1 name n 0 NC 1 YU1l nA lt cr gt lt l1f gt Channel 1 units YCF1 0 01 lt cr gt lt lf gt Channel 1 calibration factor V units YAG1 1 0 lt cr gt lt lf gt Channel 1 gain factor YZ1 1024 lt
36. nt means take the integer part of The exponential shape of the typical ion channel dwell time distribution is clearly apparent in a linear histogram 200 150 100 No Events 50 0 0 0099 0 0 20x103 40x103 60x103 80x103 10 2 0 012 0 014 0 016 0 018 Open times s Logarithmic histograms with variable bin widths Many channels however produce open or closed distributions consisting of more than one type of state with radically different mean dwell times In such circumstances it proves difficult to adequately represent the whole distribution of times using a single fixed bin width For example a closed time distribution composed of two types of states one with a mean dwell time of 1 ms and the other with a mean of 40 ms cannot be easily represented on a linear histogram as shown in the example histogram 100 bins Ims bin width The Ims bin width is too large to adequately represent the distribution of the short closures and too small to accumulate significant numbers of the long events in any one bin 103 800 600 No Events 400 200 0 0043 2 0 0 01 0 02 0 03 0 04 0 05 0 06 0 07 0 08 0 09 Closed times s One solution to the binning problems encountered with multi state distributions is to begin with narrow bin widths for brief events and to progressively increase bin width for subsequent bins An approach like this developed by Sigworth amp Sine 1987 has found widespread use Dwell times a
37. number of components necessary to fit a dwell time distribution can be determined by fitting a series of exponential p d f mixtures with increasing numbers of components until no significant improvement in fit is observed For example the figures below show the results of fitting mixtures of 1 2 and 3 exponential components The single exponential p d f can be clearly seen to be a poor fit with marked deviations between the fitted line and the data 200 150 100 50 No Events 10 4 10 3 10 2 10 1 100 101 Closed times 5 The mixture of two exponentials provides a qualitatively better fit accounting for both peaks in the distribution The residual standard deviation has also been reduced indicating a better quantitative fit 200 150 100 50 No Events 10 4 10 3 10 2 10 1 100 101 Closed times s5 The mixture of three exponentials c also provides a qualitatively good fit with a residual standard deviation similar to the two exponential fit Two of the components appear to be very similar mean values 200 150 100 50 No Events 10 4 10 3 10 2 10 1 100 101 Closed times s It is usual to choose the p d f function with the least number of parameters sufficient to provide a good fit Additional components should only be included if they can be demonstrated to significantly improve the quality of fit A quantitative estimate of the improvement in the quality of the fit obtained by adding addi
38. o Freqs 1 Save To Log To plot a power spectrum 1 Select the All Records option to include all available variance records in the spectrum or select Range and enter a specific range of records Note Records marked as Rejected and Background records are excluded Records C Allrecords Range 110 342 2 Freq Averaging Select the type of averaging to be applied to adjacent spectral frequencies Select None for no averaging Select Logarithmic to logarithmically increase the number of adjacent frequencies averaged with increasing frequency Select Linear to average a fixed number of adjacent frequencies throughout the frequency range the number entered in the No Freqs box M Freq Averaging C None Logarithmic Linear No Freqs 1 3 Time Window Select the time window to be applied to the data in each variance record Select None for no time window Select 10 Cosine to taper the first and last 10 of data points using a cosine bell function M Time window C None 10 Cosine 4 Options Tick the Subtr t Backg d option to subtract the averaged power spectrum of the Background records from the spectrum of the Test records The background spectrum is also plotted symbols Tick the Subtr t Trends option to remove any linear trends from the data points within each variance record before computation of its power spectrum Tick the Remove Harm
39. option to use all sample points contained in the recording or select Range and enter a time interval to scan only that region of recording 2 Set the position of the zero current level zero single channel current level unit c and transition threshold threshold indicated by the horizontal cursors on the display The levels can be adjusted by dragging the cursors up or down or in the case of the single channel level and threshold by entering appropriate values in the Single Chan Current and Threshold level boxes 3 Click the Detect Transitions button to begin scanning through the recording During transition detection an idealised noise free current signal representing the detected sequence of open closed channel states is superimposed in red on the recorded current signal This process can take several minutes with large data files and or slow computer progress being indicated by the progress bar at the bottom right of the window If necessary transition detection can be aborted before completion by clicking the Abort button Note The half amplitude threshold method works well when applied to signals with good signal noise ratios where the channel unitary current level is well separated from the zero level and the recording bandwidth is such that brief events are fully resolved However the method is prone to error when applied to channels with sub conductance states close to the 50 threshold Analysing Single channel Current
40. over the specified range of times The type face used to print text can be selected from the Typeface list and the type size entered into the Size box The thickness of the lines used to draw the signal traces can be set using the Line Width box Vertical and horizontal calibration bars are added to the plot to indicate the units and scaling of the plotted signals You can define the size of the bars by entering values into the calibration bars table You can set the size of the plotted record on the printed page by adjusting the size of the page margins To initiate printing click the Print button Displaying Records Stored on File gt Page View gt Copying Page View The currently displayed page can be copied to the clipboard as an image by selecting Edit Copy Image to open the dialog box x Typeface Line Current Page Arial C Whole record na z C Range Size 12 pixe 0 0 10 0 Line Width 2 pixel Use colour J Show labels JV Calibration bars Horiz 0 00999 s Vert 0 129 n amp Image size idth 600 pixels Height 500 pixels The dimensions of the image can be set using the width and height image size boxes Calibration bars zero levels and text font size and line thickness can be set in the same way as for a printed image To copy the image to the clipboard click the Copy button Detecting amp Analysing Events gt Event Detection WinEDR s even
41. points to the Windows clipboard The numerical values of the X Y data points which generate the plot can be copied to the clipboard by selecting Edit Copy Data The data is placed on the clipboard as a table of data values in tab text format defining the histogram There are 4 values per row and one row for every bin in the histogram Each row has the format lt X gt lt tab gt lt Y gt lt cr gt lt lf gt Copying an image of the plot to the Windows clipboard An image of the histogram plot can be copied to the clipboard by selecting Edit Copy Image to open the dialog box Copy Image x Typeface Image Size Arial width 600 pixels Size 12 pixels Height 500 ixels Line width 2 pixels i Marker size 5 pixels Use colour E The dimensions pixels of the bit map which will hold the image can be set using the Width and Height image size boxes The size and style of the typeface can be set using the Typeface and Size boxes When the image parameters have been set click the OK button to copy the image to the clipboard Data Files gt Opening an EDR Data File WinEDR uses its own custom data file format for storing digitised signal records These files are identified by the file extension EDR To load a previously created EDR data file select File Open to display the dialog box 2 x Open File Rob roelfsema Rocco Pizzarelli re ER
42. pulse on this input triggers the start a stimulus program which has been set up with the External Stimulus Trigger Y option Note 2 The Digidata 1320 Series only supports 4 digital output lines Troubleshooting When multiple analog input channels are being sampled and the sampling interval is greater than 10 ms samples get mixed up between channels This problem can be seen to occur also with AxoScope suggesting a bug in the Digidata 1320 firmware or AXDD132X DDL library The only limited solution at present is to increase the number of samples per record to ensure that the sampling interval is less than 10 ms Getting Started gt Laboratory Interfaces gt Molecular Devices Digidata 1440A Molecular Devices Corporation www moleculardevices com The Digidata 1440A interface consists of self contained mains powered digitiser unit with BNC I O sockets attached to the host computer via a USB 2 0 port The 1440A supports sampling rates up to 250 kHz 16 bit resolution on up to 16 channels It has a fixed input and output voltage range of 10V and supports 4 analog output channels and 8 digital output channels Software Installation WinEDR uses Axon s standard software library AxDD1400 DLL for the Digidata 1400 Series Details for steps 1 5 can be found in Axon s Digidata 1440A Manual 1 Install the AxoScope or PCLAMP software supplied with the Digidata 1440 2 Reboot the computer 3 Attach the Digidata 1440A to a USB
43. the Event Rejection Criterion from the list of measurement variables M Event Rejection Criterion imits Peak z Lower jo a Add to Filter Upper bs Enter the limits of the range of values which will result in the rejection of the event in the Upper and Lower limits boxes The example shows a criterion where events with a peak amplitude gt 0 0 pA and lt 0 5pA will be rejected Click the Add to Filter button to add the criterion to rejection list M Event Filter z Event Rejection Criterion Combine Apply Limits AND Peak z Lower 0 0 OR Clear TT E O T cool Upper os 0 lt Peak lt 0 5 2 If additional criteria are required repeat step 1 selecting another variable and limits Choose the way that the criteria are to be combined selecting AND if a match to all criteria in the list is required for rejection or OR if a match to any criterion will suffice 3 Click the Apply button to remove events matching the defined criteria Saving Loading detected event lists Note Events are permanently deleted from the event list by the filter It is prudent to save the original detected event list before applying the filter so it can be restored if necessary The list of event detection times can be saved as an ASCII text file by clicking the Save List button and entering the name and location of a file into the Save Event List dialog box A list of
44. the following page Next Getting Started gt Amplifiers gt Signal Connections Table The following table shows signal connections the required for the WinEDR suppported amplifiers Amplifier outputs input in panel A are connected to the laboratory interface input outputs in the corresponding column in Janpier Input Output panel B Type of Primary Secondary MODE Vcom Amplifier Channel Channel Manual gain ImOut Vm Out Vcommand In entry Axopatch Scaled Output 10 Vm Ext Command 1D a Axopatch Scaled Output 10 Vm VC mode Mode Gain Ext Command 200 Im CC mode Telegraph Telegraph front switched See Note 1 Multiclamp Scaled Raw See Note 2 Ext Command 700A Output Output Primary Secondary See Note 2 Ext Command Output Output veso Heka EPC 7 Current Vcomm Stim Input Monitor Monitor X10 Heka EPC 8 Current Vcomm See Note 3 Stim Input Monitor Monitor X10 Heka EPC 800 Current Vcomm Mode Gain Stim Input Monitor Monitor Telegraph Telegraph X10 Cairn Gain Out Command Pin 9 Command 10 In Optopatch X10 Out Pin 2 Gnd eet Out 37 way D socket Warner Vmx10 Gain CMD In PCSOIA Telegraph Warner Im Vmx10 Gain Command PC505B Telegraph In Warner I Monitor Vmx10 Command OC725 ae In 10 NPI SECOSLX Current Potential Curr VC Command Output Output Sensitivity Input 10 Monitor AM Systems Output X10 Vm Mode External 2400 Telegraph an at 50 pa Default Laboratory Interface Inputs Outputs Amplifer 1
45. to the clipboard 0 4 0 2 0 2 04 0 6 All Points pA 0 8 Analysing Single channel Currents gt Detecting Channel Transitions gt Transition Detection While analysis of current amplitude provides information concerning the number and size of channel conductance states it provides no information about channel kinetics In order to do this it necessary to measure the time the channel spends in each state Since ion channels fluctuate randomly between open and closed states the duration of a single opening or closure provides little information It is necessary to measure the durations of hundreds or thousands of openings and closures and to analyse the distribution of dwell times spent in each state The Detect Transitions page provides tools for the detection of channel open close transitions and the determination of the sequence of channel dwell times spent in each state Single channel Current Analysis 5 5 x Amplitude Histograms Transition Detection Edit Channel States Dwell Time Histograms SummaryStability 4 gt Detect Transitions Abort Range Whole file Range 0 10 035 8s Use C0 C1 Detection parameters Channelfcn o Im 5 C current 1 pA Threshold 50 0 level Trend Removal Linear AddBlock_ o Clear Blocks Subtract trend Restore Original SS eS ee eS ee Cursors 0 342 0 344 0 346 0 348 0 35 0 352 s Get C 0
46. using the buttons at the right edge of each plot Amplifiers The Amplifier selection box indicates which amplifier is currently selected for seal test and the current and voltage input channels being monitored If two or more amplifiers are in use the seal test can be switched between amplifiers by selecting Amplifier 1 2 etc See Amplifiers The amplifier voltage current clamp mode is indicated by the Clamp Mode options When mode telegraphs are operational for the amplifier these indicate the actual state of the amplifier When telegraph information is not available the Vclamp and Iclamp buttons must be set by the user to the amplifier clamp mode The amplifier gain current channel gain in voltage clamp mode voltage channel gain in current clamp mode is indicated in the Amplifier Gain box When gain telegraphs are operational these indicate the actual state of the amplifier When telegraph information is not available the current gain setting is entered here by the user The analog output channel s to which the test pulse is applied is indicated in the Send Pulse To list of check boxes Selecting an amplifier in the Amplifier selection box automatically selects the output channel connected to the amplifier stimulus command input The test pulse output can be routed to a different output channel or to additional channels by ticking the required channels Cell holding voltage and test pulses You can control the holding voltag
47. voltage channel scaling factors and voltage current clamp mode for each amplifier channel Resetting the Multiclamp WinEDR Communications Link If Multiclamp Commander is stopped and restarted while WinEDR is running communications between the programs can be lost It can be reestablished by selecting Setup Reset Multiclamp 700A B Link Getting Started gt Amplifiers gt DCLAMP Dynamic Clamp WinEDR supports the Strathclyde Electrophysiology Software DCLAMP dynamic clamp based on the National Instruments cRIO 9076 Real Time Controller The dynamic clamp permits the addition or subtraction of a simulated voltage and time dependent ionic conductance to from a patch clamped cell in current clamp mode A Hodgkin Huxley voltage dependent ionic current is simulated with bi exponential decay kinetics Select Setup gt D CLAMP Dynamic Clamp to open the dynamic clamp control panel Max Conductance Gmax Off Reversal Potential Vrev 9 my C Fixed Initial Conductance 4 5 C Add 5 E Steps cians Subtract urrent Command Scale Or 1E 008 A V tep ize 0 2nS Enable Inhibit Input A12 No Steps 9 No Repeats 2 Activation Parameter m Inactivation Parameter h Steady state Ming 1 1 exp V V472 Ve p V4 2 30 mv Yelp 10 mv Steady state hing 1 1 expt V V1 2 Vsip V1 2 10 mv Vslip 10 mv Time constant tau V taunt taumy tau
48. 0 0 02 04 06 08 1 St Dev limit 0 pA Patlak Average pA E Mean 0 556 pA Area 100 Curve fitting Fit Curve Set Zero Level None S C 1 pA Save to Log Current To compute the histogram 1 Select Patlak Average from the Histogram Type list 2 If more than one signal channel is available select the channel to be used from the Channel list 3 Select the All option to use all sample points contained in the recording or select Range and enter a time interval to use only samples within that region of recording 4 Enter the number of sample points in the running average in the No points averaged box 5 Enter the exclusion limit for the running mean standard deviation in the St Dev Limit box Note A suitable value is the standard deviation of a gaussian function fitted to the all points current amplitude distribution around the zero or single channel currents levels 6 Enter the width of the histogram bin in the Bin Width box 7 Click the New Histogram button to compile and display the histogram The Patlak averaging method produces histograms with much narrower better defined peaks than All Points histograms It is thus useful for resolving and viewing closely spaced sub conductance levels However it should be borne in mind that the averaging process is biased in favour of longer lasting states and short lasting events tend to be excluded from
49. 0 format file produced by PCLAMP V10 are urrently supported PCLAMP V10 files must be saved as ABF V1 8 Integer forn imported into WinEDR ambridge Electronic cfs ambridge Electronic Design CFS CED Filing System format files IDesign CFS dat DR Files cdr Strathclyde Electrophysiology Software CDR program an MS DOS based electrop package data files hart Files cht Strathclyde Chart data files CHT IHEKA asc ASCII format aata records exported from the Heka PULSE software Igor Binary Files ibw BW Igor Binary Wave files produced by the IGOR Pro software package PAT Files s pat__ Strathclyde Electrophysiology Software PAT single channel analysis program data file IPhysionet WDBF ice as WDBF format data files HEA PoNehMah Files pro__ PoNehMah cardiovascular analyser software data files SCAN Files Pes sca Strathclyde Electrophysiology Software SCAN program data files A pees Electrophysiology Software SPAN spectral analysis program data files ave File wav AV format sound files CD Files a a athclyde Electrophysiology Software WinCDR program data files Data Files gt Importing from ASCII Text Files To import records from a text file containing tables of numbers in ASCII format 1 Select File Import to display the dialog box Import File 2 x Look in E Data 7 Ae Rob roelfsema Rocco Pizzarelli My Recent Smooth muscle John McCarron Documents Steve Ennion
50. 0mY 2 10 100m 15ms DigOPulseTraind Type XML Document Desktop 10my DigOPulseTrainB Date Modified 22 06 2011 1 lt 10mi x 10ms x 10 DigDstim ES OAH o gt 20 mY per ms Digi stim LeakTest 100 mY w PN Dig Sx1ms SOH2 mk 555E prot EMS N 120 x 100mV 5s with PN Dig 5x1ms S0Hz onestep ir 200 vps ramp test Dig 10ms pro double st of 20mv Dig 100ms pro double st 22110 Fix Iramp Pulse 50mY 1 iB eee 30 Jan Om ramp Ss we SEn Filename Places Save as type ML X Cancel Stimulus protocols are stored as files with XML file extensions in the directory C WinEDR vprot Protocol files can be re loaded for editing by clicking the Open Protocol button and selecting a protocol file from the list presented in the Load Stimulus Protocol dialog box The folder used to store protocols can be changed by clicking the Set Protocol Folder button to open the protocol folder selection dialog box clicking on a a folder then clicking the Open button Next Recording Experimental Signals gt Creating Stimulus Protocols gt Example Protocols A number of example protocols are installed in the vprot folder with WinEDR VSteps 10 100mV 100ms xml A family of 10 depolarising 100 msec duration voltage steps ranging from 10 mV to 100 mV ISteps 10 100pA 100ms xml A family of 10 depolarising 100 msec duration current steps ranging from 10 pA to 100 pA tail
51. 12 pax E fe 464Hz T e e I E e e e Parameter initialisation _oK Automatic Cancel Manual Initialise and click the OK button The best fitting curve is superimposed upon the spectrum and the best fit function parameters zero frequency spectral power S and corner frequency f along with the parameters standard errors and residual standard deviation are displayed in the results box The single channel current Z and channel gating time constant are also displayed Noise Analysis F Review Edit Data Records Amplitude Histogram Variance Analysis Spectral Analysis m Records Set Axes Records Allrecords Range 30 85 Time window None 10 Cosine Options Subtr t Trends l Subtr t Backg d Remove Harmonics of 0 Hz Freq Averaging New Spectrum 2 44 Hz 24 64 pA 2 1O x 4 08E003 Curve fitting Lorentzian Save To Log y Sol 1 fife S0 0 03307 0 0001532 sd pA f None fe 513 5 3 435 sd Hz C Logarithmic MEPC Frequency t 0 3099 ms C Linear l 0 4613 pA Residual D 0 001413 pA Degrees of freedom 1667 No Freqs i Lorentzian Functions The spectra of ion channel fluctuations have characteristic shapes represented by the sum of one or more Lorentzian functions eo WA 0 At low frequenc
52. 20 e Molecular Devices Digidata 1440 1550 1550A e Instrutech ITC 16 or ITC 18 e Biologic VP500 e Heka EPC 9 EPC 10 e Tecella Pico Triton Triton Plus Next Getting Started gt Installing WinEDR To install the WinEDR program on your computer 1 2 3 4 5 6 Go to the web page http spider science strath ac uk sipbs page php page software and click the WinEDR Vxxx Setup File option to download the WinEDR installation program WinEDR_Vxxx_Setup Store this file in a temporary folder e g c temp on your computer Start the installation program by double clicking the program WinEDR_Vx x x_Setup The setup program creates the folder WinEDR and installs the WinEDR programs files within it You can change the disk drive and location of the WinEDR folder if you wish To start WinEDR click the Microsoft Windows Start button and select WinEDR Vx x x from the WinEDR group in the Programs menu Install the laboratory interface unit with the appropriate device driver software and support library supplied with the device See Laboratory Interfaces Configure WinEDR to work with laboratory interface Attach analog input output signal cables between amplifier and laboratory interface see section 4 Next Getting Started gt Laboratory Interfaces gt National Instruments Interface Cards National Instruments Inc www ni com WinEDR is compatible with most National Instruments multifunction data a
53. 87 p 0 5 10 15 20 9 Seo cwsor os a A stationary random signal is one whose basic statistical parameters mean variance power spectrum remain constant during the period of the recording A semistationary signal is defined as one where the statistical parameters are not constant but varying at a rate much slower than the intrinsic fluctuations A discussion of the theory and practice of noise analysis can be found in Defelice 1981 Eisenberg et al 1984 or Dempster 1992 2001 160 Noise Analysis gt Signal Conditioning The analogue signal conditioning requirements for signals intended for noise analysis are somewhat stricter than for other applications A low pass anti aliasing filter with sharp cut off characteristics Butterworth or Chebyshev is essential Suitable filters include the Frequency Devices 901 or Kemo VBF 8 The cut off frequency of this filter should be set to slightly less than half of the digital sampling rate In some studies e g endplate noise at the neuromuscular junction the current fluctuations are a small fraction of the mean current In these circumstances it may be necessary to use a high pass filter to separate out the noise from the mean current signal amplify by X10 X100 and digitise it on a second input channel See Dempster 2001 Noise Analysis gt Selection amp Inspection of Variance Records Select the Variance Records page to select the signal channel s to be analysed d
54. A histogram of the distribution of amplitude measurements can be plotted on the Amplitude Histograms page The cursor measurements list can be saved to an Excel readable text file by clicking the Save List to File button and entering a file name Previously saved files can be re loaded into the measurements list by clicking the Load List from File button The default inter cursor spacing is defined by the Cursor spacing box Analysing Single channel Currents gt Channel Dwell Time Analysis gt Analysis of Channel Dwell Times The Dwell Time Histograms page provides tools for compiling and analysing histograms of the distribution of the channel open and closed times determined by the transition detection process Dwell time histograms can be plotted with linear or logarithmic axes and mean state durations estimated by fitting one or more exponential probability density functions Single channel Current Analysis Amplitude Histograms Transition Detection Edit Channel States Dwell Time Histograms SummanyStability Plots New Histogram Set Axes Histogram Type 30 Closed times M Events All Events 25 Range 1 911 2 20 ao gt Ww Histogram Settings 3 15 Linear No Bins Log Sqr 100 10 Range 0 0185 Save Histogram 0 f aii 0 0495 0 Closed times s I Mean 0 0206 s Events 455 Curve fitting Fit Curve None F Test SavetoLog
55. AICh 0 AICh 1 AICh 6 AICh 7 AO Ch 0 Amplifer 2 AT Ch 2 AICh 3 ATCh 14 ATCh 15 AO Ch 1 Amplifer 3 AICh4 AICh 5 AT Ch 13 AICh 12 AO Ch 2 Amplifer 4 AICh 6 AICh 7 ATCh 10 ATCh 11 AO Ch 3 Note 1 When the Axopatch 200 is switched from voltage to current clamp mode the Scaled Output signal to the primary channel AI Ch 0 for Amplifier 1 changes from membrane current to voltage To retain a current signal the secondary channel AI Ch 1 for Amplifier 1 of WinEDR must be switched manually from the Axopatch 200 10 Vm to the Axopatch 200 Im output on the rear panel Note 2 Axon Multiclamp amplifiers support two separate amplifier channels 1 amp 2 When using amplifier Ch 1 select Multiclamp 700A B as Amplifier 1 and connect the Ch 1 Scaled and Raw Secondary outputs to AI Ch 0 and AI Ch 1 respectively and AO Ch 0 to Ch 1 EXT COMMAND When using both channels select Multiclamp 700A B also as Amplifier 2 and connect the Ch 2 Scaled and Raw Secondary outputs to AI Ch 2 and AI Ch 3 and AO Ch 1 to Ch 2 EXT COMMAND Axon Multiclamp amplifiers can only be selected as WinEDR Amplifiers 1 and 2 The Axon Multiclamp Commander software must be started up and running before WinEDR is started Note 3 The Heka EPC 8 gain and mode telegraphs are TTL digital signals provided via a 50 way IDC ribbon cable on the rear of the EPC 8 The signals in the table below must be connected to the digital inputs of the laboratory interface Heka EPC 8 Di
56. Channel States Dwell Time Histograms SummaryStability Plots New Histogram Set Axes Abort Histogram Type All Points ye m Data Channel cho Im Whole file C Range 0 0s Use co C1 No bins 200 Single channel Current Analysis CS Curve fitting Fit Curve a Set zero Level None S5 C 1 667 pA SavetoLog Current The module is split into 5 pages each associated with a specific single channel analysis operation e Amplitude Histograms Provides tools for compiling and analysing the ion channel current amplitude distribution e Transition Detection Provides tools for the detection of channel open close transitions and the determination of the sequence of channel dwell times spent in each state e Edit Channel States Provides an editor for visually inspecting each detected state and rejecting artefacts from the analysis e Dwell Time Histograms Provides tools for compiling and analysing histograms of the distribution of the channel open and closed times determined by the transition detection process e Stability Plots Provides tools for determining whether the recording satisfies the necessary stationarity conditions for a valid analysis A typical single channel analysis sequence is as follows 1 Current amplitude histograms are computed to determine the single channel current conductance number of channels in the patch and the st
57. DC Offset facility adds or subtracts a DC voltage level from the input The offset range depends upon the input mode Normal Diff and Inverted Diff 0 5mV Single Ended 500mV Electrodes 0 1mV The offset facility is often used to cancel out the standing DC voltage signal from tension transducers Next Getting Started gt Amplifiers gt Tecella Patch Clamp Amplifier Control Panel The amplifier gain compensation and current voltage clamp mode of Tecella patch clamp amplifiers can be set from this control panel Select Setup Tecella Patch Clamp to display the Tecella Patch Clamp control panel FTecellaPatch Clamp eSB Tecella Pico s n 33ffd 7053058323508580343 Lib 0 119 Ses Chis ee Channel eni i Update All Channels Config VClamp 255m o Input VMode Gain he Calibrate Amplifier m Filters Low pass filter T f j7 6 kHz gt Compensation Auto Capacity Resistance Junction Pot Searevenenreanneneed Auto Compensate Junct Pot Auto Zero Clear Compensation V Use analog leak subtraction JV Use digital leak current subtraction IV Use digital artefact removal I Apply to all channels 0 Compensation coefficient All Channels Zap Cell Amplitude 11 Dur 125 Config Selects the voltage current clamp mode and stimulus current voltage limits Input Selects the amplifier input None No input Head head
58. FURA 2 fluorescence cell capacity and real time event frequency In these modes of operation certain input channels are computed on line from the others To define the input channels and computed results channels click the Config button to open Computed Channels configuration dialog box Recording Experimental Signals gt Special Modes gt Event Frequency Selecting the Event Frequency special option opens a display window showing the frequency of occurrence of signals from a selected input channel Signals are detected using a threshold based event detector which is configured on the Event Frequency page of the Computed Channels dialog box Computed channels Resistance Detect events on channel chivm Detection Threshold fiom Running mean interval fhs Dead time interval oos OK Cancel To configure event detection Select the input channel on which the signals are to be detected from the Detect events on channel list Set the threshold which the signal must exceed to be detected in the Detection threshold box Set the averaging interval for the threshold baseline level in the Running mean baseline box SO OD Set the time after detection of an event within which detection is disabled in the Dead time interval box to exceed the duration of a signals to be detected The period over which events are counted to produce the event frequency is set by the Count Interval box in the Event Frequency display wi
59. Histogram Type list Histogram Type Closed times m Fvents i Select the All Events option to use all channel state events in the recording or select Range and enter the sub range of events to be included Events All Events C Range 1 911 If Burst Length or any of the other burst related histogram types have been selected enter the critical closed time value used to distinguish between intra and inter burst closed intervals in the T critical box Histogram Settings Set the type of bin spacing to be used Set the number of histogram bins in the No Bins box Select the Linear option for fixed width bins and enter the lower and upper limits of the range of times to be included in the histogram in the Range box a Histogram Settings Linear No Bins C Log Sqr 100 Range 0 0185 OR select the Log Sqr option for logarithmically spaced bins and enter the number of bins per 10 fold change in dwell time in the No bins per decade box Histogram Settings C Linear No Bins Log Sqt 100 No bins Me per decade ul Click the New Histogram button to plot the histogram Linear histograms with fixed width bins A Linear dwell time histogram is compiled by counting the number of state dwell times which fall into a series of equal sized time bins spread over a given range of times Each dwell time tstate is assigned a bin number ibin according to the formula t Ww where w is the width of the bin and
60. Strathclyde Electrophysiology Software WinEDR V3 6 5 Electrophysiology Disk Recorder c John Dempster University of Strathclyde 1996 2015 Table of Contents Introduction Welo Ctccastecvccts st ececettatvacs a ist ences etasidte un aceeieiniue anced siehat co cdsld EEE AAS 4 Main Features of WiInEDRe sicccx santos Ait Sch id eA a a hE 5 COMGIFO MS OF O E S E E E E E E sue T eee w U ERAN E 6 Getting Started Hardware REC IETS NES 5c ia i a hh Oh OG A R E a A 7 Installing WinEDR a Sascha fat alt ct Nace te ig Yh ca acl tal haute ee ace cual dtl Oot alec ahaa 8 Laboratory Interfaces National Instruments Interface Cards c cccccccsesscsssecscssesecsesseecseeseesesesseecsecsececsecseseesecsesseeeaeees 9 Axon Instruments Digidata 1200 cosccs ss co cass aocasdasiveactevaiveasanleieeaduaavectads tataetsdate trace mvcunsaniatanevarazetss 14 Cambridge Electronic Design 1401 Series 0 0 eccccccccccsessesesseseseeeseseescseseeecseseeaeseeecsenenseeeeeaeetees 17 Axon Instruments Digidata 1320 ssc wide conlagmaatiraGinadecions onni t aa 20 Molecular Devices Digidata 1440A ceccccescssesesesseseseescseseeseseeecseseesesesenseseeecaeseeseeeeecseseeseseeeeaes 22 Molecular Devices Digidata 1550 1550A uu ccceeesssseesseseseseeseseescseseeseseseeseeeeecsesenseeeeecseseeaeseneeaes 24 jg Keyed ic a fh Oj Gee 6 18 ran aa Mee RoR tire E IOAN OOP a A ARTE Nene REST 26 Biologie VP SOO acu actecesestestaeisi arte usceied n an awe mandi aud asta
61. The values of the function parameters along with their standard errors are displayed in curve fitting results area at the bottom of the display 200 150 100 wo w in 50 0 10 4 10 3 10 2 10 1 100 101 Closed times s Mean 0 00581 s Events 3857 Curve fitting cor ca pi S412 Avtpexp tiny i wd 87 37 2 864 sd 2 Exnonentials z 11 0 001184 5 655E 005 sd s F Test Ae 22 03 2 851 sd lt t 0 02267 0 004263 sd s __SavetoLog Residual D 0 5108 Nenrees nf freednam AT zi Note Standard error values computed by the curve fitting program are not true estimates of experimental standard error since they take no account of inter cell or other variability They tend only provide a lower bound to the estimate of the standard error in a parameter value The residual standard deviation SDrs between the histogram data and the fitted curve provides a measure of the goodness of fit The smaller the value of SDres the better the fit Iterative curve fitting is a numerical approximation technique which is not without its limitations In some circumstances it can fail to converge to a meaningful answer in others the best fit parameters may be poorly defined It is important to make an assessment of how well the function fits the histogram before placing too much reliance on the parameters Determining the required number of exponential components The
62. air of a0 al analysis region cursors Define the starting point time 0 of the exponential function to be fitted using the tO cursor 0 0 002 0 004 0 006 0 008 0 01 0 012 0 014 0 01 2 3 Select the number of exponential functions to be fitted from the Curve Fitting list Curve fitting Fit Curve 4 Click the Fit Curve button and enter an appropriate set of initial guesses for the exponential function parameters Individual parameters can also be fixed at their initial values by ticking its associated Fixed option Set Fitting Parameters xj Parameters Fixed Fixed A 0 672 nA E E T 0 00319 S E E Ss 0 0325 nA E E Click the OK button to begin fitting The best fit curve us superimposed in red over the fitted region of the waveform The parameters of the best fitting equation are displayed in the Curve Fitting results area t0a0 al_t 0 0197 0 033 0 0 002 0 004 0 006 0 008 0 01 0 012 0 014 0 0160 018 s Fit Results yi Aexpi tit Ss A 1 005 0 008681 sd nA t 0 001829 1 956E 005 sd s Ss 0 04536 0 001173 sd nA Residual D 0 01214 Degrees of freedom 253 lt 0 as Exponential v 107 Analysing Single channel Currents gt Single channel Current Analysis To analyse a single channel current recordings select Analysis Single channel Current Analysis to open the window 2 8 xi Transition Detection Edit
63. ak and pipette series resistance compensation settings in use Can be adjusted by the user or set automatically using Auto Compensate Junction Pot page Electrode junction potential compensation settings in use an be adjusted by the user or set automatically using Junct Pot Auto Zero Zap Cell Click Zap Cell to apply a voltage pulse of amplitude set by Amplitude and duration set by Dur Getting Started gt Amplifiers gt Heka EPC9 01 Patch Clamp Control Panel The amplifier gain compensation and current voltage clamp mode of Heka EPC 9 or EPC 10 patch clamp amplifiers can be set from this control panel Select Setup EPC 9 10 Patch Clamp to display the EPC 9 10 Patch Clamp control panel ioixi m Amplifier Mode Amp No ji v damp x i 1mV pA CC Gain ipajmvy CC Tau 100 ms Gentle Mode Change Filters Filter 2 Bessel v 15 96 kHz Filter 1 Bessel 100 kHz Cfast Cslow Rs Compensation Leak Vpipette Range TI c Capacity s77pr Clear G series 31s 43ns 5 Amp No Selects the amplifier channel displayed on the panel when a multi channel amplifier is in use Gain Selects the amplifier current gain Mode Selects voltage or current clamp mode The CC Gain and CC Tau settings determine gain and response time of current clamp Select the Gentle Mode Change option to change mode gently Filters Selects the filter response typ
64. ake eines asec aa od wa ada este 193 SIMGIE Chan Mel Cur 2s ecsceess casatensccatecuteltet chs nnace teem eu kanes tafe astehtal E AA bee 194 COM Automation Interface COP Auto Mabon Interface vases acess Gavictiets cities nintendo N aaeaeaandiataue eat iat Mane ES 196 References OS fo a ae EE EEE NORRIE E EAA TOR OREN TT Oat AUT er RENEE CEES OL Rr A TTT 199 Strathclyde Electrophysiology Software WinEDR V3 6 5 Electrophysiology Disk Recorder c John Dempster University of Strathclyde 1996 2015 Introduction gt Main Features of WinEDR WinEDR is a data acquisition and analysis program for handling signals from electrophysiological experiments These may include whole cell patch clamp experiments single and two microelectrode voltage clamp studies or simple membrane potential recordings Whole cell signals are produced by the summation of currents through the usually large population of ion channels in the cell membrane and thus consist of relatively smooth current or potential waveforms The amplitude and time course of such signals contain information concerning the kinetic behaviour of the underlying ion channels and other cellular processes which can be extracted by the application of a variety of waveform analysis techniques WinEDR provides in a single program the data acquisition and experimental stimulus generation features necessary to make a digital recording of the electrophysiological signals and a range of wa
65. al for this channel is being acquired Load Settings Save Settings Default Settings Note Signal channel to analog input mapping is currently only available with National Instruments interface cards Amplifier Indicates whether an amplifier has been defined for this channel For example if the membrane voltage output of your amplifier supplies a signal which is 10X the measured membrane potential of the cell and the units have been defined as mV then the appropriate V Units setting is 0 01 since the amplifier voltage output is 0 01 Volts per mV Amplifier Display Grids and Time Units The Time Units options determine the units secs or msecs used to display time intervals in signal display windows Next Getting Started gt Amplifiers gt CED 1902 Amplifier Cambridge Electronic Design Ltd www ced co uk The Cambridge Electronic Design 1902 is a computer controlled amplifier with built in isolation circuits for recording ECG and EMG signals from humans and a bridge circuit for recording from tension or pressure transducers It can also be used to record extracellular electrical activity from nerve and muscle It has two inputs e The Electrodes input is an electrically isolated differential amplifier input used to record ECG EMG and similar signals Isolation makes it safe to attach recording leads to human subjects e The Transducer input is a differential amplifier input used to record from transducers such as fo
66. annel conflicts WinEDR requires DMA channels 5 and 7 to support the transfer of data to from PC memory and the Digidata 1200 Many sound cards also make use of DMA 5 and can interfere with the operation of the Digidata 1200 Getting Started gt Laboratory Interfaces gt Cambridge Electronic Design 1401 Series Cambridge Electronic Design Ltd www ced co uk The CED 1401 series consists of an external microprocessor controlled programmable laboratory interface units attached to the PC via a digital interface card or USB There are 4 main types of CED 1401 in common use CED 1401 CED 1401 plus CED Micro 1401 and CED Power 1401 They are all fully supported WinEDR with the exception that only 4 analog input channels are available on the Micro1401 and that the maximum sampling rate and number of samples sweep for the standard CED 1401 is substantially less than the others Software installation Before WinEDR can use these interface units the CED 1401 device driver CED1401 SYS support library USE1432 DLL and a number of 1401 command files stored in the directory 1401 must be installed on the computer The installation procedure is as following but see CED documentation for details 1 Install the CED interface card in a PC expansion slot and attach it to the CED 1401 via the ribbon cable supplied or attach to USB port for USB versions 2 Download the CED 1401 Standard Windows Installer program WINSUPP EXE from the CED web site
67. aseline Trend Pts Avgd 20 Gap fo Io auaa o T X decay time 5104 5106 5108 51 5112 5114 5116 Decay From 30 Peak Det F Cait Cuanto 0 Analysis Region The pair of cursors a0 and a1 define the region within the event display window containing the waveform to be analysed This region can be adjusted to exclude stimulus artefacts from the waveform analysis al al Measurements include e The time at which the event was detected Detected at e The peak amplitude positive or negative depending upon the setting of the Polarity option of the event peak e The integral of the event waveform area e The 10 90 rise time of the event T rise e The time to decay to a user set percentage 0 100 after the peak value T x e The time constant of exponential decay Tau decay The following options configure the waveform analysis Event Polarity Set the Polarity option to determine whether the signals are to be treated as positive or negative going waveforms Zero Level Select the At start option to use the average of a series of sample points at the start of the event detection window as the zero level for amplitude measurements or the At event option to use a block of points immediately preceding the event The number of samples to be averaged is defined by the Pnts Avgd box To shift the points used to compute the zero level away from the start or e
68. attached to the host computer via a SCSI Small Computer Systems Interface interface card and cable A number of versions are available including the 1320A and 1322A The 1322A supports sampling rates up to 500 kHz 16 bit resolution on up to 16 channels It has a fixed input and output voltage range of 10V and supports 4 digital output channels Software Installation WinEDR uses Axon s standard software library AxDD132x DLL for the Digidata 1320 Series Details for steps 1 5 can be found in Axon s Digidata 1320 Series Operator s Manual 1 Install the Axon SCSI card in a PCI expansion slot 2 Attach the Digidata 1320 to the SCSI card and switch on the computer and 1320 3 Install the AxoScope software supplied with the Digidata 1320 4 Reboot the computer 5 Run AxoScope to ensure that the software installed OK 6 Run WinEDR and select from the main menu Setup gt Laboratory Interface to open the Laboratory Interface Setup dialog box _ Laboratory Int Oj x Jaxon Instruments Digidata 132 Unknown AD Converter Voltage Range 5Y OK Cancel then select Axon Instruments Digidata 132X from the laboratory interface list Signal input output connections Signal input and output connections are made via the BNC sockets on the front of the Digidata 1320 Series digitiser unit Digidata 132X Series Analog In 0 os Ch 3 Analog In 3 oawon oawore C Note 1 An active high TTL
69. ch row has the format lt Bin Lower Limit gt lt tab gt lt Bin Mid point gt lt Bin Upper Limit gt lt tab gt lt Bin Count gt lt cr gt lt lf gt Copying an image of the histogram to the Windows clipboard An image of the histogram plot can be copied to the clipboard by selecting Edit Copy Image to open the dialog box Copy Image x Typeface Image Size arial width J 600 pixels Size 12 pixels Height 500 ixels Line width 2 pixels Marker size 5 pixels Use colour E The dimensions pixels of the bit map which will hold the image can be set using the Width and Height image size boxes The size and style of the typeface can be set using the Typeface and Size boxes When the image parameters have been set click the OK button to copy the image to the clipboard No Events 200 150 100 50 0 2 0x1C4 0x1C6 0x1C8 0x10 10 20 012 0 014 0 016 0 018 Open times 5 Analysing Single channel Currents gt Stability Plots gt Stability Plots The validity of the channel dwell time analysis is dependent upon the channel gating being in a stationary condition throughout the recording Stationarity means that the rate constants governing the transitions between channel states are constant and the mean open and closed state dwell times and open state probability do not vary during the recording The Summary Stability Plots page provides a set of tools for revealing t
70. cquisition cards or devices including M X and E series cards the Lab PC 1200 series and USB devices The PCI 6221 PCI card with BNC 2110 I O box and 2m SHC68 68 EPM cable or the USB 6221 BNC device is currently recommended The National Instruments NIDAQ interface library must be installed before WinEDR can use the interface card Most modern cards X M and E series are supported via the NIDAQ MxX library Older cards Lab PC 1200 series require the Traditional NIDAQ library V4 9 or earlier to be installed WinEDR supports both types of library Software installation 1 Install the NIDAQ library from the disks supplied with interface card following the instructions supplied by National Instruments 2 Install the interface card in an expansion slot or attach a USB device 3 Reboot the computer 4 Run National Instruments Measurement amp Automation Explorer program You should find the card listed under Devices amp Interfaces Note the Device number Dev1 Dev2 etc of the card NI USB 6211 Devi Measurement amp File Edit View Tools Help E gy My System w Data Neighborhood B Py Devices and Interfaces a NI DAQmx Devices 18 NI USB 6008 Dev2 PX PXI System Unidentified TH cavin o nawallal 5 Right click over the device and select Self Test to check that the device is functioning correctly 6 If the tests check out OK run WinEDR and select from the main menu Setup
71. cr gt lt lf gt Channel 1 zero level A D bits YO1 1 lt cr gt lt l1f gt Channel 1 offset into sample group in data block TU ms lt cr gt lt lf gt Time units ID Cell 1 lt cr gt lt lf gt Experiment identification line BAK T1l lt cr gt lt 1lf gt BAK amp T indicates a BAK file exist Event detector parameters DETCH 0 lt cr gt lt lf gt Event detector channel DETRS 1024 lt cr gt lt 1f gt No samples in event detector record DETYT 500 lt cr gt lt lf gt Event detector amplitude threshold A D units DETTT 5E 3 lt cr gt lt lf gt Event detector time threshold s DETDD 5 E2 lt cr gt lt 1lf gt Event detector dead time s DETBA 5E 2 lt cr gt lt lf gt Event detector baseline averaging time s DETPT 10 0 lt cr gt lt lf gt Event detector pretrigger percentage Single channel analysis parameters VARRS 1024 lt cr gt lt 1f gt No samples in variance record VAROV 0 lt cr gt lt lf gt Percentage overlap of variance records VARTR 0 0005 lt cr gt lt l1f gt MEPC rise time MEPC freq analysis s VARTD 0 005 lt cr gt lt lf gt MEPC decay time MEPC freq analysis s UNITC 1 0 lt cr gt lt lf gt Single channel current DWTTH 50 lt cr gt lt lf gt Transition detection threshold Note It should not be assumed that the keywords will follow any particular order Data block The data block contains the digitise
72. d by the computer is not truly linear but consists of a staircase of fine steps These steps can be smoothed out by low pass filtering the voltage stimulus signal before it is fed into the patch clamp es eee Initial Delay Delay at the holding level before the pulse begins Amplitude Amplitude at start of ramp End Amplitude Amplitude at end of ramp Digitised analog waveform A digitised analog waveform loaded from an external data file Initial Delay Delay at the holding level before the pulse begins File Name Name of text file containing digitised waveform D A update interval Time interval between digitised waveform points No Points No of digitised waveform data points to be used in the stimulus Starting point increment Increment to be added between records to the first data point of the digitised waveform to be used in the stimulus Digitised waveforms are loaded into the stimulus protocol from text files containing the digitised data points The waveform data can be formatted either as a single column of amplitude data or a pair of columns of time in seconds and amplitude in the stimulus units of the output channel to contain the waveform data points separated by lt tab gt characters Le To Vo Ti Vi etc To load a digitised waveform from a text file click the zl button next to the File Name table entry and select the file containing the digitised waveform After loading the data the No Poi
73. d signals stored in the form of 16 bit binary integers Each A D sample takes up 2 bytes of space The size of the data block is determined by the number of channels and number of samples per channels in the record N pres 2 N bytes channels N somites A 2 If there is more than one A D input channel samples are interleaved within the data block For example for 2 channels YOI YII YO2 Y12 YOnsamples YInsamples A 3 Different laboratory interfaces supported by WinEDR return multi channel A D samples in different orders The channel interleaving order for a data file is specified by the YOn channel keyword in the file header block Scaling from A D unit to physical units The calibrated signal level ycai in the appropriate channel units can be reconstructed for channel n using information stored in the header block using YZn x AD YCFnx YAGnx ADCMAX 1 Veal Yaa z where AD is the maximum positive limit of the A D converter voltage range ADCMAX is maximum A D sample value YCFn is the channel calibration factor YAGn amplifier gain and YZn zero level Simulations gt Postsynaptic Currents The post synaptic current module generates series of post synaptic currents PSCs which can be used to test WinEDR s signal detection module and the MEPC frequency estimation procedure in the noise analysis module The PSCs are modelled as signals with a rapid exponential rise and slower exponential decay occurring
74. display record can be copied to the clipboard by selecting Edit Copy Data The data is placed on the clipboard as a table containing the scaled values for each sample in the record in the measurement units defined for each channel The table is stored in tab text format allowing the data to be copied into programs such as spreadsheets and graph plotting packages using an Edit Paste command Note that due to limitations in the capacity of the Windows clipboard data points may be skipped to keep the size of the copied record within clipboard storage limits Copying the displayed image The signal record s on the display can be copied to the clipboard as a bit mapped image by selecting Edit Copy Image to open the dialog box Copy Image j xj m Calibration Bars Typeface Time 0 1000000014 Im 0 2 nA Size 12 pixels Line Width 2 pixels Show zero levels Vv Use colour i Show labels Iv arial Image size Width 600 pixels Height 500 pixels OK Cancel The dimensions of the bit map which will hold the image can be set using the width and height image size boxes The more pixels used in the bit map the better the quality of the image Calibration bars zero levels and text font size and line thickness can be set in the same way as for a printed image When the image parameters have been set click the OK button to copy the image to the clipboard Displaying Record
75. ds sets the duration amplitude S of the seal test pulse e g W SealTestPulseDuration 0 01 SealTestSmoothingFactor R W Property Set cell parameters smoothing factor 0 1 1 0 1 no smoothing 0 1 maximum smoothing equivalent to averaging over 10 pulses Read Only Reads the most recent cell holding Property potential V measurement computed by the seal test Read Only Returns the most recent cell holding Property current A measurement computed by the seal test Read Only Reads the most recent cell access Property conductance S measurement Read Only Reads the most recent cell membrane Property conductance S measurement Read Only Reads the most recent cell capacity F Property measurement Rseal Read Only Reads the most recent pipette seal Property resistance measurement A file WinEDR VBSCRIPT Example vbs containng VBSCRIPT example code can be found in the cAwinedr folder References gt References Brown K M amp Dennis J E 1972 Derivative free analogs of the Levenberg Marquardt and Gauss algorithms for non linear least squares approximation Numerische Mathematik 18 289 297 Clements J D amp Bekkers J M 1997 Detection of spontaneous synaptic events with an optimally scaled template Biophys J 73 220 229 Colquhoun D amp Sigworth F H 1995 Fitting and statistical analysis of single channel records In Sakmann B amp Neher E 1995 Single Channel Recording 2 ed
76. e x SetZeroLevel None 5 C 1 pA SavetoLog Current To compile an All Points histogram 1 Select All Points from the Histogram Type list 2 If more than one signal channel is available select the channel to be used from the Channel list 3 Select the All option to use all sample points contained in the recording or select Range and enter a time interval to use only samples within that region of recording 4 Enter the width of the histogram bin in the Bin Width box 5 Click the New Histogram button to compile and display the histogram Analysing Single channel Currents gt Current Amplitudes gt Amplitude Histograms gt All Points in State Histogram This histogram displays the current amplitude distribution of the sample points contained within a selected set of channels states closed open and all Note This option can only be used after transition detection has been completed Single channel Current Analysis oj x Amplitude Histograms Transition Detection Edit Channel States Dwell Time Histograms SummarnyStabi 4 gt New Histogram unit c Set Axes Abort Histogram Type All Points in State z Data Channel cho Im Whole file Range 1 911 Use co c1f No bins 200 Bin width 0 015 pA Lower Lim 1 453 pA Upper Lim 1 547 pA 0 4895 0 0 369 0 0 369 0 738 1 11 State fan All Paints in State pA Mean 0 0882 pA Area 100
77. e and cut off frequency of the the two low pass filters in the current recording pathway Filter 1 has 4 fixed settings Bessel 100 kHz Bessel 30 kHz Bessel 10 kHz and HQ 30 kHz Filter 2 can be selected to have either a Bessel or Butterworth response and a cut off frequency between 0 1 and 16 kHz A Bessel response minimises signal distortion ringing after step changes in the filtered signal whereas a Butterworth response provides a sharper cut off of high frequencies Cfast Sets the amplitude and time constant of the fast Le pipette capacity compensation Click the Auto button to automatically set the Cfast compensation Click the Clear button to cancel compensation Cslow Sets the working range amplitude and time constant of the slow ie cell capacity compensation Click the Auto button to automatically set the Cslow compensation Click the Clear button to cancel compensation RS Compensation Sets the response speed and fraction of series resistance compensation Click the Auto button to automatically set the RS compensation Click the Clear button to cancel compensation Leak Sets the amount of leak conductance subtracted from currents Click the Auto button to automatically set the leak subtraction Click the Clear button to cancel leak subtraction Vpipette Sets the pipette and liquid junction potential compensation and holding voltage Click the Auto button to automatically set the pipette compensation Click the Clear b
78. e applied to the cell and the amplitude and duration of a test voltage pulse by selecting one of three available test pulses Pulse 1 2 3 The size of each pulse type is set by entering an appropriate value for holding voltage and pulse amplitude into the Holding voltage or Amplitude box for each pulse The width of the pulse is defined by the Pulse width box You can switch between pulses by pressing the function key associated with each pulse Pulse 1 F3 Pulse 1 F4 Pulse 1 F5 Current and voltage readouts A readout of the cell membrane holding current and voltage and test pulse amplitude appears at the bottom of the monitor window Clicking the Save to Log button saves the current Pipette or Cell readings to the log file During initial formation of a giga seal the Pipette option displays pipette resistance computed from V ays pipette pulse where Vpuise and Ipuise are the steady state voltage and current pulse amplitudes The Cell G page displays the cell membrane conductance Gm capacity Cm and access conductance Ga and the Cell R page displays the cell membrane resistance Rm 1 Gm capacity Cm and access resistance Ra 1 Ga Gm Cm and Gaare computed from I p 0 G pulse C I pulse m I V pulse ulse i G where Io is the initial current at the start of the capacity transient and is the exponential time constant of decay of the capacitance current See Gillis 1995
79. e fluctuations and about the steady state mean current Channel gating is simulated using a simple two state model The simulated current noise can be used to test the operation of the noise analysis module To create a data file containing simulated ionic current noise 1 Create a new data file to hold the records by selecting File New and entering the name of a new data file 2 Select Simulations lon Channel Noise to open the window E Ion Channel Noise Simulation mfe x 60000 M Time periods Background 100s Onset 50s a h 2 Steady state 200s Sampling 0 05ms Interval 8088 m loni c Current _ 5 c current 1 pA r T T D AS AG No channels 100 plOpen 0 100 Tau open M Recording conditions R 1 0ms Random Noise 1 00 pA 3 Enter the duration of the initial period before the application of agonist during which background noise is recorded in the Background box 4 Enter the onset time during which agonist concentration is rising in the Onset box 5 Enter the period of time that the agonist concentration is in a steady state in the Steady state box 6 Enter the single channel current amplitude in the S c current box 7 Enter the number of ion channels in the cell in the No Channels box 8 Enter the steady state probability of a channel begin open in the P Open box 9 Enter the mean channel open time in the Tau open box 10 Enter the standard de
80. e number to skipped in the No of title lines to ignore box Sample times If the first column in the table contains samples times select the Time Data in Col 0 option to derive the sampling interval from the times of successive rows Select the units that the time data is expressed in from the Units list If no sample time data is available select User Defined and enter the sampling interval into the Sampling Interval box Time Units Select the units of the time data column secs msecs mins from the Units list Channels Enter the names and units for each channel into the Channels table Click the OK button to import the data when the import settings are complete Data Files gt Importing from Raw Binary Data Files To import records from a text file containing tables of numbers in ASCII format 1 Select File Import to display the dialog box Import File 7h xt My Recent Documents Desktop My Documents on BL My Computer File name lava dat gt a Nocera Files of type Raw Binary dat raw 7 Cancel Places J Open as read only If the type of data file being imported does not match any of the known formats the import module reverts to its general purpose Binary import mode The import module assumes that the data has the general format File Data Header block At the beginning of the file there is a block of file header data which contains the information on the number of records
81. e optimal template matching algorithm developed by Clements amp Bekkers 1997 An exponentially decaying waveform template defined by the equation y di e ost C Tise T decay is slid point by point along the digitised signal its amplitude A and offset C being adjusted at each step using least squares methods to obtain the best fit The ratio between best fit template amplitude and residual sum of squares is used as a detection criterion See Clements amp Bekkers 1997 for details of the method The rate of rise and amplitude threshold methods are applicable to any shape of signal while the template matching method is specifically designed for decaying exponential signals such as postsynaptic currents or potentials To scan a segment of the continuous digitised recording for event 1 Source Select the Whole File option to scan the complete file or select Range and enter a time interval defining the section of the recording to be scanned 2 Source If there is more one channel within the recording select the channel to be scanned for signals from the Channel list 3 Mode Select the event detection method Amplitude Rate of rise or Template 4 Thresholds Amplitude mode Set the level which the signal detection criterion has to cross for an event to be detected in the Amplitude box or adjust the threshold cursor in the Det Criterion display Set the period of time which the signal has to remain above the ampl
82. e ratio of two input channels Four channels are required two fluorescent input channels the computed ratio of these two channels and computed ion concentration Computed channels chom x sd onim 73 Recording Experimental Signals gt Special Modes gt Resistance Selecting the Resistance special option opens a display window showing cell resistance or conductance obtained from a voltage test pulse applied to the cell The test pulse properties and the selected current and voltage channels are configured on the Resistance page of the Computed Channels dialog box Computed channels Plot Fluorescence Capacity Event Frequency FR Channels Current eno Im Voltage fon vim gt m Test Pulse Amplitude 10 mY Duration 20 ms Interval 025s Resistance v OK Cancel To configure the resistance plot 1 Select the input channel on which the current signals are to be detected from the Current channel list Select the input channel on which the voltage signals are to be detected from the Voltage channel list Set the amplitude of the test pulse in the Amplitude box 2 3 4 Set the duration of the test pulse in the Duration box 5 Set the interval between pulses in the Interval box 6 Select the type of plot to be produced from the Plot list Plots of cell resistance conductance steady state pulse current and voltage can be produced Dist O
83. e resolution of changes in variance The size of the record also determines the low frequency limit of the power spectrum Optional Enter the record overlapping factor in the Overlap box 0 25 50 75 The default setting an overlap factor of 0 corresponds to contiguous independent records Increasing the overlap factor increases the number of available records improving the temporal resolution at the expense of sharing samples between records 4 6 Record Size 1024 Overlap 0 Inspect each individual variance record and reject those containing artefacts from analysis by ticking the Rejected tick box _ I Type Test l Rejected Classify each record according to what type of signal it contains Background or Test by setting its Type Type Test l Rejected Records containing only noise from sources other than the ion channels under study e g instrumentation noise are classed as Background Records containing the ion channel noise under investigation are classed as Test Background and Test records are processes separately allowing the averaged variance power of the background noise to be subtracted from the ion channel noise Records can either be classified individually or in blocks by clicking the Set Block button to open the Set Block dialog box x Record state Type Rejected Range 1 342 OK Cancel If necessary set the DC Cha
84. e to be modified Closed Open or All for both types of state from the State list 2 Define the criteria to be used for selecting the events to be changed To restrict changes to events with a specific range of durations select Duration and enter a range of times in the box To restrict changes to events with a specific range of amplitudes select Amplitude and enter a range of amplitudes in the box To restrict changes to events with a specific range of current standard deviations select St Dev and enter a range of amplitudes in the box All selected criteria must be met before a change is made 3 Select the Ignore State Ignored Not Ignored setting to be applied when an event is found to match the selection criteria 4 Select the All Events option to apply the criteria to all events or select Range and enter a specific range of events 5 Click the OK button to begin the changes The number of event matching the criteria are indicated in the status box as the search progresses Note Events marked for exclusion from analysis remain within the channel state list and consequently continue to separate adjacent states It is sometimes useful to completely remove Ignored events from the list and merge adjacent states into one This can be done by clicking the Delete Ignored button Analysing Single channel Currents gt Detecting Channel Transitions gt Exporting the Channel State List To export the data stored in the channel state
85. eady state probability of a channel being open 2 Once the single channel current has been established the transition detection procedure is used to determine the location of channel open close transitions and the channel dwell times spent in each state 3 Dwell time histograms are then be computed and exponential probability density functions fitted to determine the number and mean duration of channel open and closed states A detailed discussion of single channel analysis techniques can be found in Sakmann amp Neher 1995 or Ogden 1994 Analysing Single channel Currents gt Current Amplitudes gt Analysis of Current Amplitudes Select the Amplitude Histograms page to compute and plot histograms of the distribution of current amplitudes within a single channel recording Single channel Current Analysis loj x Amplitude Histograms Transition Detection Edit Channel States Dwell Time Histograms SummaryStabi 4 gt New Histogram Set Axes Abort Histogram Type fan Points Data Channel cho Im Whole file Range 0 10 0352 Use co C1 No bins 20 Bin width 0 015 pA Lower Lim 1 453 pA Upper Lim 1 547 pA fo 0 5495 0 0 369 0 0 369 0 738 1 11 All Paints pA Mean 0 0956 pA Area 100 Curve fitting Fit Curve z Set Zero Level None 5 C 1 015 pA SavetoLog Current Current amplit
86. ed Clicking the Stop button terminates the stimulus and returns the analog outputs to the holding levels The holding voltages applied to analog outputs can be changed by entering a new voltage then pressing the Enter key Note Stimulus voltages waveforms generated during a voltage protocol are added to the existing holding voltages Analog Outputs A0 A00 aon Holding Level 100 mv _ Stimulus protocols can consist of a series of one or more pulses incremented in amplitude or duration to create a family of pulses Complex stimulus waveforms can be produced including series of rectangular steps ramps and digitised analogue signals Individual protocols can also be linked together to automatically apply a series of different protocols during an experiment Protocols are created using the Stimulus Protocol Editor and stored as protocol files xml files Note Stimulus voltage outputs are only supported with National Instruments and Cambridge Electronic Design laboratory interface units Recording Experimental Signals gt Creating Stimulus Protocols gt Opening the Stimulus Recording Protocol Editor To create a stimulus protocol select Setup Stimulus Recording Protocol Editor to open the stimulus editor window Protocol F Delphi Projects WinEDR 32 Stim ramp 5s xmil Files Waveform New Protocol 100 m Open Protocol Save Protocol 400 Save Protocol As Set Protocol Folder 100 m DO 0
87. ed segment can be increased or decreased in duration by clicking the arrow buttons or by entering a specific value and pressing the return key 40 1563 s gt Vertical Magnification The vertical magnification of each plot can be expanded by moving the mouse to the upper limit of the region pressing the left mouse button drawing a rectangle to indicate the region and releasing the mouse button The vertical magnification and position of the displayed region within the recording can also be adjusted using the oopa buttons at the right edge of each plot Individual channels can be added removed from the display by clicking the Im a n button at the left edge of each channel The vertical area of the display devoted to each channel can be adjusted by dragging the top left edge of each channel Y axis up or down All channels can be set back to minimum magnification by selecting View Zoom Out All Displaying Records Stored on File gt Compressed View gt Measuring Signal Levels To measure the signal at any point on the displayed record use the mouse to drag the vertical readout cursor to the desired part of the trace Fine positioning of the cursor can be achieved by pressing the left or right arrow keys with the mouse pointer over the selected cursor t 1 1787 0 8739 n 16 1 18 1 The signal level of the trace s at the cursor position is displayed at the bottom of each channel Time measurements are made relative to
88. eeaees 162 Analysis of Signal Variantes esnan E nave TE 164 VarlahGe MeanCurrene PO tS shies dats ci nes ono beds A E E vaaaeteen 166 ROwer Spectra Derg re CR aOR ra eet ec Ee AN CPE Ee ON COPE AOR SOURIS CRETE EE 168 Fitting Curves to Power SpeCth a ccztstecsedfagp cass eveciaaasGacacy activdeds toieer Dantes E TEENE taeda 171 Printing Plots OF SD OCU elicit ocarriz tance ies nctese toca see hatin a ented Soca ag has eate Coa beh etrte ti ctu tie tat 174 Copying Plots to the ClipBDOard aise ence ease erect civist etoile are arnt eaves tomate tlic 175 Data Files Opening ar EDR Data File ira ciccaty enh Suites ee tes Reni ve OR eo BA aai 176 Appending an EDR Data File wasiecon ursan in en a nas Ea ONEN it EEr COE Sa teanen ta CENA 177 Importing Other Data File OFM GUS kenene a a e ii 178 Imp rt ng frm ASCI Text FUSS tivesccsasdacouss seal louie lekad cers nated cask ea EREE R E E AEREE 180 Importing from Raw Binary Data Fil sS c ec eccseeseeseescseeseescseeseeseseesecaesecaeeaeecaeeaeesceeeseeseneeaeeaeees 182 Exporting to Other Data File Formats eesesesesesesesssessesesesetesrtrertrtrererernrerenseseeetsesesesesesesesesesrereret 184 Inspecting File Properts ninn n igne E o ea e S ideea 186 EDR File StrUcture ee a E E RE enn A O DoT RD aT ope C OE 189 Simulations POStSYMapU eC UMeMnts saves vay sscasssycaerwavetearvunvaceies E RNR RE eee A T caevraeReE 191 TORT Channel NoiSeisimcedsesiacanacenii i n Cased Gy ceca eatin d
89. efine the size of the variance record and to visually inspect edit and classify the recorded signals Se att Review Edit Data Records Amplitude Histogram Variance Analysis Spectral Analysis Records 135 342 25 im 6hUm E Type Test I Rejected SetBlock_ idia AC Channel cho Im rE AEEA E ET CT EEEE E EE E A N DC Channel 5 cho Im Meroni giz 13 74 13 76 13 78 13 8 13 82 1024 i i i i as Overlap 0 Data Mean DC 11 3 pA variance AC 10 7 pA 2 Variance records are displayed individually on the screen selectable using the Records scroll bar or box The record mean signal level and variance are displayed at the bottom of the screen Set up for a variance or power spectrum analysis using the following procedure 1 If there is more than one input channel select the channel containing the basic DC coupled current signal with the DC Channel list DC Channel cho Im 2 If the current noise has been acquired in an additional high gain AC coupled channel separate from the DC coupled channel select this channel with the AC Channel list AC Channel cho Im Enter the number of samples in the variance calculation record in the Record Size box 32 8192 Note The record size is constrained to be a power of 2 The larger the size of the variance record the more accurately the variance can be calculated This is however at the expense of the tim
90. el Im The amplifier membrane current output should be connected to this input channel AI Ch 0 for Amplifier 1 Enter the measurement units of the current pA nA uA mA A into the Units box and the amplifier current scaling factor of the amplifier at minimum gain in V units into the Scale factor box The scaling factor can typically be obtained by reading the minimum gain setting of the rotatable current gain switch found on most patch clamp amplifiers e g a minimum gain settings of 1lmV pA is equivalent to a scaling factor of 0 001V pA Secondary Channel Vm The amplifier membrane potential output should be connected to this input channel AI Ch 1 for Amplifier 1 Enter the measurement units of the current mV in the Units box and the amplifier voltage scaling factor in V Units into the Scale factor box Most amplifiers have a fixed scaling factor of 0 01 V mV Voltage clamp command channel Analog output AO Ch 0 for Amplifier 1 should be connected to the amplifier command voltage input Enter the voltage clamp command potential scaling factor membrane potentia command voltage into the Scale factor box A label specifying the command voltage scaling factor can usually be found beside the command voltage input socket on the amplifier Typical values are 0 1 V V or 0 02 V V Current clamp command channel E input Channels amp Amplifiers Setup EEx Input Channels Amplifiers Amplifier 1 Amplifier 2
91. equations can be combined to provide a parabolic relationship between and J I 2 n Estimates of Z and n can thus be obtained from plots of current variance vs mean current by fitting the above using non linear curve fitting Examples of the use of variance mean plots and a discussion of the issues involved can be found in Cull Candy et al 1988 Dempster 1992 2001 Noise Analysis gt Power Spectra The power spectrum is a measure of the frequency distribution of the signal variance Analysis of the amplitude and shape of the power spectrum provides a means of estimating both single channel current amplitude and channel gating kinetics The averaged spectrum is obtained by computing the individual power spectrum for each variance record using the fast Fourier transform averaging the corresponding frequency components from each record and optionally averaging adjacent frequency components By convention the power spectra is then plotted using log log axes Select the Power Spectrum page to compute and plot the averaged power spectrum for a series of variance records Noise Analysis oOo ioj x m Records New Spectrum Set Axes Records All records Range 1 85 M Time window None 10 Cosine m Options Subtr t Trends Subtr t Backg d Remove Harmonics of 0 Hz Curve fitting M Freq Averaging i None Eeuwe C Logarithmic None a plats MEPC Frequency
92. es of No Repeats recording sweeps for a total of No Steps After No Steps increments Gmax returns to the initial value Activation Parameter m Defines the dependence of steady state and time constant of the conductance activation parameter m on membrane potential Inactivation Parameter h Defines the dependence of steady state and time constant of the conductance activation parameter h on membrane potential Fast and slow kinetics are defined and the ration of fast to slow kinetics by the Fast Fraction field Load Save Settings Click the Save Settings button to save the dynamic clamp conductance settings to a DCS settings file Click the Load Settings button to load settings froma DCS file Update Dynamic Clamp Updates dynamic clamp with current settings Note Contain j dempster strath ac uk for details of the cRIO 9076 hardware and firmware required to implement DCLAMP Recording Experimental Signals gt Monitoring Input Signals amp Patch Pipette Seal Test You can monitor the signals appearing on each channel using the signal monitor pipette seal test module which provides a real time oscilloscope display and digital readout of the signal levels on the cell membrane current and voltage channels A test pulse can also be generated for monitoring pipette resistance in patch clamp experiments To open the monitor seal test module select from the menu Record Pipette Seal Test Signal Monitor An oscilloscope trace s
93. event times can be re loaded replacing the existing list by clicking the Load List button and entering the name of a file in the Load Event List dialog box m Event List Save List Load List 97 Detecting amp Analysing Events gt Exporting Events The digitised waveforms of the detected events can be extracted from the data file and exported as individual records for analysis by WinEDR s companion program WinWCP which has features for signal averaging curve fitting and more detailed waveform analysis Export Events Export Events Abort Export Gaps Events E 128 Exporting Events To export detected events 1 Enter the range of events to be exported in the Events box 2 Click the Export Events button and select the name of a WCP data file to hold the data Export to wcp rie zx Save int E My Data e aae fe My Recent Documents Desktop 4ndor Ixon X3 O LineScan 10082008 0831 867 Anselm Paul Turko Bernd Zinselmeyer Plymouth Sep 2011 D BrainMLData Problem CD Damian Cummings Rat cardiomyocyte plymouth data Plymouth Sep 2011 Rat LFP Delia Beleli 2 Richardson Leao C Denise Padget Rocco Pizzarelli DDr Demspter Shangwei hou L Guy Bewick Tony Workman John McCarron O WinFluor Atrial myocyte D Pau Ba Sciatic nerve intact sheath 30th June 2010 STREX DHEA test 1 128 wep Laurens Bosman Ba test_o01 Cp test _002 wcp amp My Doc
94. exported file click the Change File Name button and select a new folder name 5 Click the OK button to create the export file The currently supported export file formats are listed in the table below Export Data File Formats Axon Instruments ABF Axon Binary File V1 8 format files ambridge Electronic cfs ambridge Electronic Design CFS CED Filing System format files Design CFS dat ASCII Text ab delimited columns of ASCII text WCP S Sswepp Strathclyde Electrophysiology Software WinEDR data file format edr Strathclyde Electrophysiology Software WinEDR data file format Binary Ro bw BW Igor Binary Wave files produced by the IGOR Pro software package Matlab mat Matlab MAT data file 185 Data Files gt Inspecting File Properties To inspect or change the data scaling units and sampling rate properties of a data file select File Properties to display the File Properties dialog box Properties Page IMiFicPropertics o Properties Calibration Table File Header Sampling Interval 4 ms A D Voltage Range 0 00512 Y 4 D sample data max value 1024 File version 6 10 Date Created 14 08 2012 11 49 21 ID No samples in file 45000 No of channels 2 No of samples channels 22500 File header size bytes 2048 Sample value range 1025 1024 The Properties pages shows the no of samples sampling interval no of data channels and other parameters of
95. f state Closed Open its time duration average current and signal standard deviation within the state are displayed in the status box Individual channel states within the state list can be selected for display using the state selection scroll bar or by entering a state number into the box Events 3 911 gt The Exclude Edges box determines the number of zero or more samples at the beginning and end of each state which are excluded from the state average current to eliminate the parts of the signal which are in the process of transition between states Analysing Single channel Currents gt Detecting Channel Transitions gt Editing the Channel Event List Artifactual channel transitions arising from parts of the current recording corrupted by external interference or invalid for other reasons often have to be excluded from the channel dwell time and amplitude analysis Individual states can be excluded from analysis by ticking the Ignore State tick box for that event M Edit State Ignore state Cee eed Loree ie To automatically exclude a block of events click the Set Block button to open the dialog box Set Block x Events Selection criteria All Events m State s C Range 1 aa a Oms lt Duration lt 0 ms E 0 pA lt Amplitude lt 0 pA Setto Ignored a 1E 030 pA lt St Dev lt fo pA C Not Ignored OK Cancel 1 Select the type of stat
96. f x Resistance Record Stop 9 a Rm MOhms a af25s o The data displayed on the selected plot is stored in text file in the data folder 75 Displaying Records Stored on File gt Compressed View gt Displaying records Compressed View To review a recording after it has been acquired and stored in a data file select one of the signal viewing options from the View menu The Compressed View option displays the complete recording in compressed into a single window with the facility to zoom into selected regions The Page View option displays the un compressed recording within a single channel as a sequence of lines on page To display the recorded signal in the compressed view select View Compressed View to open the Compressed View window iew Recording i E gt Mark Chart Ident Cortical Day 18 300nM 5a3a then 10uM bicuculline Mark Chart A 2 m Cursor Avg t 0 t Calculate Average Im X k ni 4 2 5 t 1 1815 0 17487 n 1 12 1 14 1 16 1 18 1 2 1 22 1 24 Cms s C min Gett 0 Cursor Start Time 1134s EE E Mois The display window shows a segment of the recorded signals The displayed segment can be scrolled through the recorded signals using the display scroll bar or moved to a specific time point within the recorded file by entering a value into the Start Time box and pressing the return key Start Time 110345 4 J The size of the display
97. for signals such as single channel currents spontaneous synaptic currents or any signal where synchronisation with an external event is is not possible or required When the trigger mode is set to External Trigger recording only starts after a trigger pulse is received on the External Trigger input of the laboratory interface External Trigger mode is used when it is necessary to synchronise recording with an external event such as the pulse from a stimulator ideally so that the sweeps starts shortly before the cell is stimulated If the 5V Trigger Level option is selected recording will be triggered by a 0V to 5V transition on the Ext Trigger input If the OV Trigger Level option is selected recording will be triggered by a 5V to OV transition NOTE Some laboratory interfaces support only one or other of the two trigger polarities The No Sweeps box determines the number of externally triggered sweeps to be collected during a recording session Recording Experimental Signals gt Recording Modes gt Stimulator A wide variety of stimulus voltage or current waveforms can be generated on analag output channels 0 amp 1 and 5V TTL digital waveforms on up to 8 TTL digital output channels To generate a stimulus waveform select a stimulus protocol from the Stimulator list and click the Start button m Stimulator ramp 5s Start Stop Stimulus protocols are also started automaticaly when the Record button is press
98. frequency to be plotted Detecting amp Analysing Events gt Detecting Events To detect events within a continuous record select the Detect Events page The recording to be scanned is displayed in the upper panel and the detection criterion derived from it is displayed in the lower Event Detection 4 Detect Events ReviewEdit Events Y Plot Histogram Average Detect Events Abort Source Channel cho Im Whole file C Range D 10 0096 Detector Mode C Threshold Rate of Rise C Template M Thresholds Amplitude 1772 82 nAls Set Ampl 4 x S D Dead Time 20 ms Rising Edge 1 ms Window 1 4 0 4 1 15 1 2 1 25 1 3 2 Det Criterion 0 1 0 15 Three different types of event detection method are supported Threshold The amplitude threshold method uses a baseline tracking amplitude threshold algorithm the detection criteria being that the signal exceeds the threshold level for more than a predetermined period of time The signal baseline level is computed from a running average used to automatically adjust the threshold for slow changes in baseline level Rate of rise The rate of rise method uses a signal detection criterion based upon the signal rate of change computed using a 5 point Savitsky Golay differentation algorithm Template The template matching method is an implementation of th
99. g file can be used like an experimenter s notebook to keep a written record of the experiment A new log file is opened on a daily basis with a name in the form dd mm yy log and stored in the WinEDR program directory Select File Inspect Log File to display the experimental log Log File C Program Files Borland Delphi WinWCP 32 06 07 201 1 log 08 55 58 WinvVWCP Started 08 56 07 New data file C Data File_O09 wep created 08 57 51 Recording Started 08 57 51 Voltage Program C vvinvv CPwprotiSteps 10 100m 100ms xml 08 58 17 Stopped 10 records 08 59 34 Recording Started 08 59 34 Voltage Program C wvinvv CPwprotiSteps 10 100m 100ms xml 08 59 53 Stopped 18 records 09 00 42 Recording Started 09 00 42 Voltage Program C WwvinvvCPwprotiSteps 10 100m 100ms xml 09 01 31 Stopped 38 records 09 01 35 New data file C DataiFile_010 wep created 09 01 36 Recording Started 09 01 36 Voltage Program C Winvv CPWwprotiSteps 10 100m 100ms xml 09 02 26 Stopped 20 records 09 25 19 New data file C Data File_011 wep created 09 25 20 Recording Started 09 25 20 Voltage Program C Wvinv CPwprotiSteps 10 100m 100ms xml 09 26 10 Stopped 20 records Add Note Additional notes can be added to the log file by entering text into the Add Note box and clicking the Add button Recording Experimental Signals gt Special Modes gt Special Modes WinEDR has a number of special features for processing of
100. g the Stimulus Recording Protocol Editor ee cseseescnsescneesessesseecsesaeeecseeaeeenseeens 55 Analog amp Digital Output Channels wevicicctrercnntee scan Gnnacingl ame cae dnaabude 56 Protocol Settings merne occ cesass eth pacar a N eaten sma a 57 Adding Stimulus Waveforms to the Protocol cccccceceseesesesseseseeseseeesescseeseseeeceesenseeeeecaeeeeaeseenaes 58 Saving and Loading Stimulus Protocols 2 ce A eee eh Oa 63 Example Proto Stenn rece tae cr cate ohare Ra acre cata lao aid eal 64 D fa lt Output Seths en toccts arent cscs teceaty eco rsstecect te tceatt pase ee aces oce tate cranial aerial ict 65 Amplifier Gain SCM Ges ce 23a 8s sac gcr tte i acco a Casee cee s nokta tee yi he Seen te Bats A A tes od 8 eee toa 66 Experiment Identification COMMENUS ccccccccscsescsesssescssesssesesseeseseesesesssesceseseesseeceeseesesseseecseseesaeens 67 Special Modes Special MOGES resres raean o enaa E A E E EEEa EASE 69 Event Frequency nord enea a a eo acetate a nck E E a 70 Cell Capacity Modes cea eu eeein na ia EE O eo a ee 72 Cell IUGR ESCEN CE nire tiamcatsl cs E a E E A anes A Monel cad R a E cus 73 RESISTANCE cats cacsecciccavacintestteetsed E AE A ved KEE EEA CEEE E AEE A 74 Displaying Records Stored on File Compressed View Displaying records Compressed View ccecsceseesescesceeeeescecesecseesesecaeeecaeeseeecaeeseeseeeesecseeaeeetaeees 76 M as ring Signal EVENS casas ass 2s cnesas 2osd iaicta pecs
101. ge command inputs are also displayed If required the Gain and Voltage current clamp mode telegraph inputs can be changed to another analog input channel However if this is done the physical connections between the amplifier and the laboratory interface must be adjusted to match Telegraphs can also be disabled by selecting Off as the input channel When telegraphs are disabled gain and or voltage current clamp mode can be entered manually by the user If necessary primary and secondary channel scaling factors units voltage and current clamp command scaling factors can be altered by the user This is most likely to be case for the command scaling factors where where a range of division factors are available on most common patch clamp amplifiers The default settings are set to the most commonly used stimulus scalings 0 02 or 0 1 for the amplifier In the case of amplifiers where the primary secondary channels switch output signals in current or voltage clamp mode two different primary and secondary channel scaling factors can be entered by selecting the ICLAMP orVCLAMP options Note that in the case of amplifiers Manual amplifier configuration If the amplifier in use is not specifically supported by WinEDR current and voltage scaling information can still be entered manually by the user To enter up a manual amplifier configuration select Manual Gain Entry as the amplifier type and enter the following information Primary Chann
102. gital gain telegraph connections OOOO fe e e e pe e Next Getting Started gt Amplifiers gt Configuring Amplifier Support in WinEDR To configure amplifier support select Setup Input Channels amp Amplifiers to open the Input Channels amp Amplifiers Setup dialog box E input Channels amp Amplifiers Setup TER Input Channels Amplifiers Amplifier 1 Amplifier 2 Amplifier 3 Amplifier 4 Axopatch 200 v Input Channels amp VClamp IClamp Primary channel Scaled Output Scale factor 0 0005 VipA Units pA On analog input ai Ch 0 Secondary channel 10 Vm Scale factor 0 01 VimV Units mv On analog input ai Ch 1 Voltage clamp command channel Scale factor 0 02 VN Output ao Ch 0 Current clamp command channel Scale factor 2E 009 AN Output ao Ch 0 Telegraph channels Gain Jaich7 Voltage current clamp mode Al Ch 6 Load Settings Save Settings Default Settings Select the Amplifiers tab and choose the amplifier to be configured Amplifier 1 if only one amplifier is in use then select your type of amplifier in the amplifier list The amplifier configuration table shows the default primary and secondary input channels voltage and current command output channels and gain and mode telegraph input channels if these are required by the amplifier The basic at minimum gain scaling factors and units for the current and voltage outputs and the current and volta
103. he presence of trends in the single channel current amplitudes or dwell times Single channel Current Analysis a loj x Amplitude Histograms Transition Detection Edit Channel States Dwell Time Histograms SummarwStability Plots New Plot Summary Set Axes Abort 0 35 Plot Type Mean current 0 3 Events All Events es C Range amp 0 30 003 s E 0 2 Use C0 C1 i T Block Size 1 50015 2 0 15 0 1 0 05 Save to Log j 15 751 0 32063 0 5 10 15 20 25 Time s To create a stability plot 1 Select the type of plot to be created form the Plot Type list 2 Select the All option to use all sample points events contained in the recording event list or select Range and enter a time interval event range to define a selected region 3 Enter the number of averaging blocks that the analysis region is to be divided into in No Blocks box 4 Click the New Plot button to display the plot The values of the x y points in the plot can be read out using the blue readout cursor The following parameters can be plotted e Mean current Displays trends in mean currents The digitised current signal is divided into a series of contiguous blocks the mean current for each block is computed and plotted vs the mid point time for each block e Open probability Displays the probability of a channel being open and is derived from the mean current plot by dividing the mean block current by the si
104. howing the current signal on each input channel is displayed Pipette Seal Test Signal Monitor No channels A 8000 Cle Amplifier fe Heka EPC A Im Current cho im p in Saab EEA S SEEE EE inane lt 0 Voltage cn vim Clamp Mode 4000 6 Vclamp Iclamp Amplifier Gain 60 0 001 VipA Send Test Pulse Ta V AOO vim 20 m Test Pulse Pulse 1 F3 Y Hold 10 m 0 5 10 15 20 25 ms Amplitude 50 mY 000e C Pulse 2 F4 V Auto scale Y Hold o my mYVoltage _ _ r Current Holding Holding Pipette Cel G Cell Fi Amplitude f 100 mv F403 mV 1093 0 pA C Pulse 3 F5 Pulse Pulse Resistance Y Hold 0 my 49 1 mV 4963 2 pA 9 833 MOhm Amplitude 0 my Save to Log Pulse width 20 ms ist estimate from C Peak Exp Amp Timer Reset 00 21 Sweeps Averaged 10 Display scaling The vertical display magnification is automatically adjusted to maintain a visible image of the test pulse within the display area Automatic scaling can be disabled by un checking the Auto scale check box allowing the vertical magnification for each channel to be expanded to a selected region by moving the mouse to the upper limit of the region pressing the left mouse button drawing a rectangle to indicate the region and releasing the mouse button The vertical magnification can also be adjusted
105. http www ced co uk upu shtml and run it to install the CED1401 SYS device driver and 1401 commands 3 Ensure that the CED 1401 is switched on and then reboot your computer 4 Test the CED interface by running the program c 1401 utils try1401w exe and clicking the button Run Once 5 If the tests check out OK run WinEDR and select from the main menu Setup Laboratory Interface to open the Laboratory Interface Setup dialog box __ Laboratory Interface Se la x CED1401 16 bib 0v Device Not Initialised AD Converter Voltage Range 5 OK Cancel If you have a CED 1401 with standard analog 5V output voltage range select CED 1401 16bit 5V from the list of laboratory interface options If you have a CED 1401 with a 10V output voltage range select CED 1401 16bit 10V Signal input output connections Analog signal I O connections are made via BNC sockets on the front panel of the CED 1401 units CED 1401 Series Analog Input I O Panel Notes xe were e were xe were www e S w eo S C Trigger Inputs Inputs Trigger In Micro 1401 Power 1401 O a C powo O O O oe C E fee ws o a fee C C fe NOTE1 STANDARD CED 1401 ONLY Events Inputs 2 3 and 4 must be connected together and connected to DAC Output 2 to synchronise A D sampling D A waveform generation and digital pulse output for WinEDR s Seal Test option and recording with stimulus pulse protocols Note 2 A TTL pulse on the
106. ich input is in use When using the Transducer input there are 11 gain settings X1 X3 X10 X30 X100 X300 X1000 X3000 X10000 X30000 X10000 When using the Electrodes input X100 X300 X1000 X3000 X10000 X30000 X100000 X300000 X1000000 X3000000 X1000000 Note When CED 1902 is selected as the Amplifier in the Recording setup dialog box the gain setting is automatically included in the scaling of input channel 0 Low Pass Filter Sets the cut off frequency of the CED 1902 s built in low pass filter The filter can be set to None out of use 1000Hz 500Hz 100HZz The low pass filter removes signal frequencies signal higher than the cut off smoothing the signal High Pass Filter Sets the cut off frequency of the CED 1902 s built in high pass filter The filter can be set to None out of use 50Hz 100Hz 200Hz The high pass filter removes signal frequencies signal lower than the cut off removing steady and slowly changed components of the signal AC Coupled Check this box to make the CED 1902 input AC alternating current coupled In this mode only variations in the signal are allowed through to the amplifier constant DC levels are blocked AC coupling should beused with EMG and ECG recordings but not with force or pressure transducer recordings 50 Hz Filter Enable disables a notch filter which selectively removes frequencies around 50Hz Used to remove 50Hz interference from mains power lines DC Offset The
107. iers Setup JE oj x Amplifiers WinEDR displays the signals stored in each signal channel in the units appropriate to each channel The names units and scaling information for each channel are entered into the channel calibration table which can be displayed by selecting the Input Channels tab in the Input Channels Ch Name YJUnits Units AlCh Amplifier ap Oo 0 0005 A 0 1 Heka EPC 7 amp Amplifiers dialog box 1 vm 0 01 mv 1 1 Heka EPC 7 2 Ym2 1 m 2 The channel calibration table settings for the patch 3 m2 1 m 3 clamp current and voltage channels are automatically 4 Ch4 0 001 mv 4 configured with appropriate names units and V Units 5 chs 0001 mY 5 scaling when an amplifier is selected from the 6 Ch 0 001 mv 6 Amplifiers list However when no amplifier is selected 7 ch 0001 i 7 for use i e Amplifier None the calibration information for a channel must be entered directly into the table There are 5 entries in the table for each channel Name A 1 4 letter name used to identify the source of f the channel e g Vm Im Display Grid Time units V Units The scaling factor relating the voltage level at f msecs the inputs of the A D converter in V to the actual C secs signal levels in each channel in the units defined in the Units column Units The measurement units of the signal e g mV pA etc ADC Ch The analog digital converter input channel from which the sign
108. ies the function tends to a constant value So units I Hz At high frequencies the spectrum decays in proportion to 1 f The rate of ion channel opening and closure determines the corner frequency f at which the power has declined to So 2 Channels which rapidly flicker between short open and closed states produce higher frequency noise and spectra with higher corner frequencies For a channel with two states open and closed f is related to the ion channel gating time constant and the mean open and closed times topem teloseas by the formula 1 1 T oue mm 27 fe Vtopen It eased 2 If totosea gt gt topen then topen If a channel has more than two states then its noise spectrum will consist of a mixture of n Lorentzian functions for n channels state The relationship between channel dwell times and corner frequencies of the Lorentzian components is more complicated in these cases See Ruff 1977 If the spectrum has been recorded under conditions where the open channel probability is small e g p 0 1 the single channel current and conductance can also be computed from the power spectrum For small p values the second term in the eqn 2 can be neglected leading to a direct relationship between current variance and mean current o 1 I u m 3 Since the spectrum is the frequency distribution of the variance the variance can be calculated from the integral of the Lorentzian function 2 S 0 2 4
109. interface card 3 Locate the calibration files SCALE nnnnn EPC and CFAST nnnnn EPC for your EPC 9 10 patch clamp where nnnn is your patch clamp serial number and copy them to the WinEDR program folder c program files winEDR 4 Run WinEDR and select from the main menu Setup Laboratory Interface to open the Laboratory Interface Setup dialog box Laboratory Interface Setup l 10 x Heka EPC 10 E JEPC 10 sin 520177 Board ITC 1600 epc dil V 960 AID Converter Voltage Range 4 40 V OK E then select your device from the list Heka EPC 10 Heka EPC 10plus Heka EPC 10 USB Heka EPC 9 Heka ITC 16 Heka ITC 18 Heka ITC 1600 Heka LIH 88 5 EPC 9 10 Panel Connections If you have an EPC 9 or EPC 10 patch clamp connect a BNC cable between Filter 2 and A D Input 0 6 Instrutech ITC 16 18 I O Panel Connections Signal input and output connections are made via the BNC sockets on the front of the ITC 16 18 units ADC Input 3 ADC Input 4 ee ee ns w few o E Ch 5 ADC Input 5 Cx oo e pem ooo C e a Note 1 An active high TTL pulse on this input triggers the start of a stimulus program which has been set up with the External Stimulus Trigger Y option Getting Started gt Amplifiers gt Patch Voltage clamp Amplifiers One of the most common WinEDR applications is recording from and controlling a whole cell patch or voltage clamp experiment Two analog channels are normally recorded by WinEDR
110. ion KIE Export Gaps Eene 128 Event Filter EEEE vent Rejection Criterion pee Apply fime gt Limits m Event List G mea Clear Lower t The detected event is displayed in the upper panel with the detection point displayed as a vertical red line in the lower Each detected event can be selected for display using the event selection scroll bar or by entering the desired event number into the box Event 57 128 r Event Analysis 4 Regions before and after the event can be inspected by shifting the display window using the scroll bar at the bottom of the display The period of time displayed around the detected event can be set by entering a value into the display Width box The position of the detected event within the display window can be adjusted by setting the Pre detection percentage Width Pre detection 4 20 ms DIE F Note When measurements are being made the display width should be adjusted to display one event only Detecting amp Analysing Events gt Event Analysis The Event Analysis box displays a set of waveform measurements computed by analysing the signal waveform within the display window Event 57 128 K m Event Analysis Detected at 5 10555 Peak a aj 0 10758 nA Area a aj 1 9317 nA ms Ttrisej 0 05 ms T 90 0 1 ms Tau decayj 0 ms Duration 0 2 ms Event polarity C Positive Negative m Zero level Atstart At event Subt B
111. iser unit Digidata 1440A Analog Input 1 O Panel Ss Analog Out 0 esl E LSS Lo a Ss Analog Out 1 Analog Out 2 Analog Out 3 aa 3 Digital Output 7 Note 1 An active high TTL pulse on this input triggers the start of a stimulus program which has been set up with the External Stimulus Trigger Y option Getting Started gt Laboratory Interfaces gt Instrutech ITC 16 18 Instrutech Corp www instrutech com now handled by Heka Electronik GmbH The Instrutech ITC 16 and ITC 18 interfaces are self contained 19 rack mountable mains powered digitiser units with BNC I O sockets attached to the host computer via a digital interface card and cable Both the ITC 16 and ITC 18 support 8 analog input channels 4 analog outputs and 8 digital outputs Note The Instrutech ITC 1600 LIH8 8 and USB 18 are not currently supported by WinEDR Software installation WinEDR uses the Instrutech device interface libraries for the ITC 16 18 family Details for steps 1 3 can be found in the Instrutech Data Acquisition Interface user manual 1 Install the Instrutech interface card in an expansion slot 2 Attach the ITC 16 or ITC18 unit to the card 3 Install the Instrutech Device Driver software supplied with the card or downloaded from www instrutech com 4 Reboot the computer 5 Run the Instrutech test program installed with the device driver to test whether the software installed OK 6 Run WinEDR and select from the mai
112. ission of the author Except where otherwise specified no warranty is implied by either the author or the University of Strathclyde concerning the fitness of the software for any purpose The software is supplied as found and the user is advised to verify that the software functions appropriately for the purposes that they choose to use it An acknowledgement of the use of the software in publications to which it has contributed would be gratefully appreciated by the author John Dempster Strathclyde Institute for Pharmacy amp Biomedical Sciences University of Strathclyde 161 Cathedral St GLASGOW G4 ORE Scotland Tel 0 141 548 2320 Fax 0 141 552 2562 E mail j dempster strath ac uk Next Getting Started gt Hardware Requirements To run WinEDR you will require an IBM PC compatible personal computer with at least 16Mbyte of RAM a 66MHz 80486 or better CPU and the Microsoft Windows 95 98 NT V4 2000 XP 32 bit Vista or Windows 7 32 or 64 bit operating systems Note Windows 7 64 bit support not available for Digidata 1200 and 1320 interfaces A laboratory interface unit is required to perform analog digital A D and digital analog D A conversion of the signals and stimulus waveforms The following families of laboratory interfaces are supported e Cambridge Electronic Design 1401 1401 plus Micro 1401 Power 1401 e National Instruments laboratory interface cards e Axon Instruments Digidata 1200 13
113. itch set to NRSE the WinEDR A D Input Mode should be set at Single Ended NRSE Getting Started gt Laboratory Interfaces gt Axon Instruments Digidata 1200 Axon Instruments Inc now owned by Molecular Devices www moleculardevices com The Digidata 1200 1200A and 1200B interface boards fully supports all WinEDR features They have a 330 kHz maximum sampling rates and 4 programmable input voltage ranges 10V 5V 2 5V 1 25V Inputs to and outputs from the board are via BNC connectors on an I O box connected to the board via a shielded ribbon cable In order to use WinEDR with a Digidata 1200 the following computer system resources must be available for use by the Digidata 1200 e I O port address 320 33F Hex e DMA channels 5 and 7 Software Installation 1 Install the Digidata 1200 card into an ISA computer expansion slot and attach it to its BNC I O panel using the shielded ribbon cable supplied with the card 2 Install the WinEDR Digidata 1200 driver software for your Windows operating system by running the appropriate installation batch file If you are running Windows 95 98 or Me select WinEDR Digidata 1200 Drivers Install Digidata 1200 driver Win95 98 Me from the Programs menu If you are running the Windows NT 2000 or XP select WinEDR Digidata 1200 Drivers Install Digidata 1200 driver Win NT 2000 XP Note WinEDR does not use the standard Axon Instruments Digidata 1200 device driver 3 Reboot the computer
114. itude threshold before detection is accepted in the Time box Set the period of time over which the baseline tracking average is computed in the Baseline Tracking Time box The smaller the time period the faster the tracking rate of the baseline M Detector C Template Thresholds Amplitude DnA Set Ampl 4 x S D M Baseline Tracking Time 1000 ms Thresholds Rate of Rise mode Set the level which the signal detection criterion has to cross for an event to be detected in the Amplitude box or adjust the threshold cursor in the Det Criterion display m Detector Mode C Threshold Rate of Rise C Template M Thresholds Amplitude 1702 32 ns Set Ampl 4 x S D Thresholds Template mode Set the level which the signal detection criterion has to cross for an event to be detected in the Amplitude box or adjust the threshold cursor in the Det Criterion display Enter the rising and decaying time constants which define the shape and duration of the template waveform into the Tau rise and Tau decay boxes Suitable values can be obtained by detecting a typical signals using one of the other detection methods exporting the record to WinWCP and fitting an MEPC curve to it m Detector Mode Threshold Template M Thresholds Amplitude 55 78 Set Ampl 4 x S D M Template Tau rise 0 1 ms Tau decay 10 ms Note The Set Amp
115. ity of ionic conductances by generating records containing the whole cell membrane currents evoked in response to a series of voltage steps to different membrane potentials Oooo ee Initial Delay Delay at the holding level before the pulse begins Amplitude Amplitude of the first pulse in the series Amplitude increment Increment to be added to amplitude between records mamin foem o E Family of rectangular voltage pulses varying in duration A rectangular voltage pulse whose duration is automatically incremented between recording sweeps This element is most commonly used as a variable duration preconditioning pulse in 2 or 3 step protocols for investigating inactivation kinetics of Hodgkin Huxley type conductances OOOO pee SOS omon omean O Series of rectangular voltage pulses A train of rectangular voltage pulses of fixed size This element can be used to produce a series of stimuli to observe the effect of repeated application of a stimulus at a high rate It can also be used to produce a train of pre conditioning stimuli for a subsequent test waveform Oooo ee Initial Delay Delay at the holding level before the pulse begins Amplitude Pulse amplitude Repeat period Interval between pulse in train Voltage ramp A linear voltage ramp between two voltage levels Voltage ramps provide a means of rapidly generating the steady state current voltage relationship for an ionic conductance Note that the ramp generate
116. latte e a E lta ap doc adga Totes 78 Printing Records Compressed View ccccescsccssessessseesesseseecesceseceeeeeeecaseecaeeseeeeseeseeseseeaeeaeeeeeeaeers 80 Copying Records Compressed View s c0 naw ea ene ae 82 Page View Page View is cri occas Gp Saag r Seeing r OB aks A RID cae onda AAN 83 Printing Page VIEW scaicecet aces iasesevssteveceenaets a e E E E RE erally tte O does nearer 85 Copying Page VieWjnsiesnrenunine a a a E E EA aa 86 Detecting amp Analysing Events Event Detection rrei Sots an n E A E E GRO NEEE RGU a h E 87 Detecting Events veccevesceiaxsceeneccussensiane eaae aaa e Eae o o EE E EEE E Ee Ea a 89 Reviewing Sc Eding EVeEntS i meae E E E EEE E A aw asateiaes 92 Event Analys Saen eee a EE E E RE E a ERE ER een RARER RRR 94 Adding R moving EV GIES sxc csces sacs Seu eas te ctl ts n a he cal E a E E hue Si 96 Exporting Events i225 savasasdsesacasestataeves tha a E A ea eI aie 98 Plotting X Y Graphs of Event Analysis Measurement c ccc ese cseeeeeecsessesesetscsesessesessnaeseees 100 Plotting Histograms of Event Analysis Measurement c cece cesseeeecscsssseseescsssenseessnaeseses 102 Averaging Events os situate a e e E E KR dah E cad nada mnie ERR 105 Analysing Single channel Currents Single channel Current Analysis eaivccecine Ao eecede pach al weak eek edd he td ere 108 Current Amplitudes Analysis OF Current A Tip EGOS ennai tress sea cecdten ees sees wre a toret A ieee wena ola 110 Am
117. list Labels for the X and Y axes can be entered into the Labels boxes The style of rectangle used to plot the histogram bins can be changed using the Bin Style options Select No Fill to display bins as rectangular outlines Solid Fill to fill the bins in with a solid colour and Hatched Fill for bins filled with a diagonal lines You can define the colour used for the solid fill by clicking the Colour box and selecting a colour from the palette The Full Borders check box determines whether the outline is drawn completely around each bar or just where bars do not overlap Analysing Single channel Currents gt Channel Dwell Time Analysis gt Printing Histograms To print the displayed histogram select File Print To open the dialog box Typeface Page Margins Arial Left 5 0 cm Top 50cm Line width 2 pts Botomf50cm Marker size 5ps Use colour a cence Click the OK button to print the graph Analysing Single channel Currents gt Channel Dwell Time Analysis gt Copying Histograms to the Clipboard Copying the histogram data points to the Windows clipboard The numerical values of the X Y data points which generate the histogram can be copied to the clipboard by selecting Edit Copy Data The data is placed on the clipboard as a table of data values in tab text format defining the histogram There are 4 values per row and one row for every bin in the histogram Ea
118. list to a text file 1 Select Open Times or Closed Times to export the sequential list of channel open and closed dwell times produced by transition detection Select Event List to export a list of channel dwell times with associated channel state O Closed 1 Open Export Events Event List Closed Times C Open Times Save To File m Murenr hdaaciramant 2 Click the Save To File button and enter a file name in the Save To File dialog box which appears ave datane ij 2x Save in e Data z 4a c E My Recent Documents Desktop cell 2 slice 4 Full z series Helene Widmer July 2010 STREX DHEA Damian Cummings winwep Human Atrial myocyte D Pau Daphnia Tony Campbell John Mec Data LineScan 10082008 0831 867 Data Chart Liver 2P images David Wokosin Data Dundee Metamorph Demo Images Data Miller Lite Michael Ferber Data Plymouth Neuron Sandy Harper Data Ross Images origin Files Data T Cells Plymouth Data Data Tony Rabbit C S Susan Coker Data WinFluor Rat cardiomyocyte Plymouth DRG data Plymouth Richardson Leao ne My Documents oe A a My Computer K File name test fevent list ty My Network Save as type Text Files txt Cancel Places Analysing Single channel Currents gt Detecting Channel Transitions gt Cursor Measurements Two pairs of linked cursors a a and Z Z can be used to make manual measureme
119. ls check box to display the signal zero level as a horizontal dotted line on each line Vertical Magnification The vertical magnification of each line can be expanded by moving the mouse to the upper limit of the region pressing the left mouse button drawing a rectangle to indicate the region and releasing the mouse button The vertical magnification and position of each line within the recording can also be adjusted using the N 8068 buttons at the right edge of each plot Displaying Records Stored on File gt Page View gt Printing Page View To print a section of the digitised recording as a series of lines select File Print to open the dialog box Print S Typeface Line Arial Current Page Whole record C Range Size 12 pts 0 0 10 0s Line Width 2 pts Calibration bars Use colour J Horiz 0 001 s Show labels JV Vert 0 129nA Page Margins Left Rig Print 5 0 cem 2 5 cem Cancel Top attom Emmeni 5 0 cm 5 0 cm oO There are 3 printing options Current Page Select the Current Page option to print out the current set of lines on display on a single printed page Whole Record Select the Whole record option to print out the whole recording on to a series of printed pages Note that a large number of pages may be required for long data files acquired at high samplings rates Range Select the Range option to print out the recording
120. mber of time points read from the file in a single operation in the Read in blocks of box usually set to 512 5 Specify the format of the analog data samples to be imported Select the Float option and No of bytes samples to 4 for 4 byte floating point samples or Integer for binary integer samples and the size of the sample in No of bytes samples Enter the upper limit of the sample value in the Max Value box 6 Enter the time interval between adjacent samples within each channel in the Sampling interval box Select the units of the time interval from the Time units list 7 Click the OK button to import the data when the import settings are complete Data Files gt Exporting to Other Data File Formats EDR data files can also be exported in a number of data file formats To export the currently open data file select File Export to open the dialog box export Fie x M Output file E Data Marko Gosak TEA glu_003 10 104 ibw Change File Range m Output format Whole file Axon CED f Range C EDR C Chart Igor C MAT m Channels V im OK Cancel 1 Output Format Select the data format of the file to be exported to from the list of options 2 Range Select the Whole File option to export all data in the currently open EDR data file or select Range and enter the range be exported 3 Channels Tick the signal channels to be exported 4 Output File To change the name folder of the
121. mixtures of gaussians can be fitted simultaneously allowing the relative proportions of each to be estimated To fit a gaussian curve to a histogram 1 Define the range of amplitudes containing the peak s to be fitted using the pair of grey region of interest cursors TIA A 0 4895 0 0 4 0 2 0 0 2 0 4 0 6 0 8 1 12 1 4 All Points pA _ Mean 0 0956 pA Area 100 _ 2 3 4 Select the number of Gaussian functions to be fitted from the Curve Fitting list vure mung Fit Curve 2 Gaussians 5 Click the Fit Curve button and enter an appropriate set of initial guesses for the gaussian function parameters i i Pki Note A set of initial guesses are computed automatically but it is often necessary to adjust these to better match the location and size of the observed histogram peaks Individual parameters can also be fixed at their initial values by ticking its associated Fixed option xi Parameters Fixed Fixed Mean 0 00825pA Peak o538 D s d1 otp D tC C lt lt iw Peak 557 D Mean 101 pA E oOo E s d 2 0 0993 pA D m Parameter Initialisation ok Automatic Cancel C Manual Initialise Click the OK button to begin fitting The best fitting gaussian function s are superimposed in red on the histogram The values of the function parameters along with their estimated standard errors are displayed in c
122. n menu Setup Laboratory Interface to open the Laboratory Interface Setup dialog box Laboratory Inl Me x Jinstrutech ITC 16 18 New drivers Unknown AD Converter Voltage Range 5 Y OK Cancel then select Instrutech ITC 16 18 New driver from the laboratory interface options list Note on systems with Instrutech s older device driver software installed it may be necessary to select either Instrutech ITC 16 Old Driver or Instrutech ITC 18 Old Driver depending upon which interface unit is installed Instrutech ITC 16 18 I O Panel Connections Signal input and output connections are made via the BNC sockets on the front of the ITC 16 18 unit Instrutech ITC 18 Analog Input 1 O Panel ADC Input 0 TS Ch 3 ADC Input 3 moms fT Note 1 An active high TTL pulse on this input triggers the start of a stimulus program which has been set up with the External Stimulus Trigger Y option Instrutech ITC 16 18 Troubleshooting WinEDR requires Instrutech s combined device driver library ITCMM DLL released late 2001 It may not work with earlier libraries Getting Started gt Laboratory Interfaces gt Biologic VP500 Bio Logic Science Instruments SA www bio logic fr The Biologic VP500 is a computer controlled patch clamp with a built in laboratory interface unit attached to the host computer via a GPIB interface bus It is supported under Windows 95 98 NT and 2000 The VP500 patch clamp function
123. ndow O x E r Event Frequency 11 apacny UOTESCENCE Fe TA M Ej 71 Recording Experimental Signals gt Special Modes gt Cell Capacity Mode In cell capacity mode activated by ticking the Capacity option real and imaginary admittance signals from a patch clamp with a phase detector can be decoded to produce a continuous measure of cell capacity membrane and access conductance Computed channels 7 Event Frequency Resistance gt Input channels r Results channels lm en o Im Gm cha Gm Ym cn 1 vim Gs Ich4Gs x M v Greal cn 2 Greal Cm f Ch 6 Cm v M Invert G real signal _ Display ranges G imag cna Gimag Gm fio nS Invert G imag signal J Gs 500 nS Cm 200 pF r Capacity Compensation Excitation freq 1000 Hz Use compensation Reversal Pot omy Series Resistance 0 MOhm Cell Capacity O pF Use gain telegraph in OK Cancel A total of 8 channels are required four input channels real admittance Grea imaginary admittance Gimg membrane current Im membrane potential Vm 3 computed results channels membrane conductance Gm access conductance Ga and membrane capacity Cm Recording Experimental Signals gt Special Modes gt Cell Fluorescence In cell fluorescence mode activated by ticking the Fluorescence option intracellular ion concentration can be computed from th
124. ngle channel current value set on the Amplitude Histograms or Transition Detection page Open times Displays trends in the channel open state dwell time The channel event list is divided into a series of contiguous blocks the mean open state dwell time for each block is computed and plotted vs the mid point time for each block Closed times Displays trends in the channel closed state dwell time Single channel currents Displays trends in the amplitude of the single channel currents The channel event list is divided into a series of contiguous blocks the mean single channel current for each block is computed and plotted vs the mid point time for each block Current vs Open times Dsplays the relationship between open state duration and average open state current Dwell time and average current for the open states in the event list are displayed as a scatter plot Cursor Meas Avg Displays vs time the average current in the the manual cursor measurements lists Cursor Meas Dur Displays vs time the average duration in the the manual cursor measurements lists Cursor Meas S D Displays vs time the average standard deviation in the the manual cursor measurements lists Analysing Single channel Currents gt Stability Plots gt Printing Plots To print the displayed plot select File Print To open the dialog box Print Typeface m Page Margins arial Left 5 0 cm Size 12 pts Right 2 5 cm Top 5
125. nnel zero current level by dragging zero level cursor up or down Note The zero current level is usually set to the mean signal level that exists within the Background records Noise Analysis gt Analysis of Signal Variance Select the Variance Analysis page to display and analyse the mean current variance and other parameters computed from the variance records lolx Review Edit Data Records Amplitude Histogram Variance Analysis Spectral Analysis Records r N Records Allrecords Range 1 342 r Plot X Axis Record No Y Axis variance AC m Options Subtr t Backgr nd Variance AC pA 0 50 100 150 200 250 300 Record No Analysis Fit Curve None Save to Log Avg Variance AC 7 672 pa No records 342 To select and plot a pair of variables 1 Define the variable to be plotted on the X axis by selecting it from the X Axis variable list 2 Define the variable to be plotted on the Y axis by selecting it from Y Axis list 3 Select the All Records option to plot all available variance records or select Range and enter a specific range of records Note Records marked as Rejected and Background records are excluded 4 Select the Subtr t Backg d option to subtract the average variable value within the Background records from the plotted data 5 Click the New Plot button to plot the graph Five basic
126. ns to operate In addition if stimulus protocols containing digital ouputs are required DAC1 amp EXTTRIG must also be connected to the digital synchronisation input PC6 Note 2 Lab PC 1200 series boards need the pulse to be 10 ms in duration or longer Troubleshooting National Instruments cards can be used with a number of different types of input output panels BNC 2090 BNC 2110 or CB 68 terminal panel and can also be configured to handle the analog input channels in a number of different ways differential referenced single ended and non referenced single ended Some combinations of settings can lead to input signals drifting or going off scale Differential mode DIFF Analog input channels are paired together and subtracted e g Ch 0 Ch 7 Ch 1 Ch 8 etc Referenced single ended mode RSE Analog input channels are used individually and measured relative to signal ground of the computer Non referenced single ended mode NRSE Analog input channels are used individually and measured relative to the electrical ground of the device being measured When using the BNC 2110 I O box the USB 6221 BNC or USB 6229 BNC USB interface device the WinEDR A D Input Mode must be set at Differential When using the BNC 2090 I O box with its SE DI switches set to DI the default setting the WinEDR A D Input Mode should be set to Differential When using the BNC 2090 I O box with its SE DI switches set to SE and the RSE NRSE sw
127. nts entry in the parameter tables indicates the number of points loaded from the file For two column data files which contain time data the D A update interval is set to the time difference between the first and second rows of data For single column data files D A update interval must be entered by the user Empty analog element Empty analog waveform element Dragging this element on to an analog output list erases the element it is placed Digital pulse fixed duration A fixed duration digital pulse This element can be used to switch open or close valves controlling the flow of solutions over a cell Multiple digital outputs can be used to simultaneously open one valve while another is closed ne Family of digital pulse varying in duration aaa A digital pulse whose duration is automatically incremented between recording sweeps a O Train of digital pulses A train of digital pulses of fixed intervals and of fixed duration This element can be used to apply a rapid train of stimuli to a cell pf Parameters Initial Delay Delay at the holding level before the pulse begins State 0 0V 1 5V Digital output state during pulse 0 0V 1 5V ES O Repeat period Interval between pulse in train Empty digital element 3 Empty digital waveform element Dragging this element on to a digital output list erases the element it is placed on D A Update Interval The D A Update Interval box displays the
128. nts of duration average current amplitude and standard deviation and save these to a list This feature is enabled by ticking the Cursor Measurement Enable option Cursor Measurement V Enable T 0 0704 The average current difference between the average signal within the Z Z and a a cursors duration time between the a a cursors and standard deviation of the signal within the a a region are displayed in the Cursor Measurement box Clicking the Save to List button saves the displayed measurements to a storage list Single channel Current Analysis Amplitude Histograms Transition Detection Edit Channel States Dwell Time Histograms SummaryStability Plots r Events 1 5 3 911 gt State Open Duration 4 ms A Avg 0995pA W ee noe mA 5 D 0 0922 pA Edit State Ignore state Set Block Delete Ignored m Average Amplitude Exclude edges 0 1 ms M Export Events Event List Closed Times C Open Times Save To File Cursor Measurement V Enable T 0 0704s Avg a a 1 088 pA 5 D a a 0 09898 pA 3 Dur a a 0 9 ms Save to List 20 Clear List Save List to File i g Load List from File 0 067 D068 0 069 oof 0071 0 072 0 073 0 074 0075 Ol Cursor spacing 10 Width 410 m The amplitude duration and standard deviation measurements stored in this list can be plotted vs time on the Stability Plot page
129. onics of option and enter a base frequency in the box to remove frequencies at multiples of X1 and X2 the base Options Subtr t Trends Subtr t Backg d Remove Harmonics of 0 Hz m Fron Averaninea A 5 Click the New Spectrum button to compute and plot the averaged spectrum The spectral power at each frequency component can be displayed using the blue readout cursor The average variance contained within a range of frequencies indicated by the l region of interest cursors is also displayed The total variance obtained by integrating the spectrum and the median power frequency frequency below above which 50 of the variance lies is displayed in the results box Noise Analysis 2502 4 0 001218 103 Median power frequency 472 3 Hz Lorentzian gt Total variance 25 25 pA A detailed discussion of the computation of power spectra can be found in Dempster 1992 or 2001 170 Noise Analysis gt Fitting Curves to Power Spectra To fit a Lorentzian function to a power spectrum 1 Select the region of the spectrum to fitted using the f f region of interest cursors 4 f 170 9 0 030844 f in in2 ins 2 Select the function to be fitted Lorentzian 2 Lorentzians from the Fit Curve list Curve fitting Fit Curve E 3 Click the Fit Curve button Set the initial parameter guesses optional Set Fitting Parameters x Parameters Fixed Fixed s0 0 04
130. ords from this file on to the end of the currently open the data file Note You can only append files which have compatible records with the same number of channels Data Files gt Importing Other Data File Formats To import records from a non EDR data file select File Import To display the dialog box Import File jx Look in o Trevor Smart gt e a EB Fe 2011_06_16_0005 GABAtest PCLAMP 9 abf My Recent 2011_06_16_0005 GABAtest integer abf Documents 2011_06_16_0005 GABAtest abf test pclamp9 int abf Desktop 2011 _06_16_0005 GAB test ABF 1 8lnteger abf 9 My Documents m Br My Computer File name hoise test 2EDR 3 ETA Files of type Jaxon Files DAT gt ABF Cancel Places J Open as read only Select the disk drive and folder from the Look In list Then select the type of data file to be imported from the Files of Type list A list of available files in that type are displayed Select one of the file names then click the OK button to import the data into a WCP format file Note The original file is not changed A new EDR format file is created with the same name as the imported file but with the extension EDR The currently supported data file formats are listed in the table below Importable Data File Formats Axon Instruments abf Axon Instruments ABF Axon Binary File format files produced by the PCLAMP ar dat AxoScope programs Note ABF V2
131. ower limits of the range of variable values to be included in the histogram in the Range boxes Values outside this range are excluded from the histogram 4 Set the number of histogram bins into which the histogram range is divided in the No Bins box 5 Tick the Percentage check box to plot the histogram vertical axis as a percentage of the total number of events rather than number of events in each bin 6 Tick the Cumulative check box to plot a cumulative histogram 7 Click the New Histogram button to display the plot 8 If you want to customise the axes ranges click the Set Axes button Fitting Gaussian Curves to Histograms To fit a gaussian curve to a histogram 1 Define the range of amplitudes containing the peak s to be fitted using the pair of grey region of interest cursors i 0 905 16 0 8 1 2 Select the number of Gaussian functions to be fitted from the Curve Fitting list Curve fitting Gaussian 3 Click the Fit Curve button and enter an appropriate set of initial guesses for the function parameters A set of initial guesses are computed automatically but it is often necessary to adjust these to better match the location and size of the observed histogram peaks Individual parameters can also be fixed at their initial values by ticking the associated Fixed option Set Fitting Parameters x Parameters Fixed Fixed u 0 89 nA E is 5 fo 1 nA E E Peak 31 E E
132. parameters are computed from each variance record and can be plotted against each other Five basic parameters are computed from each variance record and can be plotted against each other Mean DC is the mean current computed with the formula where J i is the i of the N samples in the variance record Variance AC is the current variance computed using the formula SU L near o l N 1 St Dev AC is the standard deviation the square root of Variance AC Skew AC is the current skew computed using the formula N UO Tea Skew N 1 Median Freq AC is median frequency of the power spectrum calculated from each variance record Note Variables marked AC are computed from the designated AC signal channel See Variance Records page if one exists Variables marked DC are computed from the DC channel Noise Analysis gt Variance Mean Current Plots To plot a variance mean current curve and estimate single channel current and number of channel by fitting a parabolic curve 1 Select a range of variance records from a period of the recording where the mean current is changing 2 Select Mean Current DC from the X Axis list and Variance AC from the Y Axis list 3 Click the New Plot button to plot the variance mean graph 4 Select the region of the graph to fitted using the l analysis region cursors 5 Select Parabola from the Fit Curve list to fit the parabolic equation to the plo
133. plitude Histograms All Points Amplitude Histogram cccsissccccacnszscesnsteptaccs sepceesuradeacessdedvesenqvacdssmaeeensawcemenneteelneaoes 112 All Points In State HIStO Grain xssciscessesticeesteatagiesteati rteep tau n nnn aaia 114 Mean State Amplitude HistOgram cccccecceccecesssseseeseescseeseescseeseeseeecseeecseeseeeeeeesecaeaeeecaeeaeaeeaees 116 Patlak Average Histogram sce e cies eed cel th Gt Ae a cl 118 Cursor Measurements avg HiStOGraim cccceseseseescsceseesceeeseesccesecsesecaeeseecaeeaeeecseeseeeeeeeeeees 120 Extend File Histogram renidens e ps acetal ee econ Acs net eutsnat aid a denen ta a a 121 Fitting Gaussian Functions tO HiStOGraMS cceccesceseeceseesceseeecseesesecseecseeseescseeseeseeetseeseeseaeeaeees 122 Customising Histogrant Plots i 22 c n eit et edad aor eee ai tolicd eh SARS 124 PAPUA ACH Wet STD Rala IG E E E E pcs amas ducone cee eo oeads T tac AE 125 Copying Histograms to the Clipboard vec occ cue we eure eee er as 126 Detecting Channel Transitions Transition Detection satacias tricorn sresd eset tiaceva tesa E E E a guste laee nantes tant h 128 Detecting Channel Open Close Transitions cccceseseesescseeseseeeceeseeseseeeeseeeeecaeeeeseeeescsenesseeeeeeaes 129 Removing Baseline Di itinscvcsutss d ves vacaqeceiengueeielerdy used Kah Gl Gi adeno ar ROAR 131 Viewing Editing Channel Open Close States ccccccsesecscssesecsessesscseescsecsecscseesecseseesecsesseseeaeees 133
134. pny exp V V4 2 Vsip Taumn 2ms Taum x Sms V172 30 mV Vslp 10 mV Power factor i Update Dynamic Clamp Ready Load Settings COM Port com i x Save Settings Time constant fast lt lt tau V taunt taumy taupypexp V V4 2 Vsip TaUmn 5ms Ta mx 10 ms 4 2 30m Vsip 10m r Time constant slow tau V taup an t taumy taupypexp V V4 2 Vsip Taumn 10 ms TaUm x 20 ms Vi 2 30 mV Vsip 10 mV Fast Fraction 0 5 Com Port Selects the serial port used to communicate with the cRIO 9076 controller Reversal Potential The reversal potential of the simulated ionic current is defined in the Reversal Potential Vrev field Current Command Scale Factor The current command scaling factor Amps Volt of the patch clamp is entered into Current Command Scale Factor field Enable Inhibit Input AI2 Tick this option to enable inhibition of the simulated current in real time by a 5V signal applied to the AI2 input of the cRIO 9076 controller Conductance Select the Add option to add the simulated current to the cell Subtract to subtract it and Off to disable the conductance Max Conductance Gmax The maximum conductance nS of the simulated conductance is defined in the Initial Conductance field Select the Fixed option to keep the conductance fixed at this value Select the Steps option to increment the conductance by Step Size after a seri
135. port and turn it on 4 Run WinEDR and select from the main menu Setup gt Laboratory Interface to open the Laboratory Interface Setup dialog box Laboratory In 01x Molecular Devices Digidata 1440 Unknown AD Converter Voltage Range 5 Y OK Cancel then select Molecular Devices Digidata 1440 from the laboratory interface list Signal input output connections Signal input and output connections are made via the BNC sockets on the front of the Digidata 1440A digitiser unit Digidata 1440A Analog Input 1 O Panel Analog Input 0 Analog Input 2 Ch 4 Analog Input 4 SS mw o Ext Stimulus Trigger o E aon mom maon prao paos ao Digital Output 7 Note 1 An active high TTL pulse on this input triggers the start of a stimulus program which has been set up with the External Stimulus Trigger Y option Getting Started gt Laboratory Interfaces gt Molecular Devices Digidata 1550 1550A Molecular Devices Corporation www moleculardevices com The Molecular Devices Digidata 1550 and 1550A interfaces consists of self contained mains powered digitiser units with BNC I O sockets attached to the host computer via a USB 2 0 port They support sampling rates up to 500 kHz 16 bit resolution on up to 8 channels They have a fixed input and output voltage range of 10V and support 8 analog and 8 digital output channels Software Installation WinWCP uses Molecular Device s software libraries
136. quares number of function parameters and degrees of freedom for each function that has been fitted are tabulated For each successive exponential component the F statistic comparing its quality of fit with the previous one is computed along with its associated significance probability p A function is deemed to have significantly improved the quality of fit if p lt 0 05 In the example shown here 2 exponentials provide a significantly better fit p 0 0000 than but 3 exponentials do not provide a better fit than 2 p 0 9805 Click the Print button to print a hard copy of the F test results table The table of results can be cleared by clicking the Clear Table button Analysing Single channel Currents gt Channel Dwell Time Analysis gt Customising Histogram Plots If you want to alter the X or Y axis range scaling or labels on a histogram click the Set Axes button to open the dialog box Set Axes Range Labels x Min 0433 Max 1 41 Tick 0 2 Scale X Axis Y Axis C Automatic Automatic Manual Manual Min jo Max 5 57 Tick au Scale Linear Labels X Axis All Points pA Y Axis m Bin Style Empty Colour Full borders C You can change the X or Y axis limits by selecting the Manual Axis option and entering new values into the Min Max and Tick spacing boxes An axis can be made Linear or Logarithmic by selecting the option from its Scale
137. rce and pressure transducers It is not isolated Connections In order to control the amplifier the CED 1902 serial communications cable must be connect to a serial port COM1 or COM2 The analog output of the CED 1902 should also be connected to analog input Ch 0 of the laboratory interface The amplifier gain setting is taken into account by WinEDR in scaling the signal level on this channel Control Panel Select Setup CED 1902 Amplifier to open CED 1902 control panel oI input Anika Grounded E a Amplifier Gain 0 i o0 100 X High Pass Hz I AC Coupled aaa DC Offset M 50Hz Notch 0 mY E Com COM Port The following amplifier settings can be configured using the panel COM Port Selects the serial port used to communicate with the 1902 Input Selects the amplifier input Grounded to connect the input to ground i e there is no signal Single Ended for the transducer input in singled ended i e non differential mode Normal Diff for the Transducer input in differential amplifier mode Inverted Diff for the transducer input in differential mode with the signal inverted or Isolated EEG for the electrically isolated differential input Note Normal Diff mode is used when recording from a force or pressure transducer and Isolated EEG used when making EMG ECG recordings Amplifier Gain Sets the gain of the CED 1902 amplifier The range of gain settings depends on wh
138. re allocated to bins according to the formula inn 1 Int bpd log n where bpd is the number of histogram bins per decade 10 fold change in dwell time and nd is the dwell time in units of sampling intervals This algorithm results in a histogram consisting of a series of bins which logarithmically increase in width from a bin size equal to the sampling interval by the factor bpd every decade By convention the vertical axis of the Sigworth Sine histogram is plotted using a square root scaling A typical logarithmic histogram is shown here the same two state 1 ms 40 ms closed time distribution used earlier Each exponential now appears as a peaked distribution with the peak value occurring at the mean dwell time for that state No Events 140 120 100 80 60 40 20 0 t 10 4 10 2 10 2 0 12409 0 10 1 Closed times s 100 101 Analysing Single channel Currents gt Channel Dwell Time Analysis gt Fitting exponential Functions to dwell time histograms Dwell time distributions can usually be described in terms of a mixture of exponential probability density functions where the probability p t of dwell time t being observed is given by Nexp A t pl exp 2 T T where nexp is the number of exponential components in the mixture Ai is the fraction of the total number of dwell times associated with component i and i is the mean dwell time Mixtures of up to 5 exponential p d f s
139. red into the font size box The thickness of the lines used to draw the signal traces can be set using the line thickness box Vertical and horizontal calibration bars are added to the plot to indicate the units and scaling of the plotted signals You can define the size of the bars by entering values into the calibration bars table The position of the zero level for each plotted trace is indicated by a horizontal dotted line Zero levels can be disabled by un checking show zero levels Plot labelling can be disabled by un checking the show labels check box The use of colours within the plot can be disabled by un checking Use colour When all plot parameters have been set click the OK button to initiate printing Choosing a printer and output format To choose a printer and to select the paper format select File Print Setup to open the dialog box Print Setup A4 210 x 297 mm A Auto Select v A printer can be selected from the list of currently installed printers The orientation of the plot on the page can be selected as either portrait or landscape 81 Displaying Records Stored on File gt Compressed View gt Copying Records Compressed View The displayed signal record s can be copied to the Windows clipboard in a variety of formats a data table an image a WinEDR data record Copying data values Each signal record consists of an array of digitised sample values A table of data values for the active
140. rotocol Settings Select the Protocol tab page to set the number of steps and repeat period of the protocol Protocol Waveforms Stimulus repeat period 5500 00 No stimulus increments 1 No repeats per increment 1 C Repeat Protocol Link to next protocol z Stimulus repeat period the time interval between successive steps within a protocol No stimulus increments For incremented step waveforms the number of times a stimulus is incremented within the protocol No repeats per increment For incremented step waveforms the number of times a stimulus is to be repeated without incrementing the step size Repeat Protocol Select the Repeat Protocol option to continuously repeat the protocol on completion Link to next protocol Select the Link to next protocol option to load the specified protocol on completion of the current one C Repeat Protocol Link to next protocol 100 100m ramp Next Recording Experimental Signals gt Creating Stimulus Protocols gt Adding Stimulus Waveforms to the Protocol Select the Stimulus tab page to add stimulus waveforms to the analog or digital output channels Protocol F Delphi Projects WinEDR 32 Stim ramp 5s xml lol x Files Waveform ___NewProtocol 100 mv ___OpenProtocol__ ___SaveProtocol__ 400 __Save Protocol As_ __Set Protocol Folder 100 mY DOO Analog Outputs A0
141. s and Triton 16 channels are multi channel devices Software installation 1 Download and install the Tecella device driver TecellaDriver from the Tecella downloads web page www tecella com downloads 2 Run WinEDR and select from the main menu Setup Laboratory Interface to open the Laboratory Interface Setup dialog box _ Laboratory Interface Setup t O x Tecella Triton Triton Pico Tecella Pico sin 33ffd7053058323508580343 Lib V0 1 AD Converter Voltage Range 1 OK Cancel then select Tecella Triton Triton Pico from the laboratory interface options list Tecella I O Panel Connections No I O panel connections are necessary All connections between patch clamp and computer are effected via the USB bus Getting Started gt Laboratory Interfaces gt Heka Patch Clamps amp Interfaces Heka Electronik GmbH www heka com The Heka EPC 9 and EPC 10 are a range of computer controlled patch clamp amplifiers with a built in laboratory interface unit attached to the computer via PCI interface cards or USB Heka also supply and support the Instrutech range of laboratory interface units ITC 16 ITC 18 ITC 1600 and their own unit the LIH 88 Software installation 1 Install the drivers and software supplied with your patch clamp or download and install the EPC PG drivers from http www heka com download download html 2 Attach the Heka patch clamp to the computer or install the Instrutech
142. s gain filtering capacity compensation etc can be controlled from a virtual front panel within WinEDR The current implementation of the WinEDR software supports e 2 analog input channels membrane current and voltage e Command voltage output Software installation WinEDR uses Biologic s BLVP500 DLL library supplied with WinEDR to control and acquire data from the VP500 1 Install the National Instruments NIDAQ software supplied with the GPIB interface card and reboot 2 Install the GPIB card into the host computer and reboot 3 Check using the National Instruments Measurement amp Automation Explorer program that the GPIB has been detected and is functioning correctly 4 Run WinEDR and select from the main menu Setup Laboratory Interface to open the Laboratory Interface Setup dialog box Laboratory Interfe l0 x Biologic YP500 Unknown AD Converter Voltage Range 5 Y OK Cancel then select Biologic VP500 from the laboratory interface options list Biologic VP500 I O Panel Connections No I O panel connections are necessary All connections between patch clamp and laboratory interface are internal to the VP500 Getting Started gt Laboratory Interfaces gt Tecella Pico Triton Triton Tecella LLC www tecella com The Tecella Pico Triton and Triton are USB based computer controlled patch clamp digitiser units The Pico is a single channel patch clamp and the Triton 8 channel
143. s gt Detecting Channel Transitions gt Removing Baseline Drift The accuracy of the transition detection process is crucially dependent upon the zero current baseline level remaining stable throughout the whole recording period If any drift is apparent in the signal baseline level it must be removed before initiating the detection process Trends can be removed by obtaining a series of estimates of the average signal baseline level throughout the recording period fitting an equation to the resulting line and subtracting this from the digitised signal To subtract a trend in the baseline level 1 Click the Clear Blocks button to erase any existing baseline trend line M Trend Removal Linear Add Block 0 Clear Blocks Subtract trend Restore Original Cursors Get C 0 m 2 Scroll through the displayed recording and locate a section where the channel is closed for an extended period Select the closed interval for inclusion in the baseline current line using the I l region selection cursors and click the Add Block button to add the average signal level during this period to the trend line 1 0 0 408 0 41 0 412 0 414 0 416 3 Repeat step 2 selecting baseline current periods throughout the recording until an adequate representation of the trend has been obtained 4 Select the Trend Removal function Linear Quadratic Cubic to be fitted to the trend line The Linear f
144. s Stored on File gt Page View gt Page View To view a section of a selected channel within the digitised recording as a series of sequential records on a page select View Page View to open the window Ident Ch cho Im m Start At 0 00s Page Settings Lines page js Line duration 1s l Show Line Times Show Zero Levels Cortical Day 18 300nM 5a3a then 10uM bicuculline 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 Kim One channel of the digitised recording is displayed uncompressed as a sequence of lines within the display window Dragging the scroll bar at the bottom of the display shifts the display forwards and backwards throughout the recording one line at a time The starting time of the displayed sequence of lines within the recording is indicated in the Start At box Note Entering a value into Start At moves the sequence immediately to that time The channel on display if there is more than one in the recording can be changed using the channel selection list The number of lines 1 16 per page can be set using Lines Page entry box and the number of sample points per line using the Points Line box A maximum of 32768 samples can be displayed per page which limits the number of points per line to 32768 No Lines Tick the Show Line Times check box to display the time at which each line occurs within the recording at the beginning of each line Tick the Show Zero Leve
145. s range scaling or labels on a histogram click the Set Axes button to open the dialog box Set Axes Range Labels x X Axis Y Axis C Automatic Automatic Manual Manual Min 0 433 Min fo Max fia Max 557 Tick fo2 ik 1 Scale ae Scale Linear gt Labels Axis an Points pA Y Axis 1 OK Bin Style Empty Colour Cancel J Full borders C You can change the X or Y axis limits by selecting the Manual Axis option and entering new values into the Min Max and Tick spacing boxes An axis can be made Linear or Logarithmic by selecting the option from its Scale list Labels for the X and Y axes can be entered into the Labels boxes The style of rectangle used to plot the histogram bins can be changed using the Bin Style options Select No Fill to display bins as rectangular outlines Solid Fill to fill the bins in with a solid colour and Hatched Fill for bins filled with a diagonal lines You can define the colour used for the solid fill by clicking the Colour box and selecting a colour from the palette The Full Borders check box determines whether the outline is drawn completely around each bar or just where bars do not overlap Analysing Single channel Currents gt Current Amplitudes gt Printing Histograms To print the displayed histogram select File Print To open the dialog box Typeface Page Margins arial Left
146. stage input VModel 100 MOhm model cell IModel 1 MOhm model cell Gain In voltage clamp mode selects the headstage feedback resistor In current clamp mode selects amplifier gain Low pass filter Slider selects cutoff frequency of Bessel low pass filter Compensation Auto page Automatic capacity leak current compensation Click Auto Compensate to automatically compensate for pipette cell capacity and cell leak conduction Click Junct Pot Auto Zero to compensate for the electrode junction potentials setting the input current to zero Click Clear Compensation to set all compensation to zero Options Tick the Use analog leak subtraction option to use the amplifier analog leak current subtraction circuits in the automatic leak current compensation Tick the Use digital leak current subtraction option to use the digital leak current in the automatic leak current compensation Tick the Use digital artefact removal option to use the digital artefact function in the automatic leak current compensation Tick Apply to all channels to apply automatical compensation to all amplifier channels only applies to multi channel amplifiers Compensation coefficient Sets the compensation coefficient factor O optimal compensation gt O under compensation lt 0 over compensation Capacity page Capacity compensation settings in use Can be adjusted by the user or set automatically using Auto Compensate Resistance page Cell le
147. t 6 Click the Fit Curve button Set the initial parameter guesses optional and click the OK button Noise Analysis Review Edit Data Records Amplitude Histogram Variance Analysis Spectral Analysis l0 x Records New Plot 30 Set Axes Records a 25 C Allrecords q Range gt 20 fi 00 130 L o 15 Plot c X Axis 10 Mean DC i 5 Y Axis variance ac 77146 gana 0 10 20 30 Options Subtr t Backgr nd mean DC BA 40 50 Analysi y Vp lx 32 No Parabola Save to Log No of iterations 8 l u 1 062 0 2226 sd pA N c 87 71 25 17 sd Vib 0 787 3 49 sd pA Residual D 2 922 pA Degrees of freedom 27 The best fit single channel current Iu number of channels Nc and background variance Vb unrelated to channel activity along with estimated standard error and residual standard deviation are displayed in the fitting results box Theoretical Background An estimate of the single channel current and the number of channels in the cell or patch can be obtained by plotting current variance vs mean current For a cell containing a population of n ion channels each capable of passing a current and a probability p of being open at any given time the mean current for the whole cell is The variance of the current fluctuations is o 1 n p 1 p These two
148. t detection module can be used to locate individual transient signals e g miniature synaptic signals actions potentials occurring within a continuous recording The frequency of occurrence and inter event time intervals can be analysed within WinEDR Detected signals can also be exported in a WCP format data file for more detailed analysis using WinWCP a program in the Strathclyde Electrophysiology Software series designed for the analysis of transient signals To scan a recording for signals select Analysis Detect Events to open the Event Detection module Ls Event Detection Detect Events ReviewlEdit Events X Y Plot Histogram Average m Mode M Thresholds Detect Events Abort Source Channel cho Im Whole file Range 0 10 0096 Detector C Threshold Rate of Rise C Template Amplitude 1772 82 nAls Set Ampl 4xS D Dead Time 20 ms Rising Edge 1 ms Window Det Criterion The module is split into 5 pages Detect Events Review Edit Events 1 4 1 15 1 2 1 25 1 3 2 e X Y Plot e Histogram e Average The Detect Events page provides the tools for setting up and running an event detection scan of the continuous recording The Review Edit Events page allows the visual inspection of detected events and the manual insertion or deletion of events The X Y Plot page allows event detection times inter event intervals and
149. test LP 1000Hz RD 1 edr My Recent Documents E Desktop Ka A My Documents g My Computer K e My Network Places Smooth muscle John McCarron Steve Ennion Sunil Logantha Testi Trevor Smart TSeries 06242009 1457 065 binarised 514Etm 1250 2250 EDR Ss EDF Files EDA 7 Il Open as read only File name Files of type Select the disk drive and folder from the Look In list A list of available EDR files will be displayed Select one of the file names then click the OK button to open the data file for display and analysis Data Files gt Appending an EDR Data File To append records from an EDR data file on to the end of the currently open data file select File Append To display the dialog box Append File 2 x Look in Data e e e My Recent Documents E Desktop i Rob roelfsema Rocco Pizzarelli B test LP 1000Hz RD 1 edr Smooth muscle John McCarron Steve Ennion Trevor Smart TSeries 06242009 1457 065 binarised 514Etm 1250 2250 EDR noise test 2 EDR noise test EDR test1 EDR test gaps 1 128 edr My Documents gt File name noise test 2 Netra Files of type EDR Files EDR 7 Cancel Places I Open as read only Select the disk drive and folder from the Look In list A list of available WCP files will be displayed Select one of the file names then click the OK button to append the rec
150. the data stored in the file The sampling interval A D Converter Voltage Range and the upper limit of the integer binary A D converter samples can be edited by the user Calibration Table Page File Properties cho The Calibration Table pages shows the name units and scaling factor in Volts unit of each data channel within the file and allows them to be edited by the user 187 i File Properties VER 6 1 CTIME 14 08 2012 11 49 21 DETBA 100 DETPF 0 2 DETAW 512 DETPOSPK T DETBASST T DETBASSUB F DETBASPTS 5 DETBASGAP 20 DETTDECP 90 DETDECFR 0 DETREW 2 The File Header pages shows the contents of the EDR file header It cannot be edited 188 Data Files gt EDR File Structure The EDR data file is designed to store up to 12 channels of 16 bit integer binary records of digitised analogue signals the associated scaling information required to reconstitute actual signal levels EDR files consists of a 2048 byte header block at the beginning of the file which contains a list of ASCII format keywords detailing the number of records in the file record size scaling factors etc It is followed by the data block containing the digitised A D samples Header Block The header block contains the information needed to allow a program to determine the number of channels samples etc in the file It is usually the first block to be read when a file is opened File parameters are stored as ASCII text in the
151. the histogram It cannot be used to compute open channel probability values Analysing Single channel Currents gt Current Amplitudes gt Amplitude Histograms gt Cursor Measurements avg Histogram This option plots the histogram of the average amplitude measurements stored in the cursor measurement list see Cursors Measurments Analysing Single channel Currents gt Current Amplitudes gt Amplitude Histograms gt External File Histogram This option plots the histogram of a list of amplitude measurements ASCII text format one amplitude per line stored in a file Analysing Single channel Currents gt Current Amplitudes gt Fitting Gaussian Functions to Histograms The peaks of the current amplitude histogram can often be fitted by a mixture of one or more gaussian probability density functions The PI y i of bin i with its mid point at current J i is given by 2 os o 2 i l page 20 where w is the histogram bin width Each gaussian component is defined by 3 parameters A the percentage of the total area under the component the mean current and the standard deviation about the mean value WinEDR uses an iterative least squares curve fitting procedure to find the gaussian function which best fits the histogram data Fitting a gaussian curve provides a convenient way of estimating the mean current for a channel state and the percentage of time spent in that state In cases where the peaks are partially overlapping
152. the location of the t 0 cursor Signal levels are measured relative to each channel s horizontal zero level cursor Zero levels The signal zero level for each channel is indicated on the display by a green or red horizontal dotted line It is initially set to the absolute zero voltage level of the analog input The zero level for a channel can be adjusted by moving the mouse over any point on the zero level line the mouse cursor changes to I holding down the left mouse button and dragging the zero level up or down The zero level can also be entered numerically by moving the mouse over the zero level line clicking the right mouse button to open the zero level dialog box ero Level x Ch 0 Im and entering a zero level value in integer A D converter units into the Level box 79 Displaying Records Stored on File gt Compressed View gt Printing Records Compressed View To print the records displayed on the screen select File Print To open the dialog box M Calibration Bars Typeface arial Size 12 pts Line Width 2 pts Show zero levels V Use colour i Show labels Iv Page Margins Left Right 5 0 cem 5 0 cem Cancel _oK Lancet Top Bottom 5 0cm 10 0 cm You can set the size of the plotted record on the printed page by adjusting the size of the page margins The type face used to print text can be selected from the font name list and the type size ente
153. the signal which are in the process of transition between states States with a duration shorter than twice the Exclude Edges settings are not included in the mean state amplitude histogram Analysing Single channel Currents gt Current Amplitudes gt Amplitude Histograms gt Patlak Average Histogram This is an implementation of a method developed by Joseph Patlak Patlak 1988 for improving the resolution of All Points histograms without resorting to transition detection procedures The amplitude histogram is compiled from the running average of a series of n 2 n 256 adjacent sample points To avoid smearing of the distribution by including averages which contained transitions between states the standard deviation of the n point average is also calculated and the measurement excluded if it exceeds a preset threshold A typical value of n would be 16 The variance threshold is normally set to be the variance of a stable open or closed state In such circumstances about 30 of all averages are exclude from the histogram Single channel Current Analysis iol x Amplitude Histograms Transition Detection Edit Channel States Dwell Time Histograms SummaryiSt 4 gt New Histogram Set Axes Abort Histogram Type Patlak Average F Data Channel cn o Im Whole file Range 0 10 0352 Use co C1 No bins 20 Bin width 0 015 pA Lower Lim 1 453 pA Upper Lim 1 547 pA 0 5045
154. tional exponential components can be obtained by computing the F statistic _ SSQ SSQ x n m a SSQ m SSQ and SSQ are the sums of squares of the residual differences between the histogram data and the expected values for two p d f functions a and b where function a contains fewer exponential component than b m and m are the number of free parameters in each function and n is the number of histogram bins SSQ can be calculated for each function from the residual standard deviation SD and degrees of freedom pee values returned as part of the curve fitting results x SD free res SSQ n Large positive values of F indicate that extra exponentials in function b result in a better fit to the data than function a small positive values indicate that the fit is little improved and negative that it is worse The significance probability of the observed F value can be determined from a standard F distribution with m and n m as its degrees of freedom Further details can be found in Horn 1987 To compare the quality of fit of the series of p d f s fitted to a histogram click the F Test button 3 Exponentials to open the F Test box Residual SSQ 28 02 No Parameters 2 Degrees ofFreedom 49 F 30 19 0 03605 p 0 0000 0 9805 A T 91 64 87 37 87 59 Tau 1 0 001298s 0 001184s 0 001188s A 2 22 03 10 83 Tau 2 0 02267 s 0 01869 s A 3 10 7 Tau 3 0 02791 Print The residual sum of s
155. to open WinEDR s module for analysing the random fluctuations in ion channel currents under stationary or semi stationary conditions atx Review Edit Data Records Amplitude Histogram Variance Analysis Spectral Analysis Records passaz o Eo ooo Type Test Rejected a Set Block ees AC Channel cho Im DC Channel cho Im Record Size 1024 Overlap 0 Data Mean DC 11 3 pA variance AC 10 7 pA 2 The module is split into 3 pages each associated with a specific noise analysis operation The Variance Records page provides tools for the partition of the continuous digitised signal into variance records rejection of artefact and classification of records The Variance Analysis page is used to plot the mean variance and other parameters computed from variance records against each other The Spectral Analysis page is used to plot the average power spectrum of the signal Noise analysis is most commonly applied to the whole cell currents activated by the opening of receptor activated ion channels The figure below shows a typical recording Recording begins before the agonist is applied in order to obtain a sample of the background noise the agonist is applied after 10 s and both the mean current and the amplitude of its fluctuations can be seen to increase to a steady state iew Recording po fa t t 17 5 18 7
156. ts list Select the At Random Intervals option to generate PSCs at random intervals or At Fixed Intervals for fixed intervals Enter the rate of occurrence of PSCs in the Frequency box At Fixed Intervals options Enter the quantal release probabililty for each stimulus into the P release box and the number of quanta available for release in the releasable pool the Rel Pool box Set the percentage depression or facilitation during the fixed frequency train of stimuli in the Depression box 0 no depression 100 100 depression during the train 100 100 facilitation and the time constant of depression in the Time Const box To randomly vary the fixed interval frequency enter a non zero value into the Frequency St Dev box To delay the start of the PSC series enter the required delay in the Delay box Enter the standard deviation of the signal background noise in the Random Noise box To apply a slow sine wave baseline drift to the recording enter a non zero value in the Sine Wave Amplitude pk pk box and its frequency in the Frequency box Click the Start Simulation button to start the simulation run Simulations gt lon Channel Noise The ion channel noise simulation generates the random fluctuations in current associated with the open close gating of a population of receptor activated ion channels The model simulates the application of agonist the rise of current during a period of increasing agonist concentration and th
157. ts the default voltage levels ON 5V OFF 0V to be output on the digital output lines Click the Apply button to apply the settings to the outputs Recording Experimental Signals gt Amplifier Gain Setting The amplifier gain current channel gain in voltage clamp mode voltage channel gain in current clamp mode is indicated in the Amplifier Gain Mode box When gain telegraphs are operational these indicate the actual state of the amplifier When telegraph information is not available the current gain setting is entered here by the user See Amplifiers Amplifier Gain Mode 1 Gain 0 0005 WipA VClamp IClamp Update Gain Recording Experimental Signals gt Experiment Identification Comments A line of of text identifying the purpose of the recording can be entered into the Ident box at the top of the recording window J Ident This line is stored in the data file When the Return key is pressed the contents of the line is written to the log file Short tags up to 8 characters can be associated with each record by entering text into the Marker box and clicking the Add button Marker Ai Add r Log File A log file of the operations initiated by the user is updated during the course of recording or analysing an experiment The names of data files created or loaded comments entered stimulus programs used and other events are stored along with the time that the event occurred The lo
158. ude histograms generally have at least two peaks associated with the closed and open states of the ion channels The horizontal distance between the peaks indicates the current passing through a single channel The width of the peaks indicate the amount of background noise degrading the current signal The area under each peak indicates the proportion of time spent in that particular channel state A channel which is predominantly in the closed state will for instance have a large peak at the zero current level and a small one at the single channel current level Channels which have than one open conductance or patches which contain more than one channel will often result in histograms with more than two peaks The height of each histogram bin indicates the percentage of samples falling within the limits of the bin The within each bin and the current at its mid point can be read out using the green readout cursor The mean current and of samples falling within a selected range of amplitudes can be computed by placing the pair of grey I l cursors at the limits of the region of interest 0 9995 0 53811 38 1 11 5 pA Mean 1 01 pA Area 8 73 Six types of current amplitude histogram can be produced All Points All Points in State Mean State Amplitude Patlak Average Cursor Measurements avg External file Analysing Single channel Currents gt Current Amplitudes gt Amplitude Histograms gt All
159. ude in the S c current box Enter the number of active ion channels in the patch in the No Channels box Enter the mean channel open time in the Topen box Enter the mean number of channel opening per burst of openings in the Openings per burst box Enter the mean intra burst closed time in the Tclosed short box and the mean inter burst closed time in the Tclosed long box 10 Enter the standard deviation of the background noise in the Random Noise box 11 Optional To apply a linear trend to the signal baseline level enter a non zero value into the Baseline drift box 12 Click the Start Simulation button to start the simulation run COM Automation Interface gt COM Automation Interface WinEDR implements a COM automation server which allows its recording and seal test functions to be controlled from VBSCRIPT batch files or from applications such as Matlab which supports COM automation The name of the WinEDR automation object is WinEDR AUTO and is opened by the VBSCRIPT command set W CreateObject winedr auto Recording functions Recording can be started stopped data files created opened and the recording trigger mode and stimulus pulse protocols selected The recording methods and properties are listed below Recording Methods amp Properties NewFile filename wcp Method Creates a new data file with the supplied name OpenFile filename wcp Opens a pre existing data file with the supplied name
160. uments oe a My Computer gt _jLineScan 04012009 1234 180 aa Mancina File name Places Save as type WCP Files WCP 7 The record size of the exported file is determined by the size of the Review Edit Events display window Exporting the Gaps Between Events The sections of the digitised recording which do not contain events can also be extracted and saved to another EDR format data file To extract and save the gaps between events 1 Enter the range of events to be exported in the Events box 2 Click the Export Gaps button and select the name of a new EDR data file to hold the data iL xxi Save in O Data z ce EB cell 2 slice 4 Full z series John Mec Damian Cummings winwep 5LineScan 10082008 0831 867 My Recent Daphnia Tony Campbell Liver 2P images David Wokosin Documents fj Metamorph Demo Images Data Chart Michael Ferber Data Dundee Neuron Sandy Harper Desktop Data Miller Lite Origin Files Data Plymouth Plymouth Data As Data Ross Images Rabbit CYS Susan Coker Data T Cells Rat cardiomyocyte Plymouth My Documents Data Tony Richardson Leao Data WinFluor Rob roelfsema DRG data Plymouth Rocco Pizzarelli Helene Widmer July 2010 STREX DHEA I Smooth muscle John McCarron Human Atrial myocyte D Pau Steve Ennion My Network File name Bltest qaps 1 128 edr Places 3 g P Save astype EDR
161. unction represents the trend as a simple straight line Quadratic and Cubic functions allow for curvature in the trend 5 Click the Subtract Trend button to remove the baseline trend from the digitised signal If necessary to restore the original signal click the Restore Original button Analysing Single channel Currents gt Detecting Channel Transitions gt Viewing Editing Channel Open Close States The individual channel open and closed states produced by the transition detection process are displayed on the Edit Channel States page Single channel Current Analysis Amplitude Histograms Transition Detection Edit Channel States Dwell Time Histograms SummanyStability Plots Events 3 911 15 gt State Open Duration 4 ms avg 0 995pA ss i iL eee IAN HA S D 0 0922 pA VY Y YN Wi Edit State Ignore state SetBlock x 2 Jo iy Delete Ignored Average Amplitude Exclude edges 0 1 ms gt Export Events Event List Closed Times C Open Times Save To File m Cursor Measurement Enable Save to List 20 Clear List Save List to File Load List from File 0 068 0 07 0 072 0 074 0 076 01 Cursor spacing 10 width a 12 8 The idealised channel open closed state is superimposed in green on the digitised current signal The time period on display can also be adjusted by increasing or decreasing the Width time value The type o
162. urrent Analysis i 0 x Amplitude Histograms Transition Detection Edit Channel States Dwell Time Histograms Summary Stabilty Plots New Histogram Set Axes Abort Histogram Type 25 mean State Amplitude m Data Whole file Range 1 911 a 15 Use CoC No bins 200 10 Bin width 0 015 pA Lower Lim 1 453 pA Upper Lim 1 547 pA State far 0 5345 0 0 0 2 0 4 0 6 0 8 1 1 2 Mean State Amplitude pA aes evel _ Mean 0 483 pA Area 100 Curve fitting 5 C 1 A Current i ___FitCurve__ None Save to Log To compute the histogram a Select Mean State Amplitude from the Histogram Type list b If more than one signal channel is available select the channel to be used from the Channel list c Select the All option to use all detected channel transition events in the recording or select Range and enter the sub range of events to be included d Select the channel state s Close Open X1 or All to be included in the histogram from the State list e Enter the width of the histogram bin in the Bin Width box f Click the New Histogram button to compile and display the histogram Note The Exclude Edges entry on the Edit Channel State page determines the number of zero or more samples at the beginning and end of each state which are excluded from the average to eliminate the parts of
163. urve fitting results area at the bottom of the display 0 4895 0 0 4 0 2 0 02 04 O06 08 1 1 2 1 4 All Points pA I Mean 0 0956 pA Area 100 FCN yOO Eero anme expe teu oA Mean 1 0 008142 0 0002367 sd pA 2 Gaussians gt s d 1 0 09985 0 0002372 sd pA SavetoLog A 91 16 0 1814 sd Mean 2 1 006 0 002509 sd pA d 2 0 1009 0 002514 sd pA Ao A TTA N 1871 fer z B The residual standard deviation SDres between the histogram data and the fitted curve provides a measure of the quality of the fit the smaller the SDres the better the fit Note The fitting procedure will generally work well when fitting gaussian curves to distributions with single peaks or two reasonably well separated peaks which do not differ by more than an order of magnitude in height In other cases such as overlapping 3 4 peak distributions the large number of free parameters can cause the procedure to fail to converge to a meaningful result In such cases it is sometimes possible to obtain a better fit using a two stage procedure First a single gaussian is fitted to the largest and best defined peak Then the resulting best fit parameters for that peak are used as fixed parameters in a 2 or more gaussian fit to the remaining peak Analysing Single channel Currents gt Current Amplitudes gt Customising Histogram Plots If you want to alter the X or Y axi
164. utton to cancel compensation Command Stimulus Select the voltage clamp command stimulus input path and enable disable low pass filtering of stimulus pulses Getting Started gt Amplifiers gt Molecular Devices Multiclamp 700A B Molecular Devices Multiclamp 700A or 700B patch clamps are controlled via the Multiclamp Commander control panel software supplied with the amplifiers which can be used to set current and voltage channel gain and voltage current clamp mode Each Multiclamp 700 supports two separate amplifiers Channel 1 and Channel 2 which requires two WinEDR amplifier channels to be defined When a Multiclamp 700 is in used both Amplifier 1 and Amplifier 2 should be defined as Axon Multiclamp 700A B CE Input Channels amp Ampl o x input Channeis Amplifiers Amplifier 1 Amplifier 2 Amplifier 3 Amplifier 4 Axon MultiClamp 700B z Input Channels Drimar channal Ch 4 Drimary utm EE Input Channels amp Amplifiers Setup O x Amplifier 1 Amplifier 2 Amplifier 3 Amplifier 4 Axon MultiClamp 700B T Input Channels Primary channel Ch 2 Primary Output Note Ifa second Multiclamp is in use Amplifier 3 and Amplifier 4 should be defined as Axon Multiclamp 700A B A communications link between the Multiclamp Commander control panel and WinEDR is automatically set up after both Multiclamp Commander and WinEDR programs are started allowing WinEDR to determine current and
165. veform analysis procedures commonly applied to such signals WinEDR acts like a multi channel digital tape recorder collecting series of signals and storing them in a data file on magnetic disk Its major features are Recording e 1 16 analogue input channels e Continuous sampling to disc at rates up to 100 kHz e Stimulus generator e Realtime event event frequency resistance capacity and fluorescence ratio measurement Analysis e Single channel transition detection and amplitude dwell time analysis e Event detection and frequency analysis e Noise analysis e Miniature synaptic current ion channel current fluctuation and single channel current simulations Next Introduction gt Conditions of Use The Strathclyde Electrophysiology Software package is a suite of programs for the acquisition and analysis of electrophysiological signals developed by the author at the Strathclyde Institute for Pharmacy amp Biomedical Sciences University of Strathclyde At the discretion of the author the software is supplied free of charge to academic users and others working for non commercial non profit making organisations Commercial organisations may purchase a license to use the software from the University of Strathclyde contact the author for details The author retains copyright and all rights are reserved The user may use the software freely for their own research but should not sell or pass the software on to others without the perm
166. vent enter a non zero number of points in the Gap box Select the Subt Baseline Trend option to subtract any upward or downward trends caused by drifting of the baseline on which the detected signals are superimposed A cubic spline interpolation between signal baseline estimates obtained from the average of 10 samples immediately preceding each event is subtracted from the each event waveform T X Decay Time Enter the required decay time percentage for the signal decay time measurement in the T X box Select the Peak option to measure the decay from the peak of the signal the 50 rise option to measure from the mid point of the signal rising phase or the a0 cursor option to measure from the position of the a0 cursor Detecting amp Analysing Events gt Adding Removing Events Manual Event Editing The currently displayed event can be removed from the event list by clicking the Delete Event button or pressing the F2 key An event can be added to by placing the vertical display cursor over the position of an event and clicking the Insert Event button or pressing the F1 key M Edit Events Insert Fi Delete F2 V Init cursor to detection point Automatic Event Filtering Events can be automatically deleted from the detected event list using a set of filter criteria based upon the event analysis measurements To delete events matching a defined set of criteria 1 Select the waveform measurement to be used as
167. viation of the signal background noise in the Random Noise box 11 Click the Start Simulation button to start the simulation run Simulations gt Single channel Currents The single channel current module simulates the currents associated with the opening and closure of a single or small number of ion channel s Channel gating is modelled using a 3 state model with one open state and two closed C 082C Channel openings occur in bursts with the kinetics of the channels determined by the mean dwell times in each of the 3 states To create a data file containing simulated single channel currents 1 O SN oO Oe Oe Create a new data file to hold the currents by selecting File New and entering the name of a new data file Select Simulations Single channel currents to open the window Single channel current simulation m Recording Duration 10 000 s Im Z Sampling 0 05 ms a aj Interval M Single channel properties 5 c current 1 00 pA 1 5 No channels 1 ae m 10 01 10 02 10 02 10 03 10 03 10 04s Topen 2 0 ms Tclosed short 1 0 ms Recording conditions Tclosed long 20 0 ms Random Noise Baseline drift 0 10 pA 0 00 p s Openings per 4 L burst Enter the duration of the simulated recording in the Duration box Enter the time interval between simulated A D samples in the Sampling Interval box Enter the single channel current amplit

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