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WINWCP User Guide - Spider

1. EE Input Channels amp Amplifiers Setup ici xi 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 WinWCP is automatically set up after both Multiclamp Commander and WinWCP programs are started allowing WinWCP to determine current and voltage channel scaling factors and voltage current clamp mode for each amplifier channel Resetting the Multiclamp WinWCP Communications Link If Multiclamp Commander is stopped and restarted while WinWCP 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 WinWCP 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 is supported Note Contact j dempster strath ac uk for details of the cRIO 9076 hardware and firmware required to
2. Z Each record in the file can be displayed and measurements made of the signal levels within each channel using a movable cursor The displayed record or superimposed groups of records can be printed out Records can be assessed for the presence of interference other artefacts and marked as rejected or assigned with particular record types Each record in the data file is numbered in the sequence that it was recorded Records can be Record selected for display using the selection bar to move back or forward through the file You can jump directly to a record by entering its number into the record number box above the selection lal B gt bar You can also use the Ctrl Plus and Ctrl Minus keys to step forward or backwards through the file To superimpose up to 200 records on the display select View Superimpose Traces to disable automatic display erasure Selecting the option again re enables auto erase Displaying Records Stored on File gt Magnifying the display The magnification of each plot can be expanded to display a selected region of the signals 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 AN amb I 2 479 n The vertical magnification and location of the displayed region within the recording can also be adjusted using the 8068 buttons at the right edge of each plot and t
3. 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 Screw terminal panel Ch 0 68 67 62 signal ground Ch 1 33 67 62 on co S762 Ch 6 25 67 62 Ch 7 57 67 62 Trigger Inputs PFIO TRIG1 NIDAQ MX 11 44 See Note 1 PFI1 TRIG2 NIDAQ 10 44 as joi Note 1 An active high TTL pulse on this input triggers the start a stimulus program which has been set up with the
4. Select Analysis Measure Waveforms to open the automatic waveform measurement window Waveform Measurements Recordings oo x Analysis xv Plot Histogram Summary Tables Type TEST M Rejected mme Analyse Do Analysis Abort Allrecords This record Range 1 18 t 0 t 0 018372 1 11 97 e ALL v Peak Absolute Points Avgd o Rate of rise Forward Diff 25 t 0 t 0 021495 _ 0 11833 100 08666 0 20 s Rise time 10 90 Tx decay time x 1 0 Time 16 16195 Cursors Averad 69 01 mV Get Cursors 0 004474 pAs 0 006714 mV s l Lock Channels Peak a 351 3 pa 97 5 mV 7091 pat2 11 84 mVA2 RiseTime 6 647E 007s 1 483E 006 s Rate of Rise 4 224E008 pAs 0 mWs e The Analysis page is used to set up the parameters of the waveform analysis and initiate the automatic measurement sequence which generates a table of measurements The X Y plot page is used to create X Y graphs of the measurements The Histogram page is used to create frequency histograms of measurement The Summary page presents a summary mean standard deviation etc of the measurements for the series of records analysed The Tables page is used to create tables of results Automatic Waveform Measurement gt Running a Waveform Analysis Waveform Measurements Recordings TA iol x Analysi
5. 10 Scale Linear Scale Linear Labels X Axis fim Peak a nA Y Axis OK Bin Style _oK Empty Colour Cancel Full borders C Axis limits and tick spacing are initially set to default values based upon the min max range of the data You can change the axis limits by entering new values for into Min Max and Tick spacing boxes for the X and Y axes An axis can be made Linear or Logarithmic by selecting the option from its Scale list Labels for the X and Y axes and a title for the plot 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 Printing the histogram To print the displayed histogram select File Print To open the Print dialog box ti ais ipharmsrvisibsh327 Ip1 Setup gt Typeface r Page Margins 4 arial Left 25cm Point Size 12 pts Right 2 65 cm Lines ia 2 5 cm Line Thickness 2 pts Bottom 05cm OK Cancel Click the Print button to plot the
6. 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 channels 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 WinWCP 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
7. 200 In blocks of 200 Type JALL gt Alignment mode 25 No alignment a 2 00m BBSBB 99 98 ms 8068 To create a series of signal averages 1 2 3 4 5 6 Specify the range of records to be averaged by entering the first and last records separated by a in the Range box Enter the number of records to be included in each average in the in blocks of box Note The default settings produce a single average record from all the records in the data file You can restrict averaging to a specific type of record by selecting a type from the Type list Select ALL to use records of any type except rejected records Set the alignment mode Select No alignment if the position of the signals do not vary within the records and alignment is not necessary If alignment is necessary select on positive rise for positive going signals and on negative rise for negative signals Set the alignment search region cursors If records contain stimulus artefacts it may be necessary to restrict the region of the record which is searched for the signal mid point in order to avoid the averages being aligned using the artefacts rather than the true signals The alignment search region is set by moving the two vertical cursors on the display to define the beginning and end of the region containing the signal To create the averages click Do Averages The averaging process now proceeds An additional digitised data f
8. I O Panel Connections Signal input and output connections are made via the BNC sockets on the front of the ITC 16 18 units eor por pee o peme C ee as Ch 4 ADC Input 4 woms 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 WinWCP applications is recording from and controlling a whole cell patch or voltage clamp experiment Two analog channels are normally recorded by WinWCP 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 WinWCP 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 cu
9. 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 WinWCP 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 Channeli E input Channels amp Amplifiers Setup SEE The amplifier membrane current output should be Input Channels Amplifiers 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 sre is a Amplifier 1 Amplifier 2 Amplifier 3 Amplifier 4 V units into the Scale factor box The scaling factor can Input Channels typically be obtained by reading the minimum gain setting k of the rotatable current gain switch found on most Primary channel Im l patch clamp amplifiers e g a minimum gain settings of Scale factor 0 001 VipA Units pA 1lmV pA is equivalent to a scaling factor of 0 001V pA EE larcho gt Secondary Channel Vm Secondary channel Vm The amplifier membrane poten
10. 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 which 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 r
11. 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 voltage 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
12. 0 cursor Signal levels are measured relative to each channel s horizontal zero level cursor oan Cursor measurements can be written to the WinWCP log file by clicking the Save F1 button The Centre Cursor button places the readout cursor to the centre of the displayed region t 20 65 9 998 mV Zero levels The signal zero level for a channel can be defined in either of two ways In From record mode it is computed as the average level from a defined portion of each record In Fixed mode it is fixed at a level defined by the user and does not vary from record to record From record mode Use the from record zero mode when you want to measure transient signals which are Zero aval xi superimposed upon a baseline level which may be varying from record to record To set the zero level from a portion of the signal record itself in from record mode Ch 0 Im 1 Move the mouse pointer over the horizontal zero level cursor of the channel you want toa Zero level modes change The mouse pointer turns into an up down arrow G From Record 2 Slide the mouse pointer horizontally until it overlies the region of the record which is to be At sample defined as the zero level 3 Click the right hand mouse button to open the zero level dialog box No averaged 4 Select the From Record option 5 The zero level is computed for each record from the average of a series of default 20 e ine Laval samples starting at the sample
13. 1 a Res n B 8 4 kaTa Sm 000m GOGG 99 98 ms Results yi Aexp tr Ss A 0 9461 0 01516 sd nA tau 10 17 0 2978 sd ms Ss 0 0009371 0 00474 sd nA Residual D 0 102 nA Degrees of freedom 2006 No of iterations 4 Curve Fitting gt Curve Fit Equations The available curve fitting equations are listed in the table below Straight line yx M x C Exponential general Decaying Exp y 2 Exponential x general y x Al exp aa A2 exp TS C 2 Decaying Exps 3 Exponential y 3 Decaying Exps Ai y x Al exp A2 exp A3 exp ae Taul Tau2 Tau3 EPC x x0 x x0 x 0 5 A l1 er exp yo A TauR P TauD The endplate current rising phase is modelled by an error function and the decay with an exponential function EPC 2 ex i P y x 0 5 en 7 4 e c A e c R Tau Tau The endplate current rising phase is modelled by an error function and the decay by the sum of two exponential function H H K y x A x ET The time course of activation of a voltage activated current with Hodgkin Huxley kinetics H H Na y x A h apf a G inf H inf Def a The time course of a current with voltage dependent activation and inactivation following Hodgkin Huxley kinetics e g sodium current Note It is assumed that initially the activation parameter m 0 and inactivation par
14. Amplifier Gains 1 Ch 0 0 0005 WpA n Ww Recording Experimental Signals gt Recording Modes gt External Trigger When the Ext Triggered mode is selected the recording sweep is synchronised to a trigger pulse applied to the external trigger input of the laboratory interface If the Active High option is selected recording will be triggered by a OV to 5V transition on the trigger input If the Active Low 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 Many kinds of electrophysiological signals are evoked by stimulating the cell or tissue using an electrical stimulator In order to record such signals the recording sweep must be synchronised with the stimulator ideally so that the sweeps starts shortly before the cell is stimulated During a stimulus cycle the stimulator sync pulse is produced first triggering the recording sweep and after a delay settable on the stimulator front panel the stimulus itself t Trigger Level Recording Mode Ext triggered No records i Record duration 3 992 3 No input channels a No samples s05 Sampling interval 0 9746 m v Active High 5V C Active Low 0V Arnplifier Gains 1 Ch 0 0 0005 WPA my 20000 s00 Vin mi Recording Experimental Signals gt Recording Modes gt Detect Events The Detect E
15. Hessian matrix by the curve fitting program and are not true estimates of experimental standard error since they take no account of inter cell or other variability In addition they only provide a lower bound to the estimate of the standard error in parameter value It can be shown by simulation that if the random noise on the experimental signals is correlated then the variability of fitted parameters may be substantially greater than suggested by the computed parameter standard error The error in parameter estimation can be a complex function of the parameter values and the signal noise ratio of the data It is therefore wise to test the curve fitting procedure using simulated waveforms with known parameters set spanning the range of values likely to be observed in the experimental data Are all the parameters meaningful It is also necessary to discriminate between functions which fit the data equally well For instance the question often arises as to whether one two or more exponential functions are needed to fit a signal waveform It is usually obvious from the residual plot when a single exponential does NOT provide a good fit However when a single exponential does fit two or more exponentials will also provide a good fit In such circumstance it is usual to choose the function with the least number of parameters on the principle of parsimony An excess of function parameters also results in the some of the parameters being ill define
16. a level defined by the user and does not vary from record to record From record mode Use the from record zero mode when you want to measure transient signals which are Faso lesa x superimposed upon a baseline level which may be varying from record to record To set the zero level from a portion of the signal record itself in from record mode Ch 0 Im 1 Move the mouse pointer over the horizontal zero level cursor of the channel you want toa Zero level modes change The mouse pointer turns into an up down arrow A ROCA 2 Slide the mouse pointer horizontally until it overlies the region of the record which is to be At sample defined as the zero level 3 Click the right hand mouse button to open the zero level dialog box No averaged 20 4 Select the From Record option _ 5 The zero level is computed for each record from the average of a series of default 20 2 fie Level samples starting at the sample indicated in the At sample box If you want to change the F number of samples averaged to compute the zero level change the value in the No averaged box OK Cancel 6 Click the OK button to use the new zero level Fixed Level mode Use the fixed level zero mode when you want to make measurements relative to a fixed ae T x absolute level To fix the zero level at a constant value to be used for all records in the file 1 Move the mouse pointer over the horizontal zero level cursor of the channel you
17. available files in that type are displayed Select one of the file names then click the OK button to open the ASCII Import dialog box ASCII Import ct Started at 05 03 2010 15 10 57 Time s Ypulse mv 0 500 042 1 5 500 041 3 064 500 036 4 562 500 044 6 062 500 048 Channels N zZ 3 D Units mi No oftitle lines to ignore 2 Column Separator Tab Comma Space lt 3 M Time Data Sample times in 1st column No sample times Setrecord size Time units secs OK Cancel which allows you to view the format of the data to be imported and to specify how it should be imported noa SO AO OF eaa Bas Sc 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 ignore 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 the 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 Sample times in first column option to derive the sampling interval from the times of successive rows Select the units that the time data is expressed in from the Time units list If no sample time data is available select No sample times and enter the sampling in
18. 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 WinWCP Amplifiers 1 and 2 The Axon Multiclamp Commander software must be started up and running before WinWCP 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 Digital gain telegraph connections OOOO fe e e e pe e Next Getting Started gt Amplifiers gt Configuring Amplifier Support in WiInWCP 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
19. 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 WinWCP 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 WinWCP 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 attached to the host computer via a SCSI Small Computer Systems Interface interface card and cable A number of v
20. file Note that the original file is not changed A new WCP format file is created with the same name as the imported file but with the extension WCP The currently supported data file formats are listed in the table below Importable Data File Formats Axon Instruments Axon Instruments ABF Axon Binary File format files produced by the PCLAMP and abf dat AxoScope programs ambridge Electronic Cambridge Electronic Design CFS CED Filing System format files IDesign CFS SCAN Files Data files produced by the Strathclyde Electrophysiology SCAN program an MS DOS SCA based electrophysiology package Igor Binary Files IBW Igor Binary Wave files produced by the IGOR Pro software package IBW Data records exported from the Heka PULSE software Data Files gt Importing from ASCII Text Files To import records from a text file containing tables of numbers in ASCII format select File Import To display the Import File dialog box i x Look in jo Data amp er Er Smooth muscle John McCarron Fe Steve Ennion My Recent Test Documents Trevor Smart E TSeries 06242009 1457 065 04 02 2009 R Desktop E binarised 514Etm 1250 2250 Gy My Documents wr oe My Computer File name My Network Files of type Sx T Open as read only Select the disk drive and folder from the Look In list Then select ASCH Text txt asc from the Files of Type list A list of
21. from and should be placed before the rise of the waveform Rate of Rise Maximum rate of change during rise from baseline level to to Peak Rising Slope Slope of rising edge of signal within the percentage range of Peak defined in Rise Time Range box default 10 90 Slope The slope of a straight line fitted to the region of the recording sweep defined by the cursors C1 C2 or C3 C4 Peak abs Largest absolute value of Peak and Peak Rise time abs Time taken for signal to rise from 10 above pre waveform baseline level to 90 of Peak abs The CO cursor is used indicate baseline level and should be placed before the rise of the waveform Rate of Rise abs Maximum rate of change during rise from baseline level to Peak abs Cursor 1 Signal level at C1 cursor position averaged over Points Avgd points around cursor position Cursor 2 Average signal level at C2 cursor position averaged over Points Avegd points around cursor position Cursor 3 Average signal level at C3 cursor position averaged over Points Avegd points around cursor position Cursor 4 Average signal level at C4 cursor position averaged over Points Avgd points around cursor position Recording Experimental Signals gt Zero levels The signal zero level for a channel can be defined in either of two ways In From record mode it is computed as the average level from a defined portion of each record In Fixed mode it is fixed at
22. graph 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 copy image dialog box Copy Image 600 pixels 500 pixels 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 127 Curve Fitting gt Summary Statistics The Summary page displays a summary report containing the mean values and standard errors of the fitted parameters for the records which have been analysed curve Fi Recordings ToT Fit Curves X Y Plot Histogram Summary Tables Records Variable Mean St Dev St Error min Max inp 1 200 Record 57 88 4093
23. in Stimulus No records 1 o e Protocol mode these are set in the protocol Record duration 3 999 lt No records Sets the number of recording sweeps to be collected No input channels 2 No input channels Sets the number of analog input channels to be No samples 4096 20000 acquired Channels are always acquired in sequence from Ch O Sampling i g interval upwards ie No input channels 1 selects Ch 0 No input 0 3746 m channels 2 selects Ch 0 amp Ch 1 etc Record duration Sets the required duration of the recording sweep Set the duration to a value that is approximately 50 longer than the Mmplifier Gains Ym time course of the signals that you intend to record Vey 0 0005 VipA my No samples Sets the number of samples to be acquired per input channel The minimum is 256 samples per channel and increments are in units of 256 The maximum ranges from 16184 No Channels 1048576 No Channels depending on the laboratory interface in use 800 Sampling Interval The Sampling interval box displays the time between digitised samples acquired from each analog input channel It is determined from R D Sampling Interval O Duration No Samples Entering a value into the Sampling interval box will result in an adjustment of the Record duration It is important to use a sampling interval which is small enough to ensure that a sufficient number of samples are acquired during the most rapidly changing ph
24. indicated in the At sample box If you want to change the T number of samples averaged to compute the zero level change the value in the No i averaged box OK Cancel 6 Click the OK button to use the new zero level Fixed Level mode Use the fixed level zero mode when you want to make measurements relative to a fixed oe aval x absolute level To fix the zero level at a constant value to be used for all records in the file 1 Move the mouse pointer over the horizontal zero level cursor of the channel you want to Ch 1 Vm change The mouse pointer turns into an up down arrow a Zero level modes 2 Hold down the left mouse button and drag the zero level cursor vertically until it is at the From Record desired level At sample N t 3 Click the right mouse button to open the zero level dialog box i T Ae m eats No averaged 4 Select the Fixed option The vertical position of the fixed baseline is indicated in A D converter units in the Level box You can set the zero level by entering a value i 5 Click the OK button to use the new zero level Fixed Level Fixed mode is typically used for the membrane potential measurements in voltage patch clamp studies of voltage activated current Note Entering a value of zero into the Level box sets the 2 lt zero level to the true zero voltage level for the channel Cancel Displaying Records Stored on File gt Copying
25. m lus Proto I sey seas ien E R N cas mead a a ta ea E R a ETE 60 Setting the Recording Sweep Size and Duration ecccceeceseeseseeseseseeseseseescseseeseseescseenseeeeecseeeeeesees 61 Creating Stimulus Protocols Opening the Stimulus Recording Protocol Editor ees sseeecneescneeeseeesesecseeseeecseeaesenseeeens 62 Analog amp Digital Output Channel s ccececcccecscesescsceseseeeescseesescseseecseseecaesesesesenseeeeecseeeseseeenaes 63 R c rding AS CCUIIGS 5 acess aise A A OA ney eer ee ene 64 Adding Stimulus Waveforms to the Protocol ccccceceseeseesseseseeseseseeseseseeseseesceesenseseeecseeeseseeeeaes 66 Global Stim l s Vata eS esens n taal te a tt aed RE APRN ies ol cate ano gs 72 Saving and Loading Stimulus ProtOCOlS ccc cccescssssesscsseseeecseesececsseseseesecseesesecsesseecseeseeecseeeeas 74 Example Protocol S s anaon ran e trea dana tease ienantaotentranmees 75 Default Q tput Set NgS si rn E ae te E TE A E E E a wet ek rah 76 Amplifier Gain Seting inene a E ec nd i E tase re Wigs es EAER Ra 77 Experiment Identification COMMENUS ccccceccescesesseseeseseesesecseeaeseceecseesesecseescseeseeaesecaesaeeecaeeaeeetaeseees 78 SEAMING a Recording savecaesicecutancect este tocies coavecdndctea EEE EA E EE ae a a 80 On IMEANAIYS Ssn e a a E E a Ea E Eaa 81 Zero levelre ate E E EA AE A A ae 84 Displaying Records Stored on File Selecting and ASP AV IETS ce caer setae a ct ch Se rescue aU al a a rt
26. minimum possible update interval for the interface Next Recording Experimental Signals gt Creating Stimulus Protocols gt Global Stimulus Variables The amplitude and duration of waveforms within a stimulus protocol can be defined in the waveform parameter tables using the global stimulus variables G1 G5 instead of fixed values This allows multiple waveform settings within a protocol or related set of protocols to be changed by editing a single value Global variables are stored independently of individual protocols and applied to all protocols referencing them when the protocol is run A waveform parameter can be linked to a global variable by entering the name of one of the five variables G1 G2 G3 G4 G5 into the field in the parameter table Files Open Protocol Save Protocol As Set Protocol Folder Analog Outputs AO No Channels 4 w Aoo p Stimulus type Current p v Holding Level 0 pA Digital Outputs DO No Channels Holding Pattern 0 5V sv ov Toolbox AO Waveforms ne ma M Global Variables Protocol et nm Amplitud Amplitud Duration D A Updal 0 1465 ms The values of the global variables can be set in the table on the Global Variables page Changing the value of the variable in the global variables table changes the waveform parameter in the protocols which reference that variable IELE WRFOL QIOUG Val
27. open the monitor seal test module select from the menu Record Pipette Seal Test Signal Monitor An oscilloscope trace showing the current signal on each input channel is displayed Pipette Seal Test Signal Monitor 10l x No channels 2 Amplifier ja Heka EPC 7 gt Current ch o Im gt Voltage ch 1 vm lamp Mode Vclamp Iclamp Sa Amplifier Gain 0 0005 VipA Send Test Pulse To V AOO M A01 Test Pulse Pulse 1 F3 V Hold 90 mv Amplitude 40 mV C Pulse 2 F4 V Hold 0 mv Amplitude 41 mv Pulse 3 F5 V Hold 0 mv Amplitude 0 mV Pulse width 0 20 40 60 80 100 120 ms 8668 IV Auto scale Voltage Holding 90 3 mV Pulse 40 1 mV Za Amplitude 210 mV Pulse width 110 ms Pipette Cel G Cel Resistance 8353 5 MOhm Save to Log Ga estimate from Peak Exp Amp Sweeps Averaged 4 100 ms Time Reset 01 20 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
28. 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 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 WmnWCP program folder c program files winwcp 4 Run WinWCP 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 Note also that the voltage channel Ch 1 Vm is mapped to ADC Input 3 instead of ADC Input 1 6 Instrutech ITC 16 18
29. records Records can also be classified according to the type of signal that they contain by selecting a type from the record s type list box 1 Piss Eight types of records are currently defined TEST LEAK EVOK MINI FAIL TYP1 TYP2 TYP3 The EVOK MINI and FAIL types are used in the quantal analysis of synaptic currents or potentials see section 15 to indicate whether a record is a nerve evoked spontaneous or a nerve evoked transmission failure event Note Types can be selected quickly using the Ctrl T Ctrl L CtrHE CtrH M Ctrl F keys TEST and LEAK are used in the digital leak subtraction process see section 13 and used to distinguish respectively normal records containing voltage activated currents and records containing leak currents to be scaled and subtracted from the TEST records TYP1 TYP2 and TYP3 are general purpose user defined record types Displaying Records Stored on File 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 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 the start of each record and in brackets relative to the location of the t
30. tate 85 Magnifying Te GISDIAV sseceeisene tociasatscssentityoaateameieenge aula E E E 86 Prinang RecordSr eed ieena na eea eda stones a a e a E E tues teenestaeccannvarcanens 87 Accepting Rejecting amp Classifying RecordS s s ssesssessesoseseseesesosertesoseseseosesesesstseserersesesesesenesessesene 89 Measuring Signal LEV els ee mreneo nnie a a o See E E EEE SSE EAEE T EEE 90 Copying Records to the Windows ClipbOard ccccccseeescsseseseeseseeeseseescseseeecseseeseseeecsesenseeeeeeseeeeeaees 92 Smoothing the Displayed RecordS s sseseseseseesssesesesesesesesesesesesesesesesesesesesisesiseseseseststntststsentsesesesenes 93 Automatic Waveform Measurement Making Waveform Measurements icccce zscstateccctsa cs cavcnsa fice nck ticeaseesscedts cu ssre ual eyeecs ctavseuca thsceanendennateetecesd 94 Running a Waveform Analysis 2 ccsece iscecssscteiietiei idle eateine cic aceinenesia aden aeddneneieunelees 96 Plotting Graphs Of M aSUreMeNts ccecccseceseesesesseseseeseseseseescseeeesesecsesessesesensesesecsesenaeseeecsesesaeeeeetaeeeees 99 Classifying Records Using Waveform Measurement Criteria eee ccseeeeescesetseseteceetensenees 102 FIRING a Curve toia Graphe denan arity eeu eed enn ears 103 Plotting Waveform Measurement HiStOGramMs ccccceeccseseescsseseeeceeseeecseesceeesecsesaesecaeeaeecaeeaeeetaeeeees 105 Fitting Gaussian Curves to HiStOGrams ccccceccesesseseeeeseeseeecseeseeecseeseeecseeses
31. underlying ion channels and other cellular processes which can be extracted by the application of a variety of waveform analysis techniques WinWCP C Data File_002 wcp miol File Edit Yiew Record Setup Analysis Simulations Windows Help iew Recordings iol x r Record Ident FO 20720 i E EE m Marker Time ft 9 19s Type TEST Do my z Group 20 Rejected J 200 Low pass filter P Active lt 3 8068 cO 89 921 my 1000 Cursor Get Cursor Get c0 Cursor Save F1 Im a 5 1000 t 102 4 759 25 p Zero Level Baseline c0 00m BBSB Stopped 20 records in file WinWCP 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 waveform analysis procedures commonly applied to such signals WinWCP acts like a multi channel digital oscilloscope collecting series of signal and storing them in a data file on magnetic disk Its major features are 204 75 ms Recording e 416 analog input channels e 256 8388608 samples per recording sweep e 2 billion records per data file e 2 4 channel stimulus voltage waveform generator 4 8 digital output lines for operating solenoid controlled valves or other experimental devices External trigger input to synchronise recording sweeps with external events e Sp
32. want to Ch 1 Vm change The mouse pointer turns into an up down arrow a Zero level modes 2 Hold down the left mouse button and drag the zero level cursor vertically until it is at the From Record desired level At sample 3 Click the right mouse button to open the zero level dialog box No averaged 4 Select the Fixed option The vertical position of the fixed baseline is indicated in A D converter units in the Level box You can set the zero level by entering a value Fixed Level 5 Click the OK button to use the new zero level ps Fixed mode is typically used for the membrane potential measurements in voltage patch clamp studies of voltage activated current Note Entering a value of zero into the Level box sets the zero level to the true zero voltage level for the channel Cancel Displaying Records Stored on File gt Selecting and displaying records To view signal records stored in a data file select from the menu View Raw records to open the record display module WinWCP C Data Data test wep loj x File Edit View Record Setup Analysis Simulations Windows Help ioj xi Record aeni oo o 9 10 A ge Time Qs Type TEST Group 9 z M Rejected Low pass filter P Active amiss 1 9375 n Cursor Get Cursor Get c0 Cursor Save F1 Zero Level Baseline c0 cursor z c0 t 22 0 mY 00m BBB 43 96 ms
33. 1 200 57 88 4093 1 200 Type ALL Time s 5 57 88 4 093 1 200 AMA 0 01672 0 001182 0 9283 1 022 Variables tau ms 0 2723 0 01926 9 366 10 87 V Record lv Group 0 0003001 0 004494 0 0003178 0 01287 0 01352 iv Time ResSD na 0 1 0 001533 0 0001084 0 0951 0 1036 vA DegF 0 2006 0 0 2006 2006 Iv tau IV Ss V RessSD IV DegF To display the summary of results 1 Select the Summary page by clicking on its page tab 2 Select the variables to be included in the summary by ticking or un ticking the appropriate variable tick box Curve Fitting gt Tables of Fitted Parameters The Tables page is used to display lists of fitted parameters in tabular form Curve it Recordings T Fit Curves XY Plot Histogram Summary Tables Records Recora orun mime 7 200 ees eras ae 1 1 1 0 9975 Type JALL TA 2 2 2 0 9604 Variables j ise 3 3 3 0 9646 a 4 4 4 0 9651 M Time 5 5 2 EE WA 6 6 6 0 9644 4 z 7 7 7 0 9798 v Ss 8 8 8 0 9873 M RessD vee 9 9 9 0 9765 Na a on gt o aa aa wn so wes oe ox on o o 9 9 9 3 wo wo wo Sane aia sis wo N P z t p fre n Clear All Set All wn EN EN wn aga wn oo o wo nm oo a Ss Bad fits 7 17 17 0 9859 F Include 18 18 18 0 9652 Bad data flag o o i o 4 gt 4 0 0 To display tables of measurements 1 Select the Tab
34. 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 Starting a Recording After selecting a recording mode and if necessary configuring the recording sweep size and duration click the Record button to begin recording M Record Record stop Erase Screen V Save to File Incl stim protocol in file name Note make sure that the Save to File box is checked to enable writing to the data file Select the Incl stim protocol in file name option to append the name of sti
35. 12 YOnsamples YInsamples Different laboratory interfaces supported by WinWCP return multichannel 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 The calibrated signal level in the appropriate channel units can be reconstructed using information stored in the file header and the record analysis blocks using Veal ADE 1G max where V the maximum positive limit of the A D converter voltage range from record analysis block ADCmax is maximum A D sample value at V ax header block and YGn is the calibration factor Volts channel units for channel n header block Simulations gt Nerve evoked EPSC Simulation The nerve evoked EPSC module generates a series of nerve evoked excitatory post synaptic currents EPSCs or potentials EPSPs When the nerve is stimulated a random number of transmitter quanta are released from a pool of size n with each quantum having a probability p of release The number of quanta released per stimulus follows a binomial distribution The EPSC waveform can be made to decay following a single or double exponential function Random background noise with a gaussian distribution can be added to the signal EPSPs can also be simulated including the effects of non linear summation of quantal potentials To create a data file containing simulated EPSCs Create a new data file to hold the records by sele
36. 2 5 my Vslope 30 0 m Vslope 15 0 mY L J Use Inactivation Set the simulated EPSC properties Voltage Protocol Enter the voltage clamp holding voltage into the Holding voltage box the number of simulated voltage steps to be created in the No of steps box and the increment in voltage between steps in the Voltage step box Leak Subtraction If leak subtraction records are to be created select Divide by N and enter the P N divide factor Noise Enter the standard deviation of the gaussian background noise to be added to the signals in the Background noise box Conductance Enter the maximum conductance for the voltage activated current being modelled in the Max conductance box Enter the reversal potential for the voltage activated conductance in the Reversal potential box Enter the cell s non voltage dependent leak conductance in the Leak conductance box Enter the access conductance of the patch pipette used to patch clamp the cell in the Pipette conductance box Note that if the pipette access conductance is less than 5X the cell membrane conductance then pipette series resistance artefacts will occur Enter the cell capacity in the Cell capacity box This determines the size of the capacity current artifact at the beginning and end of the voltage step Activation m The Activation m settings group determines the voltage and time dependence of the Hodgkin Huxley activation parameter m Ente
37. Alignment mode NSVTYPR 0 NSVS2P F T Scale average current to peak before subtraction NSVCURO 0 First analysis cursor position NSVCUR1 0 Second analysis cursor position ID Note that it should not be assumed that the keywords will follow any particular order Record Analysis Block The record analysis block containing a series of internal format variables It varies in size between 1024 and 16380 byte depending on the number of channels in the file according to the formula RABsize Int N 1 8 1 4 1 x 1024 chans where Nehans is the number of data channels and Int means round down to the nearest integer The analysis block structure is detailed below Time recorded 4 byte floating point s Sampling interval 4 byte floating point s Max positive limit of 4 byte floating point x Nehans V A D voltage range Values 4 byte floating point Waveform measurement and curve fitting x Int Nehans 1 8 1 data storage array x28 Data block The data block contains the digitised signals stored in the form of 16 bit binary integers Each A D sample takes up 2 bytes of space Its size depends upon the number of data channels in the file RDBsize 2 X Nehans X Nsarples per chan where Nehans is the number of data channels and Nsanpies per chan is the number of A D samples per channel 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 Y
38. Amplitude of the first pulse in the series Amplitude increment Increment to be added to amplitude between records mamin fowm 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 i pee OSOS psn omean o Series of rectangular voltage pulses incrementable interval A train of rectangular voltage pulses at fixed time intervals incrementable between records and of fixed duration 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 Ooo Pe Initial Delay Delay at the holding level before the pulse begins Amplitude Pulse amplitude mamin fpem o Interval between pulses in train Repeat period incr Increment in repeat period between records No repeats Number of pulses in train Series of rectangular voltage pulses incrementable rate A train of rectangular voltage pulses at a fixed frequency incrementable between records and of fixed duration This element can be used to produce a series of stimuli to observe the effect of repeated application of
39. 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 type 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 an
40. Ch 0 Ch 0 we es 2 Time 1 00s Group fj 4000 Samp Int 0 25 ms E a pico ADRange 10V 0 20 40 60 80 100 120 ms Rejected 668 Fix Zero Levels Edit Options 5 An cs X Shift Artefact Removal This record lej TO gt Se paed neoion 5 Range Y Shift saison 1 00 5 00 i 4 88 pico amp Blank opico Y Scale Value Undo All Edits Scaleby 0 Remove Artefact Selecting records for editing Records can be displayed using the Record selection slider bar Select the signal channel to be edited from the Channel list Select the This Record option to apply editing operations to the currently displayed record only All Records to change all records in the data file or select Range and enter a specific range of records Shifting the signal horizontally To shift the signal leftwards or rightwards enter the distance to shifted in time units in the X Shift box and click the Left arrow or Right arrow button to shift the signal Shifting the signal vertically Enter the distance to shifted in the units of the selected signal channel in the Y Shift box and click the Up arrow or Down arrow button to shift the signal Scaling the signal Enter the scaling factor in the Y Seale box and click the Scale By button Note Scaling by 1 inverts the signal Stimulus artefact removal Select the region of the signal record containing the artefact using the region selection cursors The limit
41. Define the range of acceptable values by entering appropriate values into the Upper Limit and Lower Limit entry boxes 3 Define the Action to be taken when a record matches the filter criterion To set the record type classification tick the Type box and select TEST LEAK EVOK MINI FAIL TYP1 TYP2 TYP3 from the list To set the record status tick the Record status option and choose Accepted or Rejected When the Apply button is clicked of all records in the data file which match the criterion set by 2 are set to the type and or status defined in 3 Automatic Waveform Measurement gt Fitting a Curve to a Graph Linear single double exponential functions or Boltzmann functions can be fitted to an X Y graph using non linear least squares curve fitting To fit a curve to the displayed X Y graph 1 Select the type of curve linear exponential two exponential or Boltzmann to be fitted from the fitting equations list Analysi Boltzmann 2 Define the region within the graph to which the curve is to be fitted using the pair of f f vertical analysis region cursors The selected region is indicated by the horizontal bar at the bottom of the display gt 0 42006 38 958 f Note For exponential functions also define the initial starting point of the fitted curve using the x0 cursor 3 Click the Fit Curves button to start the curve fitting process The initial parameter guesses are displayed in the Set F
42. 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 Al 2 2 signal ground Al 3 3 Signal ground mew p o PFIO PFI1 Gnd signal ground PFIO PFI1 and P1 0 MUST be connected together for Stimulus Protocols to be produced and the test pulse to generated in the Seal Test window Digital Stimulus protocols are not supported by these devices Lab PC 1200 Series Cards The input output connections for 50 pin Lab PC and 1200 series boards are tabulated below Lab PC 1200 Cards Analog Inputs Screw terminal panel Ch 0 1 9 signal ground Analog Outputs rl on 0 io 12 11 See Note 1 Ext Sweep Trigger 38 50 See Note 1 Ext Stimulus Trigger PB7 29 50 See Note 2 Digital Synch Input PC6 36 13 See Note 1 Digital Outputs 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 WinWCP waveform generation functions to operate In addition if stimulus protocols containing digital ouputs are required DAC1 amp EXTTRIG must also
43. In Single channel recording 2 ed ed B Sakmann amp E Neher Plenum Press McLachlan E M amp Martin A R 1981 Non linear summation of endplate potentials in the frog and mouse J Physiol 311 307 324 Sigworth F J 1980 The variance of sodium current fluctuations at the node of Ranvier J Physiol 307 97 129 Standen N B Gray P T A amp Whitaker M J eds 1987 The Plymouth Workshop Handbook The Company of Biologists Ltd Cambridge Traynelis S F Silver R A amp Cull Candy S G 1993 Estimated conductance of glutamate receptor channels activated during EPSCs at the cerebellar mossy fibre granule cell synapse Neuron 11 279 289
44. Miniature EPSCs to open the simulation window W Miniature EPSC BME mEPSC No mEPSCs 100 Im 5 Record Duration 100 ms pA Unitary current 1 pA No channels 100 6 St Dev 0 Transmitter decay 100 us 0 00 ms 99 98 ms Recording conditions _ Mon Channel Model Backg Noise 0 0 pA 200000 s 1 10000 s 1 0 0 s 1 Low pass filter 1000 Hz A R Koin gt AR Kopen gt gp K aes 4p On Off Kyysind K tose Kya Drift Max 0 pA 1000 0 s 1 1000 0 s 1 0 0 s 1 Set the simulated mEPSC properties mEPSC Enter the number of simulated mEPSC records to be created in the No mEPSCs box Enter the single channel current amplitude for the post synaptic ion channels in the Unitary current box Enter the average number of ion channels activated when a quantum of transmitter is released in the No channels box and its standard deviation in the St Dev Box Enter the transmitter release decay time constant into the Transmitter decay box Ion Channel Model Enter the rates constants which define the ion channel gating properties Note Models which permit entry into the desensitised state Kaes gt 0 produce mEPSCs with biexponential decays If kaes 0 monoexponential decays result Recording Conditions Enter the standard deviation of recording background noise in the Backg Noise box If low pass filtering is to be applied to the mEPSC selec
45. N 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 gt Default Output Settings to open the Default Output Settings control panel Sar erT ve amp m Analog Output Holding Levels Default File Name gt A00 0 mV V Include Date AO 1 0 mV Prefix Test_ Apply 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 sets the default voltage levels ON 5V OFF 0V to be output on the digital output lines Default File Name Select the Include Date option to include the current date in auto created default file name Enter text into the Prefix field to add prefix text to the file name Click the Apply button to apply the settings to the outputs Recording Experimental Signals gt Amplifier Gain Setting L The amplifier gain current channel gain in voltage clamp mode voltage c
46. Records to the Windows Clipboard The displayed signal record s can be copied to the Windows clipboard in a variety of formats a data table an image a WinWCP data record Copying data values Each signal record consists of an array of digitised sample values A table of data values for the active 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 Copy Image 0B a bit mapped image by selecting cony Taage x Calibration Bars Typeface arial Size 12 pixels Line Width 2 pixels Show zero levels V Edit Copy Image to open the copy image dialog box 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 i the bit map the better the quality of the image Calibration bars zero i f Us
47. Strathclyde Electrophysiology Software WinWCP V5 1 1 Whole Cell Electrophysiology Analysis Program c John Dempster University of Strathclyde 1996 2015 Table of Contents Introduction NAV ICON Ctccastecvccinst eoccck te ocho aaa a iat acces cetasecte ue aceieintue cance siehat EEAS 4 Main Features of WinWCP on 20x eaten Ait Oa ed Ae aed a i BEDI D 5 COMGIFO MS OF SE E E E E E E ee eee eee EAE E 7 Getting Started Hardware Requirements i i st a hh he OG ty a A a YO CT a SE eld 8 FAS tell Vir WAVER eases tesa Aas at ait ct Nace tig Yh ca al tot le Aout tant taal a dle it EN 9 Laboratory Interfaces National Instruments Interface Cards cccccccccssesecsesecscsseseesesseecsseseeseessseeecsecsececsecseseesecsesseeeaeees 10 Axon Instruments Digidata 1200 cosccs ss co cogs aocacdasivaactevaiveasanleieeaduoadectads tatactsdats trace mveunsanateantaaaees 15 Cambridge Electronic Design 1401 Series ceccccceccscsseseseeeeseseeseseescseseeecsesensesesecseneeseeeeeaeetees 18 Axon Instruments Digidata 1320 s s c08 wide conlagmaatiaGinadecions onn a 21 Molecular Devices Digidata 1440A ccceccccescssesesesseseseescseseeseseecsesensesesenseeesecsesenseseeeeseeeeseseeetas 23 Molecular Devices Digidata 1550 1550A cccceeesssseeseeseseseeseseescseseesesesenseeeeecaesenseeeeecseseeseeeneeaes 26 Istutech Oj Ges 6 18 ran a ERENT Mee RoR Tre E POAN UO T Rar tine tn Ener AER TE ener RES 28 Biologie VP SOO acu actecesestescaeistartd sce ie
48. a stimulus at a high rate It can also be used to produce a train of pre conditioning stimuli for a subsequent test waveform remem Dentin Pie E 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 generated 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 OOOO i e O OS Digitised analog waveform A digitised analog waveform loaded from an external data file es eee Initial Delay Delay at the holding level before the pulse begins 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 chara
49. ad 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 interleave the records from this file with those of the open the data file Note you can only interleave files which have compatible records with the same number of channels and samples per channel Data Files gt Importing Other Data File Formats To import records from a non WCP data file select File Import To display the Import File dialog box import riie axl Lookin E Data J e ex Fe I 4level E ecg iS spikes01 abf A 09903000 abf emg01 abf E synaps01 abf EAE E 09903001 abf E hlvchco1 ral tails patua 99903002 abF ei hlvchco2 fi totalcot E 09903003 abF e kchancO1 ed ttxco1 E 11622000 abf Ed minis01 abf E vMTestA abf Desktop avi muscle E vMTestB abf El be3hifO1 printerspeedtestO1 abf Ei VMTestC abf gt E burst rampchan fro calcicO1 shakrcO1 My Documents E cavi singles demoicO1 sodiucOl wr E demoftx sodiuc02 S My Computer on ea i Netra Files of type axon Files DAT ABF x Cancel Places T 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
50. agging 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 o Pees S O Initial Delay Delay at the holding level before the pulse begins Amplitude Pulse amplitude Duration Pulse duration 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 sensitivity 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 Ooo Pe Initial Delay Delay at the holding level before the pulse begins Amplitude
51. ain setting is entered here by the user The amplifier voltage current clamp mode is indicated by the VClamp IClamp 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 Recording modes In general recording sweep s must be synchronised with the start of the signals under study to ensure that the signal is captured within the record and always appears in the same place The trigger mode determines how this synchronisation takes place There are 4 recording modes e Free Run e External Trigger e Event Detector e Stimulus Protocol You must select a recording mode appropriate to the type of signal to be recorded and the configuration of your recording system Recording Experimental Signals gt Recording Modes gt Free Run The Free Run trigger mode is used for unsynchronised recording ruuuu i M Recording Mode Recording sweeps start immediately after the Record button is m pressed and continue until the required number of records have been ts collected No recors ah Choose the free run mode for simple tests of the laboratory Record duration 3 992 5 interface and for signals where synchronisation is not possible or i l No input channels required p 2 No samples 4096 20000 Sampling interval 0 9746 m 800 m
52. alculated from N stimuli QC log failures 3 Binomial analysis The transmitter release process can often be modelled as a pool of n quanta available for release with each quantum having a probability p of being released when the nerve is stimulated If both evoked and spontaneous signals are available it is possible to calculate estimates for n and p on the assumption that the number of quanta released per stimulus follows a binomial distribution Avg Peak reat n Avg Peak reat Avg Peak nit Var Peak rea 4 Avg Pedk sca n Avg Peak nini 5 Correction for non linear summation of potentials Unlike currents recorded under voltage clamp conditions synaptic potentials do not summate linearly Therefore the size of the synaptic potential is not directly proportional to the number of quanta released However given certain assumptions it is possible to correct for the effects of non linear summation using the eqn Peak Peak rea 1 f Peak crea i V V 6 where Peakevoked is the measured peak amplitude of the evoked synaptic potential Vm is the cell resting potential Vr is the reversal potential for the post synaptic ion channels and f is a correction factor for the effects of the cell membrane time constant on synaptic potential amplitude A discussion on non linear summation and its correction can be found in McLachlan amp Martin 1981 Quantal Analysis of Transmitter Release g
53. ameter h 1 Curve Fitting gt Classifying Records Using Fitted Parameter Criteria The Filter Records option on the X Y Plot page Filter records I Filter can be used to automatically categorise records as particular types see 8 6 or rejected from analysis based upon best fit equation parameters To classify the records in a data file 1 Click the Filter Records button to open the Filter Records dialog box Filter Records xX M Match criteria Result on match C All Records Set record status ES Accepted Variable Rejected Record m Set record type v Ch Ch0Im fe El lt Upper limit boo Apply gt Lower limit _Cancel el 2 Define the record Match criteria a Click the Variable radio button and select the measurement variable to be used as the record matching criterion from the variable list b Select the input channel for the variable c Define the range of acceptable values by entering appropriate values into the Upper Limit and Lower Limit entry boxes 3 Define the Action to be taken when a record matches the filter criterion To set the record type classification tick the Type box and select TEST LEAK EVOK MINI FAIL TYP1 TYP2 TYP3 from the list To set the record status tick the Record status option and choose Accepted or Rejected When the Apply button is clicked of all records in the data file which match
54. an also be exported in a number of data file formats To export data files select File Export To open the Export File dialog box Export File x Files E Data Data Eddie control1 1 10 wcp Output Format Axon ABF V1 6 m Channels V im JV Ch 1 Output Format Select the data format of the file to be exported to from the list of options Record Range Select the All Records option to export all records in the currently open WCP data file or select Range and enter the range of records to be exported Channels Tick the analogue signal channels to be exported Selecting files for export The currently open data file is selected for export To select more files click Select Files to open the Select Files to Export dialog box hold down the Ctrl key and click on the files to be selected then click Open when complete The list of files selected for export are displayed in the Files box Note A list of files to be exported can also be pasted into this box Recor MM san atcha cn ss i x Wh i 5 C Ral Lookin ji Eddie e 0ce i Date modified controli 1 10 08 06 201509 12 WinWCP Data Files Chann E controli 05 06 2015 16 25 WinWCP Data Files IV im IV Ch J OK Onn Cc gt File name control1 wcp controll 1 10 wep gt Open Files of type Files wcp 7 Cancel When the export settings are complete click the OK button to create the export f
55. ance is defined in the Conductance 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 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 Parameter Incrementing Conductance model parameters can be incremented at the end of a stimulus protocol by selecting the At End of Protocol option or after a number of records by selecting the After No of Records option and entering the required number of records in the associated box Increments both positive and negative can be applied to the maximum conductance Gmax half maximal voltages of the activation m and inactivation h voltage sensitivity curves and activation and inactivation time constants by entering non zero step sizes into the increm
56. and releasing the mouse button The vertical magnification can also be adjusted 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 chann
57. ard errors for the records which have been analysed Waveform Measurements Recordings z Oj x Analysis XY Plot Histogram Summary Tables Records Mean St Dev St Error Min Max 1 200 Record 100 5 57 88 4 093 1 200 Type JALL Group 100 5 57 88 4 093 1 200 Time s 100 5 57 88 4 093 1 200 Variables Average nA 0 09783 0 001519 0 0001074 0 09411 0 1013 Area nA ms 9 398 0146 0 01032 9 041 9 731 7 Peak a nA 1 103 0 04602 0 003254 0 9912 1 26 Saar Variance nA 2 0 04584 0 000635 4 479E 005 0 04402 0 04738 Time Rise Time ms 0 4676 0 2011 0 01422 0 2147 1 501 Average Rate of Rise nAims 20 99 1 853 01311 17 76 27 68 Area Latency ms 10 44 0 04029 0 002849 103 10 52 Paaka T 1 ms 0 1321 0 2994 0 02117 0 04924 2 428 LaPamS T 90 ms 9 958 1 274 0 09009 547 1316 Rise Time Rate of Rise Interval s 0 995 0 07071 0 005 0 1 IV Latency Baseline nA 0 002124 0 02272 0 001606 0 06882 0 07315 IV T1 Conductance mS D D 0 0 0 M T 90 IV Interval V Baseline V Conductance To display the summary of results 1 Select the Summary page by clicking on its page tab 2 Select the channel to be summarised from the Channel list 3 Select the variables to be included in the summary by ticking or un ticking the appropriate variable tick box Automatic Waveform Measurement gt Tables of Measurements The Tables page is used to display lists of measurements in tabular form Wavefo
58. ases of the signals being recorded For most types of signal 2048 samples channel and a record duration approximately 50 longer than the signal time course provide satisfactory results However note that some signals such as cardiac ventricular action potentials can combine long time courses 200 300 ms with very rapid rising phases 1 2 ms In such circumstances 8192 or more samples channel might be required to accurately represent the rising phase To avoid aliasing artefacts the analog signals should be low pass filtered to remove frequency components greater than half of the sampling rate i e reciprocal of the sampling interval 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 module Protocol C Program Files Borland WDelphi WinWCP 32 vprot Steps 10 100mV 100ms xml Files Waveform New Protocol 109 9 m Open Protocol goog Save Protocol 100 m7 Save Protocol As A01 Omy Set Protocol Folder DOO Analog Outputs A0 Se 7 No Channels 2 Recording Stim Aoojao1 Toolbox gt Protocol AO 0 Wav AO Waveforms AO 0 iS i Stimulus type r4 i l LyM voltage im Holding Level 0 08 mV m IN pacamo fro Holding Pattern 5V C D A Update OV e 0 2734
59. ate the variance vs mean plot s Non stationary Yariance Analysis NN cy oh Variance pA 2 oh O records 100 8 To fit a parabola to the curve a Select Parabola from the curve fitting list to fit the equation y x Vb Iu x x Nc where Ju is the single channel current Nc is the number of ion channels and Vb is the background variance b Define the region of the variance vs mean plot to be fitted to using the analysis region cursors f f 46 828 20 621 n 20 40 60 an c Click the Fit Curve button set optional the initial parameter guesses and click the OK button to fit the curve 141 The best fitting parabola is superimposed in red on the variance vs mean plot The estimated single channel current Ju and number of channels Nc are displayed in the curve fitting results box along with an estimate of the background variance Vb 30 nN O Variance pA 2 ho o Mm o mn i GQ Vb lx 32 No Lu 0 8247 0 005228 sd pa Parabola E 194 441 196 sd V b 0 8044 0 06931 sd pA Residual S D 1 516 pA Degrees of freedom 1803 No of iterations 5 142 Quantal Analysis of Transmitter Release gt Introduction The quantal analysis module can be used to estimate the quantal content of neuromuscular nerve evoked endplate currents or potentials and other forms of synaptic signal using the either the direct method variance method and met
60. 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 WinWCP 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 WinWCP 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 switch set to NRSE the WinWCP A D Input Mode should be set at Single Ended NRSE Getting Started gt Laboratory Int
61. be significantly greater than the variance of the random background noise on the signal The distribution of the variance as displayed in the residuals plot is also important Deviations should be randomly distributed over the fitted region of the record If the fitted line is consistently higher than the data points in some parts and lower in others this indicates that the signal is not well represented by the chosen equation Are the parameters well defined The aim of most curve fitting exercises is to obtain a well defined set of function parameters e g exponential time constants which characterise the part of the signal being fitted The standard errors of the best fit parameters provide an indication of this A large standard error indicates that a parameter is poorly defined by the data and can be varied significantly with little effect on the goodness of fit Such a situation typically arises when there is insufficient information contained in the signal waveform to adequately define the function For instance in the case of exponential functions the waveform data must be of sufficient duration to contain at least one time constant of the exponential function before an accurate estimate can be obtained Similarly it proves difficult to accurately estimate the time constants of multiple exponential functions when they differ by less than a factor of 5 It is worth noting that the parameter standard errors discussed above are computed from the
62. button to open the Set Fitting Parameters dialog box Set Fitting Parameters 7 xj Parameters Fixed Fixed A 0 883 nA A z tau 12 2 ms a r Ss 0 0662 na D E Parameter Initialisation f Automatic C Manual Initialise If you want to keep a parameter fixed at a set value enter the value into the appropriate parameter box and click the Fixed check box 10 When all curve fitting settings have been made click the Do Fit button to initiate the curve fitting sequence The iterative curve fitting process now begins The SSQMIN routine iterates through a variety of trial parameter sets until no more improvement can be obtained The number of iterations are displayed as fitting progresses The best fit is usually found within 10 20 iterations Fitting is aborted if the process has not converged to a suitable answer within 100 iterations and can also be aborted by clicking the Abort button For each record fitted the best fit curve is indicated by a red curve superimposed on the blue signal trace A residuals trace is shown below indicating the difference between the fitted curve and the data The parameters of the best fitting equation are shown in the Results table along with the parameter standard error the residual standard deviation between the fitted and data points statistical degrees of freedom in the fit and the number of iterations it took to find the best fit a Im f n Oo 4
63. by selecting Copy Image x Typeface Image Size Edit Arial Width Copy Image 600 pixels Point Size 12 pts Height to open the Copy Image dialog box The dimensions of the bit map which will Lines 500 pixels hold the image can be set using the width and height image size boxes The ae 2 bts more pixels used in the bit map the better the quality of the image When the image parameters have been set click the OK button to copy the image to the clipboard Automatic Waveform Measurement gt Classifying Records Using Waveform Measurement Criteria The Filter Records option on the X Y Plot page M Filter records _ Filter can be used to automatically categorise records as particular types HE oi rejected from analysis based upon waveform measurements To classify the records in a data file 1 Click the Filter Records button to open the Filter Records dialog box Filter Records Ea M Match criteria Result on match C All Records Set record status E Accepted Variable Rejected Record r i record type v fv lt Upper limit b oo i n gt Lower limit Cancel C 2 Define the record Match criteria a Click the Variable radio button and select the measurement variable to be used as the record matching criterion from the variable list b Select the input channel for the variable c
64. cords Scaling Subtract Range 1 32 From group From voltage fcap Ileak C From whole file C Fixed fo C Ileal M Save Leak records To digitally subtract the leak current in the records 1 Select the channel containing the cell membrane potential from the Voltage channel list 2 Select the channel containing the cell membrane current from the Current channel list 3 Select the source of the Leak records f records are grouped into LEAK TEST record pairs select From Group This option should be used for P N protocols If the LEAK records are not grouped with the TEST records select From whole file Whole file mode is used when one or more records at the beginning or end of a data file are to be used as LEAK records Note that these records will have to be manually classified as LEAK 4 Set the current Scaling mode Select From Voltage to use the ratio between the TEST and LEAK voltage pulses as the current scaling factor default Select Fixed if you wish to use the fixed scaling factor entered in the box below Fixed mode is required when the record does not contain a voltage channel 5 To subtract both capacity current and ionic leak current select the I cap I leak option To subtract ionic leak current only select I leak The I leak option produces subtracted records with lower background noise but does not remove capacity current transients 6 If you have selected the From Voltage scaling
65. cters Le To Vo Ti Vi sate 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 Points 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 th 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 es Initial Delay Delay at the holding level before the pulse begins State 0 0V 1 5V Digital output state during pulse 0 OV 1 5V Family of digital pulse varying in duration pene A digital pulse whose duration is automatically incremented between recording sweeps Ooo O Pee Initial Delay Delay at the holding level before the pulse begins State 0 0V 1 5V Digital output state during pulse 0 OV 1 5V Duration increment Increment in duration between records Train of digital pulses
66. cting File New and entering the name of a new data file Select Simulations Nerve evoked EPSCs to open the simulation window Nerve evoked EPSC Simulation PTT ED Start Simulation Abort 8 _ No Records 200 Record Duration 100 ms Display Range 10 n amp Im f Currents C Potentials Restingthalding potential 90 0 mv Transmitter Release Release pool n 10 10 Release prob p 0 200 Quantal event properties 68068 0 00 ms 99 98 ms gt Amplitude __ M Rising phase __ _ r Decay phase _ Background 0 1 nA variability noise R M S Set the simulated EPSC properties No Records Enter the number of simulated EPSCs to be created Current Potentials Select the Currents or Potentials option to determine whether simulated currents or potentials are to be created If you have selected potentials enter the resting potential of the cell in the Resting holding potential box Transmitter Release Enter the number of quanta available for release in the Release pool n box and the probability of a quantum being release when the nerve is stimulated in the Release prob p box Quantal event properties Amplitude Set the average peak amplitude of the miniature quantal current in the Peak box and its standard deviation in the St Dev box Enter the standard deviation of the background noi
67. d a an a R RTE ener LHS 30 Te lla Pico Triton Triton 8 cess lose og Ba eo ag Gs RNR RNG ceca Be cas 31 Heka Patch Clamps amp Interfaces ate ae el ha eh ee Pla ed aad oF ib el aes 32 Amplifiers Patch Voltage claMp Amplifiers cc ccscsccscssesessesesscssscsscecesscsseseescseescsessesscsessessceseessseaesseaeess 34 Sighal Connections ablescxs shescee cs ade hake rane ae aa enw 35 Configuring Amplifier Support in WINWCP 0 cccceecceseeecseeseeecseeseeeceeeseeeeseeseseesecaeeaesecaeeaeeeneeeens 37 C hannel Calibration ab lesstecsice cee earache ce al oes ae cal at ak ara at R dik alan iio cul ieee 40 CED LOO 2 A UPS secectsk oles nn e e ae a e a a e 41 Tecella Patch Clamp Amplifier Control Panel cccccccscsseseseeseseseeseseseeseseeecseseeseeeeecseneeseseeeeaes 43 EPC 9 10 Patch Clamp Amplifier wicca 50a oveie eohe wenden asi adiadeonauiadwiae 45 Molecular Devices Multiclamp 7OOA B cccccccsseceessesesseseseseeseseeecseseesesesenseseeecseenseeeeecseeeaeseeetaes 47 DCLAMP Dynamic Clam Perecesen senan a R a A a uae 48 Recording Experimental Signals Monitoring Input Signals amp Patch Pipette Seal TeSt eee eseeecneesceeeecseesesecseeaeecsseaeeenaeeeens 51 Recording SIG Mall Scorch ccssereceutentsncosessurts e eeen Ea EEE a ear E Ea wien 54 Recording Modes FeS RUM ee ace Reso an a a a a a A tinct A a E A A 57 Darena DAN 01 asec AE E A E E E S E Sines 58 BISS E a E E A E E E E Ace Rec E E E EA Reece 59 S
68. d 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 print setup 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 88 Displaying Records Stored on File gt Accepting Rejecting amp Classifying Records Rejecting flawed records Digitised records cannot all be assumed to be perfect Consequently the visual inspection of records and the elimination of flawed records is an important part of the analysis process If automated waveform measurement procedures are to be applied a mechanism is required for excluding flawed records from the analysis Checking a record s rejected box marks a record as being flawed Rejected records are excluded from automatic waveform curve fitting signal average or leak subtraction calculations Note Pressing the CtrHR key combination is a quick way of toggling the rejected check box on and off T Rejected Classifying
69. d 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 button 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 WinWCP 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 Drimaru channal Ch 1 Drimaru Outnuts
70. d with standard errors values often larger than the parameter values themselves A more detailed discussion of the above issues can be found in Dempster 1992 and 2001 Signal Averaging gt Introduction Many electrophysiological signals have poor signal noise ratios making it difficult to obtain accurate measurements from individual records However if a signal can be made to occur repeatedly digital signal averaging techniques can recover the signal waveform from the background noise The signal average of a series of records is generated by computing the average of each corresponding sample within the records For a set of N records consisting of samples yi i 1 n the average record consists of n samples and is given by 1 N Avg 7 gt The location of signals within the record sometimes varies from record to record due to imperfections in the detection of spontaneous signals or fluctuations in stimulus latency In such circumstances averaging corresponding sample points within the record would result in a distorted signal average This problem can be avoided by aligning the signals by the mid points of their rising phases before averaging Signal Averaging gt Creating Signal Averages Select Analysis Signal Averaging to open the signal averaging window Signal Averager ia 15 x M Average Record 200 200 Ki E 2 Type mini Rejected Do Averages Im _ g nA Abort Range 1
71. data 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 WinWCP 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 WinWCP 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 http www ced co uk upu shtml and run it to install the CED1401 SYS device driver and 1401 commands 3 Ensur
72. e Append File dialog box Append File i x Look in Test gt t c EJ My Recent Documents E Desktop e 080603c ITO activation dep_001 My Documents im dd Netra Files of type WEP Files WCP Cancel Places T 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 records 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 and samples per channel Data Files gt Interleaving Records from a Data File Two WCP data files can be merged by alternately interleaving records from one file with the other This feature can be useful for leak subtraction when leak records have been acquired in another file To interleave records from a WCP data file with records of the currently open data file select File Interleave Data File To display the Interleave File dialog box Interleave File 21x Look in L Test J acm a 080603c ITO activation Y dep_001 My Recent test_001 Documents test 002 Q Shows the files Folders program shortcuts and other items on the desktop Desktop at My Documents wa BE My Computer ra File name Open i S EEE Files of type WCP Files WCP 7 Cancel Places T Open as re
73. e Format cceeccceescesceseseeseecseesesecseeseeecsessceeeseceeeeesecaeeaeeecaeeaeeeeaeesens 161 WGP File SHUCtURexc cGrecy banc a E SRNR Bene Ge a ERT OTS 163 Simulations Nerve evoked EPSC SimUulation ccccccssssesessescsesseseesseesesececsceeseseceeseaesesenseaesecaeaeeaeseeecaeesaeeesenaeaeees 166 Voltage activated Current Simulation ccececceeccssesescseesesseecseseeseseeecseseeseseseeseseeecseenseeeeecseeeaeseeeeaes 168 MiniatGreEPSE SiniU lati OM scaise stew oes syiaseiisecas A E ene a 171 COM Automation Interface COM Automation Interface i i n ied ave A RN idaeas ina Cadac aed ae ERAEN 173 References P E EEN E E E E E NEA EE E EAT E E O E E 175 Strathclyde Electrophysiology Software WinWCP V5 1 1 Whole Cell Electrophysiology Analysis Program c John Dempster University of Strathclyde 1996 2015 Introduction gt Main Features of WinWCP WinWCP is a data acquisition and analysis program for handling signals from whole cell electrophysiological experiments whole cell patch clamp experiments single and two microelectrode voltage clamp studies 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
74. e a relationship between t and J t fal n 3 o t i I m The single channel current i and number of channels n can thus be calculated by fitting the above parabolic function to a plot of t vs I t during a current transient where p t is changing I t can be computed as the average current of a series of transient current records repeated M times all evoked by the same stimulus S M 4 1 t The variance t at each sample point t can similarly be computed from M y 7 0 o t M 1 s The method was developed by Sigworth 1981 for voltage activated Na currents It has also been used to study the fluctuations during the rapidly desensitising currents induced by high concentrations of acetylcholine Dilger amp Brett 1990 With modification it can also been applied to synaptic currents The basic non stationary variance approach assumes that the only source of variance arises from the fluctuations of the ion channels that carry the current However synaptic current amplitude can fluctuate due to both ion channels and quantal size content variation Traynelis et al 1993 found a way round this problem by scaling the amplitude of the average current to the peak amplitude of each signal before the subtraction in Eqn 5 thus compensating for the quantal variation This approach does have limitations and it is worth reading De Koninck amp Mody 1994 if considering using the scaling app
75. e colour E levels and text font size and line thickness can be set in the same Show labels Vv way as for a printed image Image size When the image parameters have been set click the Width OK OK GRA 1000 pixels Height button to copy the image to the clipboard 1000 pixels Displaying Records Stored on File gt Smoothing the Displayed Records A digital low pass filter algorithm can be used to smooth the displayed signal To enable the low pass filter tick the filter Active option and enter a cut off frequency into the Cut off Frequency box m Low pass filter IV Active Cutoff Frequency 1000 Hz The low pass filter set in the display module also acts upon the records in waveform measurement curve fitting and other modules Note that the digitised record on the data file is preserved filtering takes place when the record is read from the file Automatic Waveform Measurement gt Making Waveform Measurements The automatic waveform measurement module provides a means of automatically making series of standard measurements on the digitised signals Ten basic amplitude and duration measurements can be made on each channel and stored with each record The results for each record are displayed on screen In addition sets of measurement variables can be plotted against each other or compiled into histograms A summary report showing mean value and standard errors for the measurement sets can also be produced
76. e module is split 5 functional sections pages Curve Fitting X Y Plot Histogram Summary Table accessed by clicking on the page tab The Fit Curve page is used to select the region of the signal and equation to be fitted and initiate the curve fitting process The X Y plot page is used to create graphs of the best fit equation parameters for the series of records analysed The Histogram page is used to create frequency histograms of of the best fit equation parameters for the series of records analysed The Summary page presents a summary mean standard deviation etc of the of the best fit equation parameters for the series of records analysed The Tables page is used to create tables of results Fitting Curves to Waveforms To fit a mathematical function to a selected region of a signal waveform 1 2 3 4 5 6 7 8 9 Select the Curve Fit page by clicking on its page tab Select the equation to be fitted to the record s Select the channel containing the signal waveform to which the curve is to be fitted from the Channel list Define the range of records to be analysed by selecting All Records to analyse all records or This Record to analyses only the currently displayed record or Range and enter a range of records Select the type of records to be measured by selecting an option from the Type list Select ALL to measure records of any type except rejected records Select Manual or Aut
77. e 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 WinWCP 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 WinWCP s Seal Test option and recording with stimulus pulse protocols Note 2 A TTL pulse on the Ext Sweep Trigger input triggers the start of a recording sweep when Ext Trigger sweep trigger mode has been selec
78. ecting it from the X Axis variable and channel lists 3 Define the variable to be plotted on the Y axis by selecting it from Y Axis variable and channel lists 4 Click the New Plot button to plot the graph Conductance plots Y Ads When Conductance is selected as the Y Axis plot variable the conductance G can be computed Conductance from a pair of recording channels containing current and voltage using the formula Current from G Ix V Viev average Ch where J is the peak or average measurement from the Current from channel V is the peak or Snom average measurement from the Voltage from channel and V is the current reversal potential Yotage tom entered into the Reversal Pot box The units in which the conductance is plotted S mS uS nS or Bin od pS can be selected from the Units list Customising the graph If you want to alter the X or Y axis range scaling or labels click the Set Axes button to open the Set Axes Range Labels dialog box Axis limits and tick spacing are initially set to default values based upon the range of the data You can change the axis limits by entering new values for into Min Max and Tick spacing boxes for the X and Y axes 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 A type face can be selected for the plot from the Font list and its size defined in the Po
79. ed by splitting up the range of possible values into sets of adjacent bins counting the number of individual measurements falling within each bin then plotting the bins as rectangular bars whose height indicates the number of measurements and position on the X axis indicates the range of values in the bin Waveform Measurements Recordings JF oj x Analysis xY Plot Histogram summary Tables r Records 50 1 200 Type ALL 40 New Histogram Set Axes 20 r Histogram 4 Peak a 10 Channel 0 cho Im 0 5 0 7 0 9 No bins 50 Im Peak a nA Rangen Lower Limit Analysi 0 5 Upper limit None ft 5 fis Tal 1 3 pa J Percentage M Cumulative To plot a histogram 1 Select the Histogram page by clicking on its page tab 2 Select the waveform variable from which the histogram is to be generated from the variable and channel list boxes 3 Enter the number of histogram bins in the No of bins box max 1024 4 Enter the range of variable values which are to be included in the histogram from the lower limit in Lower box to the upper limit in the Upper box 5 If you want the histogram bar height expressed as a percentage of the total number of records tick the Percentage option 6 If you want a cumulative histogram tick the Cumulative option 7 Click the New Histogram button to compile and plot the histogram For example the hist
80. ee 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 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 WinWCP 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
81. eeseceesaesecaeeaeeecaeeaeeeeaeeeens 108 Summary Stati stts ieia on i Gain vast ach its BG a OMA le BGO 110 Tabl s f M as rementSr nuiir nteni iae A E E AEE O Ea EEEE E TEE 111 Curve Fitting TYRE CA Niece a E A A E EEE E TE ET E E E E A E 113 Fitting Curves Signal WaveformS s ssensssssesesoseesesesesresesessesestsreststssesesestssesestsrenesessssesesrenesesssrenesest 114 C rve Fit EqUatONS ener r E E A E E A a A 118 Classifying Records Using Fitted Parameter Criteria ees sesesesesesessessssssssrsssrsresesrsrsrssestsrsreresrereens 120 Plotting Graphs of Best Fit Equation ParameterS cccccecccsseseescseeseeeceeesceeesecseeesecseeaeeecaeeaeeesaeeeees 121 Plotting Histograms of Best Fit Equation Parameters ssssssssssesssesesrssesestsrsssntstsrneesesesesnesrsrsresese 124 Summary SHAN SUCS eae a eons tena A a E aa A 128 Tables of Fitted PatameterS dnaceani annen a E E E E E E R ested 129 Assessing Curve Fit Quality saccicci coscaut sve cenrcorucehost exces prercetnumaaucdopti R EAE TEn osare Eians 130 Signal Averaging Introdu OMecndan e e E a A a ee es 131 Creating Signal Averages sesesesssesesssssesssserererereeereseressssseseseseseseseseseseseseseseststststsesesesesesestststseseseseses 132 Digital Leak Current Subtraction Introductio ereenn e a a e a aa a eaaa tones 134 Stimulus Protocols for Leak Subtraction e esesseseesessessssestesesseseetestssestesessessssestestssestesessssestesess
82. els Cell holding voltage and test pulses You can control the holding voltage 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 _ pube 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 0 G 7 pulse C _ I pulse e I V pulse ulse G where Io is the initial current at the start of the capacity transient and is the ex
83. emoving 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 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 A
84. ential for the conductance Gmax is the maximal conductance Taum the activation m time constant Tauh the inactivation h time constant and p the power to which the activation parameter is raised m V and h V are the steady state values of the activation and inactivation parameters respectively defined by Boltzmann functions m V and h V are bell shaped functions To create a data file containing simulated voltage activated currents Create a new data file to hold the records by selecting File New and entering the name of a new data file Select Simulations Voltage activated currents to open the simulation window Yoltage activated Currents Simulation yea Start Simulation 6 Apon Voltage protocol g Si Holding voltage 90 0 mY voltage step 10 0 mY 6 No of steps 16 150 m Leak subtraction None Ch 1 C DividebyN 4 mv gt Noise 200 Background Ona noise R M S3 0 00 ms i Conductance Activation m Inactivation h n Smax X MP xh Y Vrey mM Minti Minti hD explttauty h hinti hinti hD explttaui h Gmax 20nS T mint e MA hinf v4 MiTeC Y1 2 slope hinta tA texpCCV V4 2 slope G 1n8 leak Vaio 1 0 mY Vaso 45 0 mi series Vslope 11 0 m slope 11 5 m P 1 TE Tmint Tima min EXPEC Y1 slope TE Tmin Tmax min EXPEL Y4 2 slope Tmi liens X CH 30 pF min 1 5 ms min 14 0 ms Tmax 3 5 ms Tmax 14 0 ms W439 0 0 mY Vaio 5
85. ents table NOTE It can take up to 45 seconds to completely update the voltage sensitivity of the conductance model and during that period the conductance model will be in an intermediate state Records acquired within the first 45 seconds after a model change should be discounted Updates to Gmax are faster and are complete within 5 seconds 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 Graphs The Graphs page shows the steady state activation and inactivation voltage and the activation and inactivation time constant voltage curves for the current set of model parameters The data points in these curves can be copied to the Windows clipboard by clicking Copy to Clipboard DCLAMP Dynamic Clamp Control Panel Steady state Membrane Pot mV Blue Activation Red Inactivation 50 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
86. er 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 WinWCP 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 port and turn it on 4 Run WinWCP and select from the main menu Setup gt Laboratory Interface to open t
87. erfaces 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 WinWCP 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 WinWCP 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 WinWCP 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 WinWCP 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 WinWCP Digidata 1200 Drivers Install Digidata 1200 driver Win NT 2000 XP Note WinWCP does not use the standard Axon Instruments Digidata 1200 device driver 3 Reboot the computer 4 Run WinWCP and select from the main menu Setup gt Laboratory Interface to open the Laboratory Interface Se
88. ersions 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 WinWCP 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 WinWCP 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 pulse on this input triggers the start a stimulus program which has been set up with the External Stimulus Trigg
89. essene 135 S btracting Leak Currents eein einne a a A aed R 136 Non stationary Noise Analysis Introductions araen A E T E A di etna yest eae E aa 138 Analysing Synaptic Current Fluctuations cc eccecesecsseseseeseseeeseseeecseseesescseescseecsesenseeeeecseeesesenesaes 139 Quantal Analysis of Transmitter Release Injo kelo oLa n a yz 55 cerca tena va na eeepc PES E PEE EE E EE E E E 143 Analysing Quantal Content of Synaptic Currents sessessssessssseeresesesrssesesrsssstststsretenrsrsesnentsrnenest 145 Synaptic Current Driving Function Analysis Computing a Driving Function sic xedscctaseetacbscestltsvecsacep cess E RE EE ERE EE EE EAE 147 Signal Record Editor Editing Digitised Signal RecordS vicreninentie edd eee Qn tnasinicentiiead aden tawinnes 150 Data Files Opening A WCP Data File xccach ware caccccisca cavacetica tive n E voyonsade Oocancy E a a r ai iioa 152 Adding Records from a Data Fllets cic cscseiceasdeeseetinniaiitiessascinedinasaaslin nsviendadicinaeianesiecinelses 153 Interleaving Records from a Data File cccececcecesescesesceeseseeecseseesesesecseseecsesensesesecseenseeeseeseeeeesees 154 Importing Other Data File Formats vers cs ivedseasssev she seeeeaicieaese ee iene epee ei oavdix iene cee eos 155 ImMpering trom ASGLE Text Files Aseria dae at antennae 157 Importing from Raw Binary Data Files evccessetscerescancncnces ecoats seeaicatenntads Coaeae eee cease Jos ee 159 Exporting to Other Data Fil
90. h record select the Keep parameters fixed option The basic deconvolution process computes a driving function which represents the rate of change of post synaptic conductance induced by the release of transmitter It is also possible to reconvolve this driving function with a different post synaptic current decay function to generate the waveform of the synaptic current that would have existed under these new conditions If you wish to create a simulated current select the Reconvolute waveform option and enter the new time constant in the Reconvolution column Click the Do Transforms button to begin the deconvolution process Driving functions are created and stored in a driving function data file DFN extension and can be viewed by selecting Driving Functions from the View menu to display them in the record display module The a driving function b computed from a simulated endplate current a is shown below ze 2 0 uS ms 1 0ms Signal Record Editor gt Editing Digitised Signal Records The record editing windows can be used to make modifications to the digitised signal records stored on file The position of signals can be shifted vertically or horizontally within the records inverted or scaled in amplitude Regions of the record containing stimulus or other artefacts can also be blanked out Select Analysis Signal Editor to open the Edit Record window Edit Record Record 1 5 4 Ch
91. hannel Amplifier Gains gain in current clamp mode is indicated in the Amplifier Gains box When gain 1 Ch 0 0 002 vipa 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 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 log 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 WinWCP program directory Select File Inspect Log File to display the experimental log Log File C Program Files Borland Delphi WinWCP 32 06 07 201
92. he Laboratory Interface Setup dialog box O x Molecular Devices Digidata 1440 Unknown lt Laboratory Inter AD Converter Voltage Range 5 Y OK Cancel then select Molecular Devices Digidata 1440 from the laboratory interface list Analog Input Offset Calibrations The offset calibration values for the Digidata 1440 analogue input channels can be modified by creating a file called Digidata 1440 adc offsets txt in the settings folder U users Public Public Documents SESLabI0O containing a list of offsets for each channel The file should contain a list of 16 offset values in mV to be subtracted from each channel signal e g 3 5 4 0 1 0 1 0 A file Digidata 1440 calibration factors txt containing the analogue input and output offset and gain correction factors in current use in the Digidata 1440 is also created in the settings folder when WinWCP is started 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 a __ eo Analog Out 3 a ae 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 25 Getting Started gt Laboratory Interfaces gt Molecular Devices Digidata 1550 1550A Molecular Devices Corporation www molecularde
93. he data is determined from the sum of the squared differences S between a set of n data points y i i n and the theoretical curve f i computed at the same sample time points i e 2 Y v 20 The best fit is found by repeatedly computing S at each iteration adjusting the equation parameters using a strategy to minimise S The best fit parameters are the ones which yield a minimum value of S WinWCP uses a modified Levenberg Marquardt least squares minimisation algorithm the SSQMIN routine developed by Kenneth Brown at the University of Cincinnatti A more detailed discussion of curve fitting algorithms can be found in Chapter 6 of Dempster 1993 Curve Fitting gt Fitting Curves Signal Waveforms Select Analysis Curve Fitting to open the curve fitting window Curve Fit Recordings Fit Curves xv Plot Histogram Summary Tables Record 200 200 Type mini M Rejected Analyse Do Fit Abort Exponential 7 Records C All records This Record C Range 1 200 Channel cho Im 7 Type jaLL v Set Parameters Data Cursors Manual Automatic Jon Decay Limits 5 0 95 0 Get Cursors t 0 33063 7S 00m BBS Results yit Aexp tt Ss A 0 9225 0 0107 sd nA tau 10 02 0 2492 sd ms Ss 0 0003448 0 004462 sd nA Residual D 0 1017 nA Degrees of freedom 2062 No of iterations 4 Th
94. he horizontal magnification and location by the 6668 buttons at the bottom edge of the plot Individual channels can be added removed from the display by clicking the a 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 Printing Records To print the records displayed on the screen select File Print To open the print record dialog box M Calibration Bars 4 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 cm 5 0 em _oK Eaha Top Bottom 5 0 cm 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 entered 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 indicate
95. he table to a text file tab separated columns click the Save to File button to open the Save Table dialog box and enter a file name Printing Tables To print out a copy of a summary report or table of results on the printer select File Print Copying Tables to the Clipboard To copy the report or table to the Windows clipboard select Edit Copy Data Curve Fitting gt Introduction A mathematical model consists of a general equation representing the time course of the signal or part of the signal under study For instance the decay of many signals e g synaptic currents can be represented by an exponential function f t Aexp T 5 where A is the amplitude of the signal and T is the decay time constant Expressed as above the equation is quite general applying to any signal depending on the values of the parameters A and T In order to determine whether the equation actually does provide a good model of the signal decay it is necessary to find the parameter values which provides the best match or fit of the theoretical curve to an actual signal This can be done using the process know as iterative curve fitting Starting with initial guesses for the parameters the theoretical curve is compared with the experimental data points the parameters are adjusted to try to improve the fit and the process is repeated until no more improvement can be obtained The quality or goodness of the fit between theoretical curve and t
96. hod of failures In circumstances where both evoked and spontaneous miniature events are available transmitter release parameters n number of available quanta and p probability of release is calculated using binomial analysis Quantal content direct method If the data file contains both evoked and miniature signals the direct method of calculating quantal content can be used Avg Peak OC irec 9 oe Avg Peak nini This is the most accurate method for calculating quantal content Quantal content variance method It is not always possible to record the miniature synaptic signals which represent single quanta In such circumstances it may still be possible to calculate quantal content from the variability of the evoked signal Avg Peak sea Var Peak Var Background evoked 2 This method is dependent upon the assumption that the number of quanta released follows a Poisson distribution This will only be the case when the probability of release is very low i e p lt 0 1 Since large errors can result if this condition is not satisfied results using the variance method should be treated with caution Quantal Content failures method If the quantal release probability is very low a nerve stimulus may occasionally release no quanta at all resulting in intermittent failures to evoked post synaptic signals Again using the assumption of a Poisson distribution controlling release the quantal content can be c
97. ice 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 WinWCP and select from the main 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 Trigge
98. icg Dies AT Files Waveform AO wee DO Oem _ _ Set Protocol Folder JY Recording Stimulus Global Variables Global Variables G1 50 G2 10 G3 G4 G5 Note that the units of the global variables applied to waveform duration parameters are seconds rather than msecs as in the protocols and for amplitude parameters the units of the stimulus type 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 30mY 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 Iram
99. igital 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 leak 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 EPC 9 10 Patch Clamp Amplifier The amplifier gain compensation and current voltage clamp mode of Tecella 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
100. ile format version number CTIME 19 05 2010 15 15 59 010 Creation date time RTIME 19 05 2010 15 15 60 000 Date time of start of recording RTIMESECS 2000 00 Time of start of recording in seconds relative to last system boot NC 2 No of channels per record NR 50 No of records in the file NBH 2 No of 512 byte sectors in file header block NBA 1 No of 512 byte sectors in a record analysis block NBD 4 No of 512 byte sectors in a record data block AD 5 0000 A D converter input voltage range V ADCMAX 2047 Maximum A D sample value NP 512 No of A D samples per channel DT 1600 A D sampling interval s NZ 10 No of samples averaged to calculate a zero level TU ms Time units ID Cell 1 Experiment identification line Channel scaling factors YNO Im Channel 0 name YUO nA Channel 0 units YGO 167E 04 Channel 0 gain factor mV units YZO 1997 Channel 0 zero level A D bits YOO 0 Channel 0 offset into sample group in data block YRO 2 YN1 Vm Channel 1 name YUL mV Channel 1 units YG1 10 0 Channel 1 gain factor mV bit YZ1 2048 Channel 1 zero level A D bits YO1 Channel 1 offset into sample group in data block YR1 0 Waveform measurement settings TXPERC 0 T x decay time settings PKPAVG 1 No of points to average before after for peak measurement Non stationary variance settings NSVCHAN 0 Signal channel to be analysed SVALIGN 0
101. ile is created containing the average record s with the same name as the original data file but with a AVG file extension rather than WCP On completion of averaging the record display module is opened to show the average records You can switch the display back and forth between the averages file and the raw data file by selecting View Averaged Records To view the averages and View Raw Records To view the original digitised signal records Digital Leak Current Subtraction gt Introduction Ionic currents recorded using the voltage clamp technique are usually composed of a variety of components mediated by different ionic channels e g Na K Ca Cletc In order to study a particular current in detail it is usually necessary to eliminate all the other currents from the signal This is often done using pharmacological agents such as TTX to block Na currents TEA to block K currents etc However even when such blocking agents are used there often still remains some residual current in addition to the one under study This current is known as the leak current It usually displays linear time independent properties In some circumstances the leak current is very small and can be ignored However in others it can be as large as the currents under study complicating the analysis of the signal waveforms unless it is removed Although the leak current cannot be removed pharmacological its linear properties permit a digital
102. ile s The currently supported export file formats are listed in the table below Export Data File Formats Axon Instruments Axon Instruments ABF Axon Binary File V1 8 format files abf ambridge Electronic Cambridge Electronic Design CFS CED Filing System format files Design CFS ASCII Text Tab delimited columns of ASCII text txt CP Strathclyde Electrophysiology Software WinWCP data file format wep IEDR Strathclyde Electrophysiology Software WinEDR data file format edr Igor Binary Files IBW Igor Binary Wave files produced by the IGOR Pro software package IBW Matlab Files MAT format array data files readable by Matlab MAT Data Files gt WCP File Structure This appendix provides a detailed specification of the internal structure of the WinWCP data file The WCP data file is designed to store digitised 16 bit integer binary records of analog signals the associated scaling information required to reconstitute actual signal levels validation information entered by the user and measurements generated by WinWCP analysis modules A WCP file can contain up to 2 separate records each record containing up to 128 channels each channel containing a multiple of 256 samples up to a total of 1048576 for the whole record Note All records within a file must be the same size i e same number of channels and samples channel Three kinds of data blocks can exist within the file The header block co
103. implement DCLAMP Select Setup gt D CLAMP Dynamic Clamp to open the dynamic clamp control panel DCLAMP Dynamic Clamp Control Panel Conductance Parameter Incrementing Off Reversal Potential Vrev 49 5 my Off C Add C At End of Protocol Conductance C Subtract _ After No of Records Current Command Scale Factor 1E 009 AN 1 Enable Inhibit Input A12 I Activation Parameter m Inactivation Parameter h Steady state AA A Steady state Minf 1 1 exp V Vi5 Veip hinti Y 1 1 exp V Vy Vsin Vin 14 6 mV Vsip 4 5 mV Viz 28 3 mV Vslp 4 4m Time constant Time constant fast tau V taumn taumx tally exP V Vpk Vsip 4 tau V t umn t t umx taumnlexp V Vpi Vsip TaUmp 2 4ms TaUm x 0 59 ms TaUmn 2 9E004 ms TaUm x 7 17 ms Vok 21 04 mV Vslp 25 mV Vpk 0 99 mV Velp 1868 mV Power factor i Time constant slow tau V tau taupy taumnlexp V Vpk Ysip DCLAMP COM Port comi Tatmn 66 6 ms Ta mx 810 ms Update Dynamic Clamp Vpk 29 6mv 6 mV Vsip 5sm mV Load Settings Save Settings Fast Fraction 9 6 DCLAMP 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 Conductance Gmax The maximum conductance nS of the simulated conduct
104. incrementable interval A train of digital pulses at fixed intervals incrementable between records and of fixed duration This element can be used to apply a rapid train of stimuli to a cell OOo o e O Train of digital pulses incrementable rate A train of digital pulses at a fixed frequency incrementable between records and of fixed duration This element can be used to apply a rapid train of stimuli to a cell OOOO o pe O OS mon omean O S Empty digital element 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 D A converter update interval to be used to produce the analog stimulus waveforms within the protocol D24 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
105. int Size box The graph can be plotted as a line unconnected markers or both by ticking the Lines and or Markers tick boxes Labels X Axis Record Y Axis imPeakfajnA Trace OK IV Lines Cancel V Markers Chi Ch 1 vm Reversal Pot 0 my Units ns m Set Axes Range Labels x 0 9 Tick Ro Scale X Axis Y Axis Automatic Automatic C Manual Manual Min Min Max Max 0 200 Tick 50 Scale Linear Printing the graph To print the displayed graph select Typeface i Arial File Print Point Size 12 pts To open the Print dialog box You can set the size of the graph on the page adjusting the Left Right Top and Bottom page margin settings Click the OK Lines Line Thickness 2 pts Page Margins _ Left 50cm Right 50cm Top 50cm Bottom 10 0cm button to plot the graph Copying the graph data points to the Windows clipboard Cancel The numerical values of the X Y data points which generate the graph can be copied to the clipboard by selecting Edit Copy Data The data is placed on the clipboard as a table of X Y data pairs in tab text format allowing the data to be copied into programs such as spreadsheets and graph plotting packages using an Edit Paste command Copying an image of the graph to the Windows clipboard An image of the graph on display can be copied to the clipboard
106. irectly 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 signal 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 b
107. is region The variance of the signal within the analysis region Rise Time The time taken for the signal to rise from lo hi 10 90 default of peak time units The maximum rate of change during the rising phase of the signal Latency The time delay between the zero time cursor and the point at which the signal has risen to 10 of peak T X The time taken for the signal to fall from its peak value to X set by user of peak T 90 The time taken for the signal to fall from its peak value to 10 of peak Baseline Signal level computed from the zero level measurement region but measured relative to true zero levels of input channel Automatic Waveform Measurement gt Plotting Graphs of Measurements The X Y Plot page can be used to create graphs of the measurements obtained from the analysis run Any measurement variable from any channel can be plotted against any other as a Y vs X graph Waveform Measurements Recordings ioj xj Analysis XY Plot Histogram Summary Tables Records 1 18 50 Type ALL New Plot Set Axes 7 x Axis Average x Ch cnt vin 7 Y Axis Average cn fcn o Im 7 10 Im Average pA 50 50 aaa a N Vin Average mi Analysis Fit Curve Y m Filter records Filter To plot a graph 1 Select the X Y Plot page by clicking on its page tab 2 Define the variable to be plotted on the X axis by sel
108. itting Parameters dialog box Set Fitting Parameters JES r Parameters Fixed Fixed Yamp 454p E Ilo O E O E mp em T E Ymi 404pa D E m Parameter initialisation Automatic C Manual Initialise p If you want to keep a parameter fixed i e not changed by curve fitting process tick its Fixed box You can also change the initial parameter guesses if they appear to be unrealistic Click the OK button to fit the curve The best fitting curve is superimposed on the X Y graph in red and the best fit equation parameters are displayed in the Curve Fitting table along with the parameter standard error the residual standard deviation between the fitted and data points statistical degrees of freedom in the fit and the number of iterations it took to find the best fit h7 Waveform Measurements Recordings ft a w faz g lt x p i 50 o0 38 58 f Vin Average m aii yG Yamp 1 exp Gexi dep Yn Yamp 46 01 0 8065 sd pA Mie 21 39 0 585 sd mY Msp 9 404 0 5064 sd m Yinn 4 166 i 0 4958 sd pA Residual D 0 8261 pA Degrees of freedom 8 No of iterations 5 104 Automatic Waveform Measurement gt Plotting Waveform Measurement Histograms The Histogram page can be used to create frequency histograms of waveform measurements representing the frequency of occurrence of different values within the total set of measurements It is compil
109. l bar at the bottom of the display 1 13 2 0 9 1 1 1 3 Im Peakfa n 3 Click the Fit Curves button to start the curve fitting process The initial parameter guesses are displayed in the Set Fitting Parameters dialog box Set Fitting Parameters x Parameters Fixed Fixed Mean Tizma I a sd oomen a Peak ar a ___Se S a Parameter Initialisation ok Automatic Cancer Manual jnitialise If you want to keep a parameter fixed i e not changed by curve fitting process tick its Fixed box You can also change the initial parameter guesses if they appear to be unrealistic Click the OK button to fit the curve The best fitting curve is superimposed on the X Y graph in red and the best fit equation parameters are displayed in the Curve Fitting table along with the parameter standard error the residual standard deviation between the fitted and data points statistical degrees of freedom in the fit and the number of iterations it took to find the best fit 50 40 30 20 10 ta 1 1 1 3 0 5 0 7 0 9 1 5 Im Peak a n e ER CUE yoo ain expt beno Mean 1 116 0 002946 sd nA G Y Gaussian s d 0 03958 0 002989 sd nA a 189 9 11 41 sd Residual D 3 675 Degrees of freedom 12 No of iterations 8 Automatic Waveform Measurement gt Summary Statistics The Summary page displays a summary report containing the mean values and stand
110. le exponential mepsc Micro1401 Digtest wep Simulated two exponential mepsc w x NS Variance Test wep test 1 wep File name Save as type WCP Files WCP 7 Cancel Select the disk and folder into which the file is to be placed using the Save In list box WinWCP data files have the extension extension wcp After a data file has been created select Record Record to disk to open the Record to disk window Record to Disk Record Record Stop Erase Screen V Save to File I Incl stim protocol in file name Single List Protocol Steps 10 100m Y 100ms Set Stimulus Protocol Folder m Amplifier Gain Mode 1 Ch 0 0 0005 WpA VClamp IClamp Recording Mode Stimulus protocol On line Analysis Window Open Close Time Marker Oh Om 10s Reset Add Ident 15000 20000 120 0 20 40 60 80 100 8666 Rec 6 Stim Steps 10 100m 100ms Step 7 10 Repeat 1 1 Fix Zero Levels The display area of the screen acts like a digital oscilloscope showing traces of the signals as they are recorded 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 gain settings of the amplifier When telegraph information is not available the current g
111. les page by clicking on its tab 2 Click the Clear Table button to erase the existing table 3 Tf results for failed curve fits are to be included in the table tick the Include Bad Fits option 4 Select the variables to be added to the table by selecting a channel ticking the variables to be added then clicking the Add Variables button Saving Tables to File To copy the table to a text file tab separated columns click the Save to File button to open the Save Table dialog box and enter a file name Printing Tables To print out a copy of a summary report or table of results on the printer select File Print Copying Tables to the Clipboard To copy the report or table to the Windows clipboard select Edit Copy Data Curve Fitting gt Assessing Curve Fit Quality 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 curve before placing too much reliance on the parameters Does the chosen function provide a good fit to the data One assessment of the goodness of fit is to compare the variance of the residual differences between the best fit function and the data with the background variance of the signal If the function provides a poor fit to the data the residual variance will
112. lipboard An image of the graph on display can be copied to the clipboard by selecting Edit Copy Image to open the Copy Image dialog box Copy Image x Typeface Image Size arial Width 600 ixels Height p Hnes 500 ikels Line Thickness 2 pts a Cancer Eh AAt 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 When the image parameters have been set click the OK button to copy the image to the clipboard Curve Fitting gt Plotting Histograms of Best Fit Equation Parameters The Histogram page can be used to create frequency histograms of waveform measurements representing the frequency of occurrence of different values within the total set of measurements It is compiled by splitting up the range of possible values into sets of adjacent bins counting the number of individual measurements falling within each bin then plotting the bins as rectangular bars whose height indicates the number of measurements and position on the X axis indicates the range of values in the bin Curve Fit Recordings a Fit Curves X Y Plot Histogram Summary Tables Records 20 1 200 Type ALL 15 co New Bistogrann Set Axes Histogram 10 tau No ofbins 50 5 Range Lower E Upper 12 0 10 53 4 I Percentage g 9 6 10 2 10 8 11 4 F Cumula
113. log box 7 Raw Binary Import pS iol xj m File Description Channels File header size bytes 612 Ch Name Units bit Units No Input Channels 2 Oo Im 0 1 pA No Samples Channels 512 o BAU 0 1 mY Sample Format C Float Integer No of bytes sample F Max value 2047 Sampling interval 1000ms msecs C secs mins OO OK Cancel Specify the format of the data in the file to be imported 7 File header size Enter the size of the file header in bytes If there is no file header enter 0 No of Input Channel Enter the number of analog input channels in the file No of Samples Channel Enter the number of A D samples per channel in the record Sample Format Select the numerical format of the sample data Float for 4 byte floating point numbers or Integer If Integer has been selected enter the size of the integer number bytes in No of bytes sample and the upper limit of the numerical data in Max Value Sampling interval Enter the time interval between adjacent samples within each analog channel Select the units of the time interval from the Time units list Channels Enter the name and measurement units for each channel and the scaling factor to convert from integer value to the measurement units Click the OK button to import the data when the import settings are complete Data Files gt Exporting to Other Data File Formats WCP data files c
114. mode use the Vret and Vno display cursors to define the measurement points on the voltage trace used to compute the voltage scaling Vuoid is placed over the holding voltage level and Vrest is placed over the mid point of the test voltage An average of 20 samples around each measurement point is used to compute the voltage levels VHold YTest VHold VYTest 7 Select Do subtraction To initiate the leak subtraction process For each group of records the LEAK and TEST records are averaged scaled and subtracted using See Introduction Each group is condensed down to one leak subtracted record that is stored in a SUB file with the same name as the data file These records can then be displayed and analysed using the View Records Waveform analysis and Curve fitting modules by selecting View Leak subtracted Non stationary Noise Analysis gt Introduction The non stationary noise module analyses the random fluctuations in the decay of ion channel currents providing an estimate of single channel current and total number of channels in the fluctuating population For a cell containing a population of n ion channels each capable of passing a current i the mean whole cell current Z t is I_ 0 i n pl where p t is the probability of a channel being open at time t The variance t of the current fluctuations at time 1 about this mean is o t i n plt 1 ptt 5 These two equations can be combined to provid
115. ms Up to 4 voltage waveform output channels AOO0 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 and the holding level the level relative to which voltage current steps are applied in the Holding Level box Analog Outputs A0 No Channels 2 A00 aot Stimulus type voltage imi Holding Level 0 08 mV Digital Outputs DO Select the number of digital output channels to be used in the digital outputs No Channels list Set the holding pattern the default digital output state between digital stimulus pulses in the Holding Pattern check boxes 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 m Digital Outputs DO No Channels Holding pattern 0 SV C OV ie Next Recording Experimental Signals gt Creating S
116. mulus protocol being applied to the file name When this option is selected a new data file is created every time a series of recording sweeps are initiated by pressing the Record button or when a linked stimulus protocol is run If you want to stop recording before the selected number of records has been acquired click the Stop button Recording Experimental Signals gt On line Analysis The on line analysis window allows a series of measurements signal level at cursor peak amplitude 10 90 rise time and maximum rate of rise to be made on the waveform recorded during each sweep A maximum of 10 measurements can be plotted 8088 8088 M Measurements Peak he Chan Im v Cursors 12 Polarity Positive heal Add to Plot For Stim Protocol v Cir Measurements Clear Plots Copy to Clipboard M Zero Level A Peak Im 1 2 pA io xi 200 150 100 50 50 100 150 200 t 19 25 189 15 20 o x To display the on line analysis window click the On line Analysis Window Open button It can be closed by clicking the Close button or closing the On line Analysis window Creating Measurement Plots To create a waveform measurement plot select 1 The type measurement from the Measurements list 2 The recording channel to be measured from the Chan list 3 For peak rise time rate of rise and slope the cursor pair C1 C2 o
117. n The default values are 4 leak records with voltage waveform divided by 4 Note that subtraction of the LEAK from TEST records is done using the leak subtraction module See section 13 for details Protocol linking Recording normally stops when the requested sequence of records within a protocol is completed Protocols can however be linked together by selecting a protocol from the Link to next protocol list so that on completion of the first protocol control is transferred to the linked protocol Link to next protocol TEES Steps 10 100m 10ms bd 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 C Program Files Borland Delphi WinWCP 32 vprot Steps 10 100mV 100ms xml Files Waveform New Protocol 409 9 m Open Protocol 0 ten Save Protocol 400 mv Save Protocol As AO 1 Om Set Protocol Folder Analog Outputs AO No Channels 2 Recording it A00 aor Toolbox gt Protocol i AO D Wave AO Waveforms A00 i E SE gE E fE JE i H Delay Stimulus type m des Amplitude Voltage mv Amplitude Holding Level nt Wi Duration 0 08 mV Digital Outputs DO pe No Channels fi Holding Pattern 5V A D A Update 0 2734 ms OV i Waveforms are constructed by dr
118. nded The National Instruments NIDAQ interface library must be installed before WinWCP 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 WinWCP 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 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 WinWCP and select from the main menu Setup 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
119. nt cell membrane Property conductance S measurement Read Only Reads the most recent cell capacity F Property measurement Read Only Reads the most recent pipette seal resistance Property measurement A file WinWCP VBSCRIPT Example vbs containng VBSCRIPT example code can be found in the cAprogram files winwcp 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 Bezanilla F amp Armstrong C M 1977 Inactivation of the sodium channel I Sodium current experiments J Gen Physiol 70 548 566 De Koninck Y amp Mody I 1994 Noise analysis of miniature IPSCs in adult rat brain slices properties of synaptic GABAA receptor channels J Neurophysiol 71 1318 35 Dempster J 1986 The use of the driving function in the analysis of endplate current kinetics J Neurosci Methods 18 277 285 Dempster J 1993 Computer Analysis of Electrophysiological Signals Academic Press London ISBN 0 12 208940 5 Dempster J 2001 The Laboratory Computer A Guide for neuroscientists and physiologists Academic Press Dilger J P amp Brett R S 1990 Direct measurement of the concentration and time dependent open probability of the nicotinic acetylcholine receptor channel Biophys J 57 723 731 Gillis K 1995 Techniques for membrane capacity measurements
120. ntains a list of ASCII format keywords detailing the number of records in the file record size scaling factors etc Signal records are stored in sequence after the header block Each record consists of an analysis block containing validation and analysis results pertaining to the record followed by a data block containing the digitised A D samples A data file thus has the form Record 1 Record 2 Record 3 4 L4 ol o o The beginning of each signal record can be determined as a byte offset from the start of the file using the formula Offset HB 7 Rec 1 RAB RDB where HBsize RABsize and RDBsize are the sizes bytes of the header record analysis and data blocks respectively and Rec is the record number Header Block The header block contains the information needed to allow a program to determine the size and number of records in the file The header block can vary in size between 1024 and 16380 byte depending on the number of channels in the file according to the formula HB Int N size 1 8 1 x 1024 chans File parameters are stored as ASCII text in the 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 lf gt are the carriage return and line feed characters A typical header block from a file with 2 channels contains the following keywords VER 9 WCEP data f
121. ocols for Leak Subtraction One of the most commonly used leak subtraction protocols is the P N protocol developed by Bezanilla amp Armstrong 1977 For each depolarising test pulse there are N additional subtraction pulses evoked by hyperpolarisng pulses 1 Nth the amplitude of the test pulse WinWCP s stimulus generator can be configured to produce the necessary sequence of test and leak subtraction recording sweeps by selecting the P N Mode leak subtraction option in a stimulus protocol See Recording Settings This causes the stimulus generator to produce additional scaled down and inverted stimulus pulse waveforms for evoking the linear leak currents without the voltage activated currents The leak current recordings are averaged and stored in a record marked as a LEAK type The test record is marked as TEST type record The TEST record with its associated LEAK record are collected together in a group i e they have the same group number Digital Leak Current Subtraction gt Subtracting Leak Currents To subtract the leak currents from a data file select Analysis Leak Current Subtraction to open the leak current subtraction window Leak current subtraction i a 5 x Record 8732 6 Ea E Oo 7 Type Test O n d Group E 4 Rejected VHold VTest m Average Do Subtraction Abort Voltage channel cnt vim VHold VTest Current channel cho Im 000m BGSS 17 50 ms m Leak re
122. ogram below shows the distribution of peak amplitudes for a series of 200 simulated endplate currents It consists of 50 equal sized bins over the range 0 5 to 1 5 nA i e a bin width of 0 2 nA The height of each bin represents the number of records containing a signal with a peak amplitude falling within that bin range 50 40 30 20 10 0 113 0 5 07 09 1 13 15 Im Peakfa nA Customising histograms If you want to alter the X or Y axis range scaling or labels click the Set Axes button to open the Set Axes Range Labels dialog box Set Axes Range Labels ES X Axis Y Axis C Automatic Automatic Manual Manual Min fos Min o Max 1 5 Max 50 Tick fo2 Tick 10 Labels X Axis lim Peak a nA Y Axis OK Bin Style _oK Empty Colour Cancel libodea __ Axis limits and tick spacing are initially set to default values based upon the min max range of the data You can change the axis limits by entering new values for into Min Max and Tick spacing boxes for the X and Y axes An axis can be made Linear or Logarithmic by selecting the option from its Scale list Labels for the X and Y axes and a title for the plot 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 fo
123. ols in the Protocol list Protocol By default protocol files are stored as files with the XML extension in the folder c winwep vprot The protocol folder can be changed by clicking Set Stimulus 10 100mv 100ms Protocol Folder and selecting another folder Set Stimulus Protocol Folder Applying Sequences of Protocols Select the List option to applying a sequence of protocols defined by the a m Recording Mode protocol execution list selected from Protocol List 7 Stimulus protocol bs qeeeeeseney To create a new protocol execution list C Single Protocol List List 1 2 Add protocols to the list by clicking Add Protocol to List and selecting a 10 100m 100ms protocol using the protocol file selection box 10 100m 1s 10 100m 10s 1 Enter a name for the new list and click New List To delete the currently selected protocol execution list click Delete List Protocol execution lists are stored as files with LST extensions in the protocols folder Add Protocol to List Delete List _New List List Name 1 Recording Experimental Signals gt Setting the Recording Sweep Size and Duration Tuuuy When Free Run External Trigger or Detect Events recording modes m Recording Mode are selected the number of analog input channels number of sweeps the number of digital samples per channel and the recording sweep duration need to be set in the recording window Note
124. omatic Data Cursors mode In manual mode the region within the signal to which the curve is to be fitted is set manually using a set of 3 cursors on the display In automatic mode the cursors are set automaticall automatically If you have selected Automatic Data Cursor mode select On Rise On Decay or Rise Decay to determine whether the cursors are to be placed on the rising phase decaying phase or complete time course of the signal waveform Then enter the levels on the waveform where the cursors are to be placed in the Limits box Default setting is 10 90 placing the cursors at 10 and 90 of peak amplitude on the selected phase If you have selected Manual Data Cursors mode define the curve fitting region by using three vertical cursors two blue marked define the region to which the equation is to be fitted the third green marked t0 defines where the zero time points for the equation is Note that the choice of fitting region depends upon the kind of curve being fitted Sometimes only part of the signal is chosen such as when an exponential curve is to be fitted to the decay phase of the signal In the example shown here the fitting region cursors have been placed on the decay phase of an endplate current Zero time t0 has been defined at the onset of the signal If you wish to change the initial values for the equation parameters or fix some parameters so they do not change during the fit click the Set Parameters
125. on 3 9925 No input channels 2 No samples 4095 Sampling interval 0 9746 m Channel cho Ch 0 Threshold 10 Pretrigger 10 Amplifier Gains 1 Ch 0 0 0005 WpA Ki Recording Experimental Signals gt Recording Modes gt Stimulus Protocol In Stimulus Protocol mode WinWCP functions as a stimulator as well as a recording device Sequences of recording sweeps are acquired at timed intervals in synchrony with computer generated stimuli applied to the cell The stimuli can be in the form of either voltage waveforms or on off TTL digital pulses for controlling valves or other devices Up to 4 voltage waveform output channels are supported and 4 8 TTL digital output channels depending on the laboratory interface Each stimulus pulse is associated with a single recording sweep and the duration or amplitude of any part of a pulse can be incremented between records A complete stimulus protocol thus consists 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 analog signals Protocols are created using the Stimulus Recording Protocol Editor Applying Single Protocols m Recording Mode Select the Single option to apply a single stimulus protocol or linked series of Stimulus protocol protocols selected from the list of available protoc
126. on defines the region within the record within which waveform measurements are to be made Note To allow the analysis region to be set for each channel separately untick the Lock Channels cursor option The t 0 cursor defines the zero time for the latency measurement ot v ww apo gaos Set the peak finding mode Select absolute to define the peak value as the largest absolute 2 amp 3 i e positive or negative deviation from the record zero level Select positive to define the peak 4 solute value as the largest positive deviation Select negative to define the peak value as the largest Points Avgd fo negative deviation Note Absolute mode should be used for I V curve measurements where n both positive and negative going signals may be found in the sequence of records being analysed Enter the number of data points to be averaged at the peak into the Points Avgd box If you want the rise time to be measured over an interval other than the standard 10 90 Rise time range enter a new range in the Rise Time box Note that this setting also determines the part 10 90 of the waveform rising edge used to calculate the slope rate of rise option Set the rate of rise mode to select the algorithm used to calculate the rate of change of the Rate of rise signal The Forward Diff Quadratic 5 Quadratic 7 options compute the maximum rate of Forward Diff change within the analy
127. ontaneous event detector Depending upon laboratory interface Analysis e Signal averaging e Digital leak current subtraction e Automatic waveform amplitude time course measurement e Mathematical curve fitting to waveforms e Non stationary current fluctuation analysis e Quantal analysis of synaptic currents e Synaptic driving function analysis e Synaptic current and Hodgkin Huxley 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 permission 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 advi
128. other artefacts within the recording sweep 499 76 vm EI mv cursor measurements are made relative to the signal level at CO when the at 0 cursor zeroj at 0 cursor level option is selected OR relative to the z horizontal zero level cursor when the at Z cursor atZ cursor The CO cursor defines the baseline level preceding the waveform of interest Peak and C1 C4 Zero sora L option is selected Measurements The available waveform measurements are listed in the table below Measurements Peak Peak positive amplitude within the region of the recording sweep defined by the cursors C1 C2 or C3 C4 Rise time Time taken for signal to rise within the percentage range of Peak defined in Rise Time Range box default 10 90 The CO cursor is used indicate the baseline level the signal is rising from and should be placed before the rise of the waveform Rate of Rise Maximum rate of change during rise from baseline level to Peak Rising Slope Slope of rising edge of signal within the percentage range of Peak defined in Rise Time Range box default 10 90 Peak Peak negative amplitude within the region of the recording sweep defined by the cursors C1 C2 or C3 C4 Rise time Time taken for signal to rise within the percentage range of Peak defined in Rise Time Range box default 10 90 The CO cursor is used indicate the baseline level the signal is rising
129. own Click the Do Analysis button to begin the quantal analysis The analysis procedure scans through the data file calculates the mean and variance of the peak amplitude of the signal records uses these to obtain estimates for the quantal content and displays the results in the report window A copy of the quantal analysis report is also written to the log file Note that you can test the operation of the quantal content analysis module using simulated endplate currents or potentials generated by the synaptic signal simulation module Synaptic Current Driving Function Analysis gt Computing a Driving Function The synaptic current driving function is a measure of the rate of evoked release of transmitter at a synapse If the time course of decay the post synaptic current is known the driving function can be computed using a deconvolution process More details of the method can be found in Dempster 1984 WinWCP s driving function module can be used to compute the driving function from synaptic current records such as endplate currents To carry out a driving function analysis a Record a series of stimulus evoked synaptic currents b Use the signal averaging module to create an average synaptic current from the set of raw records c Use the curve fitting module to fit an exponential function to the decay phase of the averaged synaptic current Fit the function from around 95 5 of the decay phase excluding the 5 around the
130. p 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 WinWCP 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 WinWCP 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 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 O
131. peak where the transmitter is still being release d Select Analysis Driving Function to open the driving function window Driving function analysis 5 x Record ii KE gt Rejected Analysis Do Transforms Abort Ch cho Im Range All records C This record C Range 000m B68 0 10 ms 1 1 8oG68 Equation Jexp tTau fp ratentiols Holding potential om Reversal potential 0 mY Keep parameters fixed 7 Reconvolute waveform 1 If there are several channels in the record select the channel containing the signal to be transformed from the Ch list 2 Set the range of records to be transformed Select All Records for all the records in the file This Record for the currently displayed record only or select Range and enter a range of records into the box 3 Enter the cell holding potential in mV that the currents were recorded at in the Holding Potential box and the 4 5 6 reversal potential of the post synaptic current in the Reversal Potential box The driving function is expressed in units of conductance unit time The holding and reversal potentials are required to convert from current to conductance The time constant computed by the curve fitting module in step 3 is used to deconvolve the current signal If you wish to use the same time constant for all records rather than using the individual value computed from eac
132. ponential time constant of decay of the capacitance current See Gillis 1995 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 Zap Clicking the Zap button to apply a single voltage pulse of amplitude and duration defined in the Amplitude and Pulse Width boxes A zap pulse can be used to perforate the cell membrane in a cell attached patch to form a whole cell path Recording Experimental Signals gt Recording Signals Creating a data file To create a new data file to hold your recordings select from the menu File New Data File to open the New Data File dialog box Save in E WinWCP 22 amp Al el prot a Power 1401 Digtest wep WinRT 95 a Power 401 Digtest wep fa Zexp100ch wep a Simulated 2 exp 50 channels wep a Micro 1401 Digtest wep a Simulated sing
133. 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 WinWCP Vxxx Setup File option to download the WinWCP installation program WinWCP_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 WinWCP_Vx x x_Setup The setup program creates the folder WinWCP and installs the WinWCP programs files within it You can change the disk drive and location of the WinWCP folder if you wish To start WinWCP click the Microsoft Windows Start button and select WinWCP Vx x x from the WinWCP 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 WinWCP 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 WinWCP is compatible with most National Instruments multifunction data acquisition 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 6341 with BNC connectivity device is currently recomme
134. r Y option Instrutech ITC 16 18 Troubleshooting WinWCP 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 functions gain filtering capacity compensation etc can be controlled from a virtual front panel within WinWCP The current implementation of the WinWCP software supports e 2 analog input channels membrane current and voltage e Command voltage output Software installation WinWCP uses Biologic s BLVP500 DLL library supplied with WinWCP 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 WinWCP 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
135. r C3 C4 defining the analysis region from the Cursors list and the polarity of the signal positive going negative going or absolute both positive and negative going from the Polarity options 4 For Cursor l 2 3 or 4 enter the number of points averaged about the measurement point into Points Avgd 5 For rate of rise the series of samples points 1 5 7 used to computer a smoothed rate of change into Smoothing 6 Optional The stimulus protocol associated with the measurement in the For Stim Protocol list When a stimulus protocol is selected measurements are only added to the plot when the protocol is running M Measurements Peak xl Chan fim Cursors h2 Polarity Positive Add to Plot For Stim Protocol Cir Measurements Clear Plots Copy to Clipboard Then click the Add to Plot button To erase the measurements list click the Clr Measurements button To clear the plot but not the measurement list click the Clear Plots button The data in the currently displayed plots can be copied to the Windows clipboard by clicking the Copy to Clipboard button Waveform measurements are also written to the log file as they are acquired Measurement cursors The two pairs of linked vertical cursors C1 C2 or C3 C4 define the region within the recording sweep where a waveform measurement is made They should be placed to include the waveform of interest but exclude any stimulus or
136. r 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 Printing the histogram To print the displayed histogram select File Print To open the Print dialog box ZL xi ipharmsrvisibsh327 Ip1 Set i gt Typeface Page Margins arial Let 25cm Right 125 cm Point Size 12 pts Top 25cm Line Thickness 2 pts Bottom 9 5 cm OK Cancel Click the Print button to plot the graph m Lines CEGE 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 copy image dialog box Copy Image xj Typeface ___ __ r Image Si
137. r the power to which the activation parameter m is to be raised to in the Activation power factor box Default 3 typical of sodium currents The voltage dependence of steady state activation is set by the Boltzmann function min V defined by its voltage of half maximal activation Vi and slope Vsiope The voltage dependence of the activation time constant tau V is set by a bell shaped function which varies the time constant between the minimum tmin and maximum tmax with tmax occurring at Vi 2 and the steepness of the cursor defined by Vlope Inactivation h The Inactivation h settings group determines the voltage and time dependence of the activation parameter h To enable the inactivation parameter check the Inactivation in use box The voltage dependence of steady state activation is set by the Boltzmann function Min V defined by its voltage of half maximal activation Vi 2 and slope Vsiope The voltage dependence of the activation time constant tau V is set by a bell shaped function which varies the time constant between the minimum tmin and maximum tmax with tmax occurring at Vi and the steepness of the cursor defined by Vsiope Enter the voltage at which the inactivation parameter is at 0 5 in the V half box Enter the inactivation time constant when the membrane potential is at V half in the Tau V half box Enter the voltage sensitivity in the V slope box large values weak voltage sensitivity Click the Start Simulation b
138. re 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 The import settings must be carefully set up to match the characteristics of the file being imported To import records using the raw binary function select File Import To display the Import File dialog box LI i zx Look in S Data gt cf EB Fe 5th Feb 07 Francisco Palma Shreena Joshi Ty annalisa bernareggi O Guy Bewick Sindy_Noise analysis My Recent Anselm Zdebik HEKA data D tecella test Documents Axon Data Helene Widmer Test Brack S IGOR datafile jdtest f Sy Tony H Desktop CED Files O Immy Dennis avg CED Micro 1401 Mk2 Tests C Janez Presern en Derek Costello Karen Mckloskey Digidata 1200 Tests O Lab PC Test Digidata 1440 Tests Murray Herd Eddie D Feter Fraser Eduard Domingeuz Riad Seddick Frances Edwards samantha pitt GS My Documents PI e My Computer File name 08 03 2010 gt eh My Network Files of type Raw Binary dat raw Cancel Places T Open as read only Select the disk drive and folder from the Look In list Then select Raw Binary dat raw 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 open the Raw Binary Import dia
139. release probability is expected to be low Otherwise choose Binomial 2 Enter the range of records to be used in the analysis Select All records if you want to use all records in the file or select Range and enter a range of records 3 If there are several channels in the signal record select the channel which contains the signals to be analysed from 4 5 6 7 the Ch list Evoked events Select the EVOK record classification type used to indicate stimulus evoked signals from the Type list Quantum events Jf the file contains miniature events select the Events in file option and select MINI from the Type list If there are no miniature events in the file but you know what the quantal signal amplitude is select User entered and enter the average peak amplitude of the spontaneous miniature signal into the Amplitude box and the standard deviation of peak amplitude in the Standard Dev box Analysis mode If the signals being analysed are currents recorded under voltage clamp conditions select the Currents option If the signals are potentials select the Potentials option and enter the cell resting potential and the reversal potential of the synaptic conductance into the Resting potential and Reversal potential boxes This data is used to apply a correction for the non linear summation effect The Correction factor should be left at the default value 1 unless the appropriate factor for the synapse under study is kn
140. rm Measurements Recordings i Analysis Xf Plot Histogram Summary Tables Peak a Records 1 200 Psy cnma Ch mins Ch 0Iminay Ch 0 Im maa Type ALL 1 0 09876 9 488 1 102 0 04718 2 2 0 09668 9 288 1 093 0 04617 Variables 3 3 0 09965 9 573 1 093 0 04617 cnom gt 4 4 0 09881 9 493 141 0 04594 5 5 0 09881 9 492 1 09 0 04622 V Record I Group 6 6 0 09908 9 518 1 198 0 04656 Time 7 7 0 09723 9 341 1 127 0 04602 Average 8 8 0 09913 9 523 1 117 0 0462 Area g g 0 09812 9 426 1103 0 04673 Peak a 10 10 0 09837 9 451 1 15 0 04626 raayre 11 11 0 09698 9 317 1 1 0 0451 Rise Time Rate of Rise 12 12 0 09728 9 346 14118 0 04583 I Latency 13 13 0 09844 9 458 0 04597 mo 14 14 0 09787 9 402 1131 0 04523 7 90 15 15 0 09963 9 572 1 062 0 04504 I Miteral 16 16 0 09823 9 437 1 003 0 04508 Baseline Gandudlance 17 17 0 09754 9 37 1 196 0 046 18 18 0 09784 9 399 1 099 0 04486 19 19 0 1002 9 624 1 088 0 04628 20 20 0 09936 9 545 14 479 0 04552 2 2 0 09586 9 21 14181 0 04577 22 22 0 0981 9 136 1 049 0 04642 __ClearTable_ 23 23 0 09657 9 278 1 083 0 04671 Save to File 1 To display tables of measurements 1 Select the Tables page by clicking on its tab 2 Click the Clear Table button to erase the existing table 3 Select the variables to be added to the table by selecting a channel ticking the variables to be added then clicking the Add Variables button Saving Tables to File To copy t
141. roach Non stationary Noise Analysis gt Analysing Synaptic Current Fluctuations The WinWCP non stationary noise analysis module can be used to compute the time course of residual current fluctuations between individual current records and the average current time course and plot the variance of these fluctuations vs mean current at each sample time point during the decay of the current The average number of ion channels contributing to the ionic current and the single channel current can then be estimated by fitting a parabolic function to the variance vs mean plot Select Analysis Non stationary noise analysis to open the non stationary variance analysis window IA Data Record X Y Plot Spectral Analysis Records 17100 Type TEST v _ Rejected 8068 Analysis _j Channel en o Im TypefaLL All records C Range 1 100 50 a 17 017 p 4 8088 Average Alignment mode On Negative Ri Scale to peak 100 No Scaling a t 31 299 0 13733 p a4 0 00 ms 99 98 ms The upper trace in the signal display panel shows a selected individual signal record blue with the average of all the records in the file superimposed in red The lower trace shows the residual difference Res between the signal record and the average To generate a variance vs mean plot and estimate single channel current and channel number from a series of synap
142. rotocol e g WCP StimulusProtocol prot01 Status Read Only Reads the current operational status of Property WinWCP 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 WCP SealTestPulse Amplitude 0 01 SealTestPulseDuration R W Property Reads sets the duration amplitude S of the seal test pulse e g WCP 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 potential Property V measurement computed by the seal test Read Only Returns the most recent cell holding current Property A measurement computed by the seal test Read Only Reads the most recent cell access Property conductance S measurement Read Only Reads the most rece
143. rrent 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 WinWCP 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 WinWCP are shown in the table on the following page Next Getting Started gt Amplifiers gt Signal Connections Table The following table shows signal connections the required for the WinWCP 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 S
144. s 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 Amplifiers Setup JE oj x Amplifiers WinWCP 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 Ch Name Units Units AlCh Amplifier 253 Oo 0 0005 A 0 1 Heka EPC 7 Input Channels 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 d
145. s xrv Plot Histogram Summary Tables Type TEST M Rejected aes Analyse Do Analysis Abort Allrecords This record Range 1 18 Peak Absolute Points Avgd o Rate of rise Forward Dif Y t 0 t 0 018372 6 11697 2501 t 0 t 0 021495 _ 2t5 11833 000 0866 0 20 s Rise time 10 90 i Tx decay time x 71 0 16 1958 Cursors r 69 01 mV GetCursors Area 0 004474 pAs 0 006714 mVs I Lock Channels z 351 3 pA 97 5 mV 7091 pA 2 11 84 m 2 RiseTime 6 647E 007s 1 483E 006 s Rate of Rise 4 224E008 pAs 0 mws i The first stage in the measurement process is to define and run a waveform analysis sequence on a selected series of records 1 Select the Analysis page by clicking on its page tab 2 Define the range of records to be analysed by selecting All Records to analyse all records or This Record to analyses only the currently displayed record or Range and enter a range of records All records C This record C Range 3 Select the type of records to be measured by selecting an option from the Type list Select ALL to measure records of any type except rejected records 4 Define the analysis region for each channel by using the pairs of linked vertical cursors t nn s t yy s 5 6 7 8 9 superimposed on the display The analysis regi
146. s of the region to be modified are indicated by a horizontal bar along the bottom of the display Enter the signal level to be substituted for the artefact in the Blank Value box then click the Remove Art button Undoing or accepting changes To undo editing changes click the Undo All Edits button to restore the data to the backup copy of the original data file A backup copy of the data file file name wcp BAK is created when the the Edit Record window is opened for the first time Data Files gt Opening a WCP Data File WinWCP uses its own custom data file format for storing digitised signal records These files are identified by the file extension WCP To load a previously created WCP data file select File Open Data File to display the Open File dialog box a ax Look in Test a e My Recent Documents E Desktop 080603c ITO activation dep_001 2 My Documents r S My Computer n File name gt Open Nien Files of type wer Files WCP x Cancel Places E 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 open the data file for display and analysis Data Files gt Adding Records from a Data File To append records from a WCP data file on to the end of the currently open data file select File Append Data File To display th
147. se on the signal in the Background noise box Rising Phase Enter the time constant of the EPSC rising phase in the Tau rise box and the variability of the time between stimulation and the event in the Latency variability box Decay Phase Enter the time constant of the decay of the EPSC in the Time constant 1 box If a double exponential decay is required tick the Double exponential decay option enter a second time constant in the Time constant 2 box and enter the ratio between the amplitudes of the two decaying exponential components in the Amp 1 Amp 2 box Click the Start Simulation button to create the simulated EPSC records Simulations gt Voltage activated Current Simulation The voltage activated currents module simulates the currents evoked in response to a series of rectangular voltage steps using the Hodgkin Huxley equations Both activation and inactivation kinetics are modelled Two channels are generated membrane potential and membrane current The model also simulates the effects of patch clamp pipette access conductance on measured currents The Hodgkin Huxley simulation can be used to test the leak subtraction module and the Hodgkin Huxley functions in the curve fitting module The currents are modelled by the equation T V V Gaex m V m V m V Jea E i ha 0 0 hV T V 7 where V is the voltage level to which the cell potential is stepped V is the initial holding voltage V is the reversal pot
148. sed 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 WinWCP 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 bit and 64 bit operating systems 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 1320 22 1440 A 1550 or 1550A Note Data acquisition is not supported with the Digidata 1200 and 132X interfaces under Windows 7 64 bit e Instrutech ITC 16 or ITC 18 e Heka EPC 9 EPC 10 e Biologic VP500 e Tecella Pico Triton Triton Plus Next Getting Started gt Installing WinWCP To install the WinWCP
149. sis region The Forward Diff option computes the rate of change from the difference between successive sample points The Quadratic 5 and Quadratic 7 options compute the nohi rate of change estimated of 5 and 7 sample point windows respectively The Linear Slope option computes the rate of change from the slope of a straight line fitted to a section of the rising edge of the signal defined by the Rise Time setting default value 10 90 If you want to change the decay for the T x decay time enter a new value into the gt decay time T x Decay Time box x 11 0 To begin the analysis of selected range of records click the Do Analysis button On completion of the analysis the measurements for each record appear in the Results table Results ae ER RN FO nlir 17 17 17 16 19s 16 19s 45 99 pA 69 01 mY O 004474pAs 0 006714 mV s 351 3 pA 97 5 mV 7091 pat2 11 84 mvA2 6 647E 007 5 1 483E 006 s 4 224E008 pAs 0 mis Measurement variables The sequence number of the record within the data file Group The number of the group to which the record belongs used by leak subtraction module The time relative to the first record in the file that the record was acquired The average signal level within the analysis region The integral of the signal level within the analysis region Peak The peak absolute positive or negative depending in peak measurement mode used signal level within the analys
150. spacing are initially set to default values based upon the range of the data You can change the axis limits by entering new values for into Min Max and Tick spacing boxes for the X and Y axes An axis can be made Linear or Logarithmic by selecting the option from its Seale list Labels for the X and Y axes can be entered into the Labels boxes A type face can be selected for the plot from the Font list and its size defined in the Point Size box The graph can be plotted as a line unconnected markers or both by ticking the Lines and or Markers tick boxes Printing the graph To print the displayed graph select File Print To open the Print dialog box Typeface Page Margins arial Left 5 0 cm Point Size 12 pts 2 pts 5 0 cm Lines _ Top 5 0 cm Line Thickness 2 pts Bottom 10 0 em Cancel You can set the size of the graph on the page adjusting the Left Right Top and Bottom page margin settings Click the OK button to plot the graph Copying the graph data points to the Windows clipboard The numerical values of the X Y data points which generate the graph can be copied to the clipboard by selecting Edit Copy Data The data is placed on the clipboard as a table of X Y data pairs in tab text format allowing the data to be copied into programs such as spreadsheets and graph plotting packages using an Edit Paste command Copying an image of the graph to the Windows c
151. subtraction approach to be used The current signal can be considered to consist of 3 components LO LO LO L where Ji t is the time dependent voltage activated ionic current under study JIk is the leak current and Ic t is transient capacity current due to the charging and discharging of the membrane capacity Ic t and Jk are always present in the signal and scale linearly with the size of the voltage step However i t only occurs for voltage steps to potentials which activate the voltage sensitive ion channels The Na current for instance is only evoked by depolarising voltage steps to potentials more positive than 60mV It is possible to obtain a record containing only leak and capacity currents by using a hyperpolarising voltage step or a small depolarising step lLQO L 1 Scaling this record to account for the differences in the size and or polarity of the voltage step and subtracting it from the test record effectively removes the leak and capacity currents V L t 1 t KOT S Since the scaling up of small subtraction records also scales up the background noise it is usual to average several subtraction records before scaling and subtracting It is also possible to average the test records WinWCP uses the following general algorithm 1 M V N I t Sy Oe a YL s j l where M is the number of test records averaged and N the number of subtraction records Digital Leak Current Subtraction gt Stimulus Prot
152. t Analysing Quantal Content of Synaptic Currents The following procedure can be used to calculate the quantal content of a series of synaptic currents or potentials which have been recorded and stored in a digitised data file a Using the record display module inspect each record in the data file and classify it as being either a nerve evoked signal EVOK a spontaneous miniature event MINI or in experiments where the probability of transmitter release is low a nerve stimulus which has failed to release any quanta FAIL You should also mark any records containing artefacts as REJECTED b Use the waveform measurements module to calculate the waveform parameters for ALL of the records with the intention of measuring the signal peak amplitude Take care to exclude the nerve stimulus artefact c Select Analysis Quantal Content to open the quantal analysis window quantal Analysis aT c Binomial Record Range All records C Range 1 200 Ch icholm Evoked event Type TEST v EEEN m Quantum event Events in file Type MINI X C User entered Amplitude OnA Standard Dey Ona Mode Currents Potentials Resting Reversal Correction otential otential Factor Fao0 mY foo my 1 To calculate the quantal content of the synaptic currents 1 Select the type of quantal analysis Choose Poisson if there are no MINI records available and the transmitter
153. t the Low p filter On option and enter the cut off frequency in the box Random baseline drift can be added to each record by entering a non zero value in the Drift Max box Click the Start Simulation button create the simulated mEPSC current records COM Automation Interface gt COM Automation Interface WinWCP 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 WinWCP automation object is WinWCP AUTO and is opened by the VBSCRIPT command set WCP CreateObject winwcp 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 Creates a new data file with the supplied name OpenFile filename wcp Method Opens a pre existing data file with the supplied name HoldingVoltage R W Property Reads sets the holding voltage in Volts applied to the cell E g WCP HoldingVoltage 0 06 TriggerMode R W Property Read sets the recording sweep trigger mode F Free run E External trigger D Event Detection P Stimulus Pulse E g WCP TriggerMode F StimulusProtocol R W Property Read sets the selected stimulus pulse p
154. ted 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 WinWCP Use the TRY 1401W program to test the CED 1401 WinWCP 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 WinWCP stimulus protocol being produced incorrectly On Off levels are inverted and pulse durations are incorrect This problem can be corrected by setting the CEDPOWER 1401DIGTIM COUNTSHIFT entry in the WinWCP lab interface xml file in c winwcp to 0 Stop the WinWCP program if it is running and open the file c winwep 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 and save the file Standard CED 1401 The performance of the original CED 1401 interface unit is very limited
155. terval into the Sampling Interval box Time Units Select the units of the time data column secs msecs mins from the Time Units list No of time points per sweep If a sample time column is present containing time values incrementing from zero for each separate recording sweep contained the data table the size of the WinWCP data record is determined automatically from when time data resets to zero The number of data points in each sweep can also be set manually by selecting the Set record size option and entering the record size into the box 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 When a specific data file format is not supported by WinWCP it can still be possible to import data using the raw binary import The import module assumes that the data has the general format File Record Data Record Data Header Header block 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 in the file size of record number and scaling of channels This is followed by one or more data blocks containing the A D converter samples 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 These details of the data file structu
156. the criterion set by 2 are set to the type and or status defined in 3 Curve Fitting gt Plotting Graphs of Best Fit Equation Parameters The X Y Plot page can be used to create graphs of the best fit equations parameters obtained from the curve fit Any variable from any channel can be plotted against any other as a Y vs X graph Curve Fit Recordings j Fit Cumes XY Plot Histogram Summary Tables Records 1 200 Type ALL New Plot Set Axes xais Record YAxis Im tau ms Filter records Filter 0 50 100 150 Record To plot a graph 1 Select the X Y Plot page by clicking on its page tab 2 Define the variable to be plotted on the X axis by selecting it from the X Axis variable and channel lists 3 Define the variable to be plotted on the Y axis by selecting it from Y Axis variable and channel lists 4 Click the New Plot button to plot the graph Customising the graph If you want to alter the X or Y axis range scaling or labels click the Set Axes button to open the Set Axes Range Labels dialog box Set Axes Range Labels E4 X Axis Y Azis Automatic Automatic C Manual C Manual Min 0 Min 0 Max s Max 200 Tick 3 Tick 50 Scale Linear Scale Linear Labels x Axis Record Y Axis Im Peak a nA Tace OK E Lines Cancel V Markers Axis limits and tick
157. tial output should be Scale factor 9 001 VimV Units mv connected to this input channel AI Ch 1 for Amplifier 1 Enter the measurement units of the current On analog input Al Ch 1 mV in the Units box and the amplifier voltage scaling factor in V Units into the Scale factor box Most Voltage clamp command channel amplifiers have a fixed scaling factor of 0 01 V mV Scale factor 1VN Output AO Ch 0 Voltage clamp command channel Current clamp command channel Scale factor Out ut Analog output AO Ch 0 for Amplifier 1 should be TaY jao cho connected to the amplifier command voltage input Enter the voltage clamp command potential scaling factor membrane potential 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 Load Settings Save Settings Default Settings Current clamp command channel 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 Setting
158. tic current records 1 If there is more than one signal channel select the channel containing the current signal to be analysed from the Channel list 2 Optional To use a specific record type only change the Type list from ALL to the selected type TEST LEAK EVOK MINI FAIL TYP1 TYP2 TYP3 3 Select the All Records option to use all records of the selected type To use a sub range of the records within the file Select Range and enter the range of records in the box 4 If synaptic currents are being analysed select the Scale to Peak option to scale the average current to the peak amplitude of each current record before the subtraction to produce the residual variance Select No Scaling if the unscaled average is to be subtracted 5 Optional If the starting time of each signal varies significantly from record to record it can be re aligned with the average current before subtraction to produce the residual Set the Alignment mode to On Positive Rise for positive going signal and On Negative Rise for negative signals 6 Select the region of the signal waveform the decay phase in the case of synaptic currents to be included in the variance vs mean plot using the pair of a a analysis region cursors a 17 017 p a a t 31 299 0 13733 a 7 Click the X Y Plot tab to switch to the X Y Plot page Select Mean from the X Axis variable list and Variance from the Y Axis list then click the New Plot buttonto gener
159. timulus Protocols gt Recording Settings Select the Recording tab page to set the number of recording sweeps to be acquired during the protocol and the number of analog channels samples channel duration and sweep repetition interval Recording duration the duration of the recording sweep Note When the Fixed sampling interval option is selected this entry is defined by the Sampling interval setting Sampling interval Time interval between samples on each channel When the Fixed option is selected the sampling interval entered here defines the duration of the Recording duration Sampling interval x No samples per channel When the Fixed option is not selected sampling interval is set by the Recording Recording Stimulus Recording duration Sampling interval Stimulus repeat period T Fixed No records No repeats per increment No input channels No samples per channel Leak Subtraction 400 only duration Recording duration No samples per amp None channel PiNmode No pulses 4 Divide by 4 Stimulus repeat period the time interval between Link to next protocol successive recording sweeps in the protocol No records The number of records to be acquired during 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 No input channels The number of analog input channels
160. timulus Trigger setting in a stimulus protocol is set to Y 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 digitiser 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 WinWCP Software installation WinWCP uses the Instrutech dev
161. tive Im tau ms L To plot a histogram 1 Select the Histogram page by clicking on its page tab 2 Select the waveform variable from which the histogram is to be generated from the variable and channel list boxes 3 Enter the number of histogram bins in the No of bins box max 1024 4 Enter the range of variable values which are to be included in the histogram from the lower limit in Lower box to the upper limit in the Upper box 5 If you want the histogram bar height expressed as a percentage of the total number of records tick the Percentage option 6 If you want a cumulative histogram tick the Cumulative option 7 Click the New Histogram button to compile and plot the histogram For example the histogram below shows the distribution of fitted decay time constants for a series of 200 simulated endplate currents It consists of 50 equal sized bins over the range 9 to 12 ms Le a bin width of 0 6 ms The height of each bin represents the number of records containing a signal with a peak amplitude falling within that bin range 20 15 10 5 0 10 53 4 g 9 6 10 2 10 8 11 4 12 Im tau ms Customising histograms If you want to alter the X or Y axis range scaling or labels click the Set Axes button to open the Set Axes Range Labels dialog box Set Axes Range Labels ES X Axis att ea C Automatic Automatic Manual C Manual Min 0 4 Min 0 Max 1 5 Max 50 Tick 0 2 Tick
162. to be acquired Channels are always acquired in sequence from Ch 0 upwards i e No input channels 1 selects Ch 0 No input channels 2 selects Ch 0 amp Ch 1 etc No samples The number of samples to be acquired per input channel The minimum is 256 samples per channel increasing in steps of 256 The maximum ranges from 16184 No Channels 1048576 No Channels depending on the laboratory interface in use External Stimulus trigger Y N Enables the stimulus protocol to be triggered by an external TTL pulse instead of the internal timer When set to Y the stimulus repeat period is ignored and the stimulus protocol begins when a TTL pulse is received on the External Stimulus Trigger Input See laboratory interface card connections tables in section 1 Leak subtraction settings A protocol can be programmed to add digital leak subtraction records using the P N mode option When this option is selected a series of additional recording sweeps are generated for each record defined in the protocol using an inverted and scaled down version of the command voltage waveform A digital average is obtained from these records and stored in the data file as a LEAK record along with the basic TEST record L J Leak Subtraction C None PNmode No pulses 4 Divide by 4 You can change the number of pulses used to compute the LEAK record and division factor by altering the values in the boxes show
163. tup 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 WinWCP only supports digital output lines 0 3 of the Digidata 1200 Troubleshooting There are two known problems which will prevent WinWCP 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 channel conflicts WinWCP requires DMA channels 5 and 7 to support the transfer of data to from PC memory and the Digi
164. uilt 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 force 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 WinWCP 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
165. uto 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 headstage 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 d
166. utton to create the simulated voltage activated current records Simulations gt Miniature EPSC Simulation The miniature EPSC module generates simulated miniature postsynaptic currents exhibiting the stochastic fluctuations associated with the current flow through the population of ion channels opened by a single quantum of transmitter The gating of a single post synaptic ion channel is represented by a simple 4 state model Binding of an agonist molecule A with receptor R to form an agonist receptor complex makes it possible for the channel to shuttle between a closed state AR an open state AR and a closed desensitised state ARP Kpina gt AR Kopen Ha K tes Ei K unbind lt k A R AR lt K na close The single channel current time course is governed by six rate constants rate of binding and unbinding of agonist from receptor Kbina Kunbina rate of channel opening and closure Kopen Kciose and the rate of entry and exit from the desensitised state Kaes Kuna The mEPSC is generated by summing the individual single channel current time courses for each ion channel opened by the brief pulse of transmitter released from each vesicle time course represented by a decaying exponential function with a time constant of 10 s To create a data file containing simulated mEP SCs Create a new data file to hold the records by selecting File New and entering the name of a new data file Select Simulations
167. vents event detector mode provides a means of detecting signals as they occur within an incoming analog signal A threshold based event detection algorithm monitors the incoming signal on one of the input channels An event is detected when the signal deviates by more than a predetermined level from the average baseline level To compensate for slow drifts in the baseline level the threshold level is maintained at a constant distance from the baseline by means of a running average calculation The event detector is configured by setting three parameters If more than one channel is being recorded select the input channel on which events are to be detected from the detection Channel list Enter the detection threshold into the Threshold box The threshold level is expressed as a percentage of the total input range with its polarity determining whether positive or negative going signals are to be detected The level should be set as small as possible to maximise the likelihood of an event being detected but without producing an excessive number of false events due to background noise triggering the detector Values of around 5 10 are often used but several trials may be necessary before the best level for a particular experiment is found The Pretrigger setting determines the percentage of the record to be collected before the detection point A typical value is 30 Recording Mode Detect events Tz No records mo Record durati
168. vices 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 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 o x DD1550A Vd100 3 1 firmware 00 AID Converter Voltage Range t0v m ae 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 WinWCP is waiting for an external START trigger pulse in the Ext Triggered recording mode or when the External S
169. ze arial Width 600 pixels Point Size 12 pts iSe Lines 500 pixels Line Thickness 2 pts i 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 Automatic Waveform Measurement gt Fitting Gaussian Curves to Histograms Gaussian probability density functions representing the distribution of discrete populations of events can be fitted to a histogram using non linear least squares curve fitting The number of events expected to be found in each histogram bin for a distribution represented by a mixture of m gaussians is given by N w a x L y x gt exp x a i l 4 200 20 where N is the total number of events records w is the histogram bin width and each gaussian i is defined by three parameters its mean i standard deviation i and the fraction of the total number of events ai contained within it To fit a gaussian curve to the displayed histogram 1 Select the number of gaussian functions 1 2 or 3 to be fitted from the equations list Analysis Gaussian 2 Define the region within the histogram to which the curve is to be fitted using the pair of vertical analysis region cursors The selected region is indicated by the horizonta

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