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AxoGraph X Data Acquisition Manual

1. float refArray 0 float dataArray 0 Check that this protocol is repeated at least twice if prRepetitions lt 1 Exit Check the analysis window number is correct It might change if a protocol is halted then Resumed if WindowTitle acgAnalysisWindow AmplitudeFileName NewAnalysisWindow AmplitudeFileName newWindow 1 Get the selected regions of the most recently acquired signal the signal displayed in the Acquisition Monitor window refArray YRange monitorWindow 1 acgRefMin acqRefMax dataArray YRange monitorWindow 1 acgAnalysisMin acqAnalysisMax Adjust the size of the global array that receives the amplitude measurements SetArraySize acgAnalysisData acqgAnalysisEpisodet1 Perform the amplitude measurement add it to the measurements array and increment the episode counter acgqAnalysisData acgAnalysisEpisode Max Abs dataArray Mean refArray acqAnalysisEpisode Pass the measurements array to the Amplitudes monitor window YData acqAnalysisWindow 1 acgAnalysisData Redraw the Amplitudes monitor window DontRecalculateYAxis acgAnalysisWindow DrawTrace acgAnalysisWindow 1 eraseFirst 69 9 The Protocol Launch List 9 1 Introduction 9 2 Edit Launch List 9 3 Default Launch Mode 9 4 Open Listed Protocols 9 5 Remove the Launch List Features 9 1 Introduction Protocol files can be opened manually v
2. Digidata 1320 http www axon com CN_Digidata1320 html ITC 16 and ITC 18 http w ww instrutech com Summary of hardware requirements Machine Digitizer Interface Card 68K Mac ITC 16 NuBus PowerMac prior to G4 ITC 16 ITC 18 PCI Bus Digidata 1320 series not required uses built in SCSI Power Mac G4 and newer ITC 16 ITC 18 PCI Bus Digidata 1320 series SCSI card shipped with Digidata Very early PowerMacs prior to the 7100 model had a NuBus not a PCI bus Check the bus type of any PowerMac 6000 or 7000 series computer before purchasing an Instrutech digitizer interface card Memory Recommendations 32 MB RAM or more for episodic acquisition 64 MB RAM or more for continuous acquisition Virtual memory is not recommended when running data acquisition package 1 3 Install Acquisition Hardware and Software This section describes five steps required to set up a computer for data acquisition 1 Connect a digitizer to the computer 2 Load the data acquisition software 3 Test the software installation 4 Connect electronic equipment to the digitizer 5 Describe the connections 1 Connect a digitizer to the computer Always shut down the computer before connecting a digitizer To connect an Instrutech ITC 16 or ITC 18 digitizer An interface card and connecting cable are supplied with the Instrutech digitizer Open the computer case and install the interface card into an internal slot usually a PCI bus slot but could be a
3. mode that synchronizes the start of each sweep with the power line frequency 50 or 60 Hz A single sweep can be captured and overlaid on later sweeps Signals on up to 8 channels can be displayed simultaneously Signal waveforms are displayed but are not saved to disk See Chapter 4 Digital Chart Recorder or Chapter 5 Protocol Driven Data Acquisition for programs that can save the acquired signals to disk In addition to the standard storage scope features outlined above AxoGraph s digital scope can average a signal over multiple sweeps and display the running average It can send a synch pulse to a Digital Output at the beginning of each sweep and a test pulse to an Analog Output during each sweep It can also measure and display the peak amplitude rise time and width of an event in each sweep AxoGraph presents a pop up menu in the toolbar at the bottom left of the screen When the Scope item is selected in this pop up menu a new window titled Scope Window will appear The following sections describe the function of each of the buttons in the ee toolbar 3 2 Run Scope The Run Scope button starts the digital scope A series of sweeps will be displayed in the scope window Hitting the space bar on the keyboard halts the scope The rate at which the traces appear will depend on how the sweeps are triggered The various trigger options are described in Section 3 7 below 19 3 3 Hot Keys The Hot Keys button
4. Describe the connections AxoGraph requires information about the connections made to the input and output channels of the digitizer Specifically it requires a signal name for each connection and the signal s units and gain for each analog connection To enter this information a Change the toolbar popup menu setting to Configuration b Click on the Full Configuration button in the toolbar A series of dialogs will appear Each of these dialogs is described in greater detail in Chapter 2 Please skip to Chapter 2 if the following outlines are too terse e The first series of dialogs ask which input and output channels on the digitizer are being used Turn on the check box next to each group of channels then each individual channel that has a cable connected e The next dialog is only relevant to electrophysiologists using a patch clamp or microelectrode amplifier from Axon Instruments Dagan or Warner AxoGraph can read and interpret the telegraph signals gain filter frequency etc that are output by these amplifiers If one or more amplifiers is connected then several additional dialogs will appear asking which digitizer channels the telegraph signals are connected to e The next few dialogs request signal names and units for the input and output channels that have cables connected The names should clearly distinguish between the various channels but should not be too long because they will be used to label the axes of the dat
5. Pre pulse Holding Level 2 Pre pulse Train 3 Test Holding Level 4 Test Pulse The analysis region is determined from the pre pulse train Its length is equal to the pulse width plus the interpulse interval The pulse is at the center of this region The EventRateSetup program name should be added to the Command dialog field Before First Repetition PonNMeasure This program works in conjunction with the previous program It performs P N leak subtraction after each episode is acquired and adds the resulting waveform to the P N results window The PonNMeasure program name should be added to the Command dialog field After Each Repetition The example protocol titled I V with P N Leak Subtraction demonstrates the use of these two programs 8 4 Writing New Analysis Programs For general information about writing an online analysis program see the chapter on programming in the AxoGraph Online Manual Online programming documentation that can be accessed by selecting Program gt Programming Help Three online analysis programs are presented below They illustrate techniques for accessing data from the most recent episode analysing it and reporting the result The first program displays the baseline noise standard deviation in the Acquisition Monitor window To activate this program save it as a text file in the Plug In Programs folder Next select Program gt Reload Plug Ins Finally enter the program name OnlineNoiseSD in
6. periodic test pulse to a single output channel In contrast the acquisition program described in this chapter takes full advantage of the digitizer s capabilities The protocol driven acquisition program can monitor and record signals from Analog Input and Digital Input channels while sending a complex series of waveforms to multiple Analog Output and Digital Output channels Data can also be analysed in real time as it is acquired Data acquisition is controlled by a set of instructions contained in a protocol A protocol is a graph file that contains one or more Analog Output and Digital Output waveforms It also contains instructions about which Analog Input and Digital Input channels to record signals from how to trigger acquisition of each episode how many episodes to acquire etc A protocol window must be open before running a data acquisition program Detailed information about creating and editing protocols is provided in Chapter 7 Example protocols are supplied in the Acquisition Protocols folder that can be found in the Data Acquisition Package folder inside the AxoGraph 4 9 folder The function of each of the example protocols is described in Chapter 10 Data is acquired in a series of separate epochs or episodes An episode can be of any length When the episode is longer than 0 5 seconds the input signal will be displayed as the data is acquired For shorter episodes the data is only displayed when acquisition of that episo
7. 10 Time m 4dd a Pulse Selecta name for this pulse o The parameters needed to define the shape of a Pulse ora Ramp are identical amplitude onset time and width These parameters are entered via a series of dialogs which is the same for both waveform types These dialogs are described in the next section A different set of parameters 1s needed to define the shape of a Pulse Train These parameters are entered via a series of dialogs which is described in a following section see Section 7 8 Define Pulse Train Shape The next dialog 1s common for all three waveform types It asks for a name to describe the pulse A default name is suggested Pulse names are useful when a protocol contains a number of pulses on several different channels and one particular pulse or ramp needs to be edited The easiest way to identify which pulse to edit in a complex protocol is to give each pulse a distinct name 50 7 7 Define Pulse or Ramp Shape The following series of dialogs will appear if a Pulse or a Ramp has been specified If the protocol contains only one output waveform then the first dialog ask for the pulse amplitude and a subsequent dialog asks for the pulse onset time and width These dialogs are self explanatory and are not shown If more than one output waveform has been specified then a series of three dialogs appears They request the amplitudes and the timing of the pulse on each wavefo
8. Command connections on the AxoPatch change their behaviour depending on the mode Before each trace is acquired the signal on Analog Input 6 will be sampled and the voltage will be used to determine the AxoPatch clamp mode When the recording mode is changed from current to voltage clamp the External Command channel switches from a current to a voltage command Select the External Command channel Command Piezo cancer Cox 15 The units and gain setting for the AxoPatch External Command connection will be interpreted as either a current command or a voltage command depending on the AxoPatch recording mode The next dialog asks which Analog Output channel is connected to the External Command signal The signal units will be adjusted to pA for current clamp or mV for voltage clamp The Mode Telegraph also adjusts the units for the signal connected to the AxoPatch Scaled Output If the gain telegraph is active then the Scaled Output channel has already been specified see Gain Telegraph dialog above so no dialog is presented However if the gain telegraph is not active then dialog appears asking which Analog Input channel is connected to the Scaled Output signal The units of the specified channel will be adjusted to mV for current clamp or to pA for voltage clamp Frequency and Capacitance Telegraphs Axo0Patch amplifiers have Frequency and Cell Capacitance telegraph outputs These
9. In practice this is only true when a cell has no processes dendrites axon etc or when the processes are very short or fine When a cell is isopotential the current response decays as a single exponential permitting and accurate estimate of membrane capacitance If large dendrites or other processes are present the current decay will follow a multi exponential time course because the membrane capacitance is distributed along the dendrites This makes it very difficult to calculate whole cell capacitance If the exponential fit 1s restricted to the first millisecond or so of the response then the reported capacitance value may represent a useful approximation of the membrane capacitance of the cell body minus the processes The reported capacitance value will depend on the time range over which the exponential or double exponential function is fit This can be adjusted via the Test Setup button as described in Section 5 7 below 5 6 Test To Log The Test To Log button activates either the Test Seal or the Test Cell scope window whichever was most recently active When the scope is halted by hitting the space bar the parameter values that are displayed in the scope window will be appended to the log window The time that the measurement was made will also be noted in the log 5 7 Test Setup The Test Setup button brings up two dialogs that adjust the behavior of Test Seal and the Test Cell programs Increasing the Pulses per Computa
10. MINIONS O RE O OS 47 TE 1 3 0 cd Vl ict a roe RE OTR Se Pen ve ee aE OOD Soe E A AAT 48 Ted Define Pus OF RAMPAS uri ted 50 RS Define Pulse Train Shapes ecesiacasoes ests ios 5I L E O a A E E E IE E A ES 53 TAC D re A A AATA A E A 53 A e E E T 54 CADA A O O E E E E 56 TS C BOW aV Cl ONIN icc ad E 57 IA A E E A A AA 58 LES PIN Leik subir ac 0 e eae nr E a e a REEERE 59 POE CEPO OC Olan a cate A A 60 PEL Conve e A OI A e a A E E EE e 60 1V S Online AA YSIS aii 61 Oud RAKOEN 110 0 E E E E TETEE A E E IA I A E TTT 61 8 2 Add Analysis Programs to a Protocol ccssscscccccccsssssssssnsasecececcesesessnnnecseceeecessseesnnsaeeeesesessnaeeeesens 62 8 3 Standard Analysis Programs IEA A bad 63 9 4 Writing New Analysis Programs iii 65 9 The Protocol Launch Eta Ea EEA E E R 69 NP RI EA A E E E 69 92 Edit Lac Sos 69 93 Detault Laune n Mode zeen dea a 70 OA Open Listed Protocols enion A E E 71 g9 5 Remove the Launch List Feature S dais 71 LO Example Protocols aaeain E ANAE EAEE OOSA OEE OSATEN 72 A ons sinan A ETA nee eee 72 10 2 Brief Ovetview OF Each PrOOCO Los ici I2 10 3 Detailed Description of Each Protocol csr a a a a danza dncndas 74 11 Precise Acouisiton TIMO lie ias 93 11 1 Stability and Accuracy of Regular Trigger ooocccccnnnnooonnonnnononcnononnnonnnnonnnnnnncnnnnnnnnnnn ono nnnnnnnnonnnnnnos 83 11 2 Hardware Limitations on the Sampling Rate cccnnonnonooocconnncnnnnnonanonnnonononnncncnonnnnnnn on
11. NuBus slot in an old 680x0 Mac Close the case and connect the cable between the interface card and the digitizer Plug in and switch on the digitizer Power up the computer To connect an Axon Digidata 1320 series digitizer An AdvanSys PCI to SCSI card and a SCSI cable are supplied with the Digidata Open the computer case and install the SCSI card into an internal PCI slot Close the case and connect the supplied SCSI cable from the card to the Digidata Plug in and switch on the Digidata Power up the computer Insert the AdvanSys CD and run the installation software Alternative connection strategy for Axon Digidata 1320 series digitizer All Mac models prior to the G4 have a built in SCSI port at the rear of the computer It is possible to connect a Digidata 1320 directly to this port or to daisy chain it with an external hard drive However the cable supplied with the Digidata will not connect to the Mac s built in port because it uses a different physical format Cables and adapters for connecting between the three main SCSI port formats are available at computer shops 2 Load the data acquisition software There are two different installers for AxoGraph 4 9 e Install AxoGraph 4 9 Acq installs a fully functional acquisition package and preloads the package The acquisition programs are automatically loaded into toolbars and the Program menu If an item named Acquisition appears under the Program menu then the acquisition
12. Subtraction El E I V with P N Leak Subtraction Episode 10110 This protocol delivers a series of 10 waveforms to Analog Output 0 Each waveform consists of a test pulse preceded by a train of 8 smaller inverted pulses that are superimposed on a long negative step The response to these 8 pre pulses is used to perform P over N or P N leak subtraction which removes the passive or linear component of the response to the test pulse see Section 7 15 P N leak subtraction is performed as follows The 8 responses to the pre pulses are averaged scaled then subtracted from the response to the test pulse The resulting signal is displayed in 100 200 a separate P N analysis window The Time ms monitor window and the data file record the raw responses to both pre pulses and test pulse The P N result window contains only the non linear component of the response to the test pulse The P N analysis programs are named PonNSetup which is set to run before the first waveform and PonNMeasure which is set to run after each waveform See Chapter 8 Online Analysis for information on how to add online analysis programs to a protocol To use these online programs the P N protocol must be set up to contain exactly 4 pulses in the following order 1 Pre pulse Holding Level 2 Pre pulse Train 3 Test Holding Level 4 Test Pulse gt E i E E E Paired Pulse AP with P N Paired Pulse AP with P
13. The gains are given in mV of voltage clamp command per Volt at the Analog Output and in pA of current clamp command per Volt at the Analog Output The fourth dialog asks which Analog Input receives the amplifier s Scaled Output signal This signal will report current when in voltage clamp mode and membrane potential when in current clamp mode If a gain telegraph is active then the Scaled Output channel has already been specified see Gain Telegraph dialog above so this dialog will not be presented The fifth dialog asks for the signal names to be used for the Scaled Output channel when in voltage clamp mode and current clamp mode 18 3 Digital Oscilloscope 3 1 Introduction 3 2 Run Scope 3 3 Hot Keys 3 4 Display 3 5 Average 3 6 Timebase 3 7 Trigger 3 8 Channels 3 9 Gains 3 10 Test Pulse 3 11 Analyse 3 12 Custom Analysis 3 1 Introduction The Scope program implements all the main features of a storage oscilloscope It is designed for monitoring an intermittent electrical signal Each time the scope is triggered an electrical signal is plotted versus time in the scope window The displayed waveform is termed a sweep Successive sweeps can be overlaid storage mode and erased manually Alternatively the window can be erased automatically before displaying each new sweep Sweeps can be triggered from an external pulse or they can be triggered at regular intervals by the computer There is a line trigger
14. This program is very similar to Amplitude Setup In cooperation with the following program it monitors two parameters of each episode the series resistance of the preparation Rs and the amplitude of a response that occurs within a specified time range during each episode The protocol should specify multiple repetition of a single output waveform on each of two channels a test pulse and a stimulus One Analog Output channel should deliver a voltage pulse that will be used to monitor the series resistance This should be the first pulse added to the protocol see Section 7 6 Add a Pulse The second pulse should be used to trigger a stimulus The RsAndAmplitudeSetup program name should be added to the Command dialog field After First Repetition It creates a new graph window titled Amplitudes and Rs that will monitor the response amplitude and series resistance while the protocol is running It then poses two dialogs that requests the time ranges for the baseline and the peak amplitude measurements RsAndAmplitudeMeasure This program works in conjunction with the previous program It measures the response amplitude and series resistance after each episode is acquired and adds these to the Amplitudes and Rs monitor window The RsAndAmplitudeMeasure program name should be added to the Command dialog field After Each Repetition The example protocol titled Pulse Stim and Rs demonstrates the use of these two programs EventRat
15. a data file is saved the parameters defining the protocol that created it are saved in its Notes field The Extract Protocol button reads these parameters and uses them to rebuild the original protocol It will only work if the front window is a data file This program may be useful for reconstructing a lost protocol or for confirming the stimulus conditions that were used to acquire a given data file For technical reasons it will not work correctly if the original protocol contained custom waveforms see Section 7 13 7 17 Convert To Protocol Almost any graph file can be converted to a protocol This permits completely arbitrary output waveforms For example a recorded signal could be used as an output waveform For a graph file to be converted to a protocol it must meet two requirements Firstly the X axis should have units of time ms or s and the data points should be regularly spaced The interval between data points determines the acquisition sample rate Secondly the traces should be organized into groups with the group numbers specifying which Analog Output channel the waveforms in that group will be directed to All groups should contain the same number of traces episodes Information on how to group traces 1s available in the AxoGraph Online Manual Section 7 4 Combine Traces into Groups If a Digidata 1320 series digitizer is in use then the group numbers should be either 0 or 1 corresponding to the two Analog Outp
16. and voltage are measured over two different time periods selected by placing cursors in the monitor window Data 8845 Current pa The slope of the I V plot is inversely proportional to the input resistance of the preparation A typical result is up bel shown Yoltage mY Note that there are two traces in the I V plot These correspond to the Ist and 2nd repetitions of the series of voltage pulses The I V analysis program is a standard part of the data acquisition package and it can be modified to perform any kind of analysis on the acquired data file see Chapter 8 Online Analysis 1 U using Command Table HH I V using Command Table Episode 10 7 10 When delivering a series of pulses to an Analog Output channel it is not always desirable to increase the pulse amplitude in constant increments For example a logarithmic series or random series of amplitudes may be required In this example protocol each pulse amplitude is specified by a table entry see Section 7 7 A series of 10 voltage command pulses is directed to Analog Output 0 and the pulse amplitudes are in mV 100 100 50 50 25 25 10 10 5 and 5 Any series of pulse amplitudes can be specified in a table See Section 7 7 Define Pulse or Ramp Shape for a description of how to add a series of pulses to a protocol with amplitudes specified in a Command mY 10 Time ms table 78 I with P N Leak
17. as waveform several limitations are imposed for technical reasons It is not possible to edit the sample rate the active Analog Output channels or the pulse parameters and pulses can not be added or deleted The Custom Waveform feature can be used to generate any standard mathematical function However some output waveforms may require the summation of several different functions To achieve more complex output waveform two or more waveforms can be added together using the Trace Math feature select the menu item Program gt Trace Manipulation gt Trace Math The resulting graph window can then be converted to a protocol by selecting Program gt Protocol gt Convert to Protocol see Section 7 17 This feature can take any waveform in any graph window and convert it into a protocol 7 14 Add a Link When a protocol terminates it can automatically initiate another protocol Several protocols can be linked together to form a chain The last protocol in the chain can link back to the first protocol forming a loop Using this mechanism it is possible to generate output waveform sequences of arbitrary complexity It also permits the acquisition of a series of episodes with different sample rates and lengths The connection from one protocol to the next 1s established by adding a link to the end of the first protocol The Add a Link button brings up a dialog that ask for the name of the next protocol to initiate following the
18. asking whether the power line frequency is 50 or 60 Hz Sweeps will be initiated at the shortest possible interval that is a multiple of 20 ms for 50 Hz line or 16 6666 ms for 60 Hz line The line trigger mode is useful when testing for contamination of a signal by power line noise It can be used in conjunction with sweep averaging to improve sensitivity see Section 3 5 above 22 When Internal triggering is selected a series of three dialogs appears that request additional information about when to initialize each sweep The first dialog not shown asks which Analog Input channel carries the signal that will be used to trigger the sweeps This can be any of the connected signals Typically one of the signals being displayed in the scope window will be used as the trigger signal Trigger Scope Either initiate a sweep when the ADC signal amplitude exceeds a threshold level or when its Ist derivative slope exceeds a threshold level Amplitude Threshold Slope Threshold cancel Cox Trigger Scope when the signal amplitude on an ADC input channel crosses the specified level Use or hot keys to adjust the level up or down while the scope is running Lf Trigger on Low to High Crossings Trigger Level pA Pre Trigger Interval ms 5S Trigger Scope when the slope of the signal on an ADC input channel crosses the specified level Use or hot keys to adjust the level up or down while the sco
19. frequency equal to half the sample rate This dialog also controls the length of the chart record The chart recorder will halt automatically when the record reaches the length specified in the Record Length field For example the above dialog requests a 20 minute recording 20 x 60 1200 sec If the Record Length is set to zero then recording will continue until all available system memory is full Record Length for continuous recording Because of limitations in the digitizer hardware it is not always possible to sample data points exactly at the requested sampling rate See Section 11 2 Hardware Limitations on the Sampling Rate for a discussion of these limitations 4 7 Channels The Channels button in the Chart toolbar brings up two dialogs The first dialog asks which Analog Input and Digital Input Chart displays one or more signals to display in the chart window Only of the ADC and TTL Input channels channels that have a cable connected are listed see Section 2 2 Connections Channels are listed by name not by number Mf Current see Section 2 3 Channel Names Chart channels Lj Voltage When two or more input channels are selected the signals will be displayed by default in separate groups within the chart window The signals from multiple channels can be overlaid by selecting either Combine or Group under the Trace menu In general channels should only be combined if their signals h
20. input channel so maximum channels 3 The protocol requests a sample rate of 20 kHz 50 us The ITC needs to process all three output channels in this interval or one channel every 50 3 16 6667 us This inter channel interval is not permitted because it is not an integer multiple of the clock tick interval e For a Digidata 1320 or 1321A the inter channel interval corresponds to 166 667 ticks The Digidata delivers the closest available sampling rate by processing one output channel every 167 ticks This gives an effective sample interval of 167 x 3 x 0 1 50 1 us which corresponds to 19 960 kHz 85 e For an ITC 16 the inter channel interval corresponds to 16 6667 ticks The ITC 16 delivers the closest available sampling rate by processing one output channel every 17 ticks This gives an effective sample interval of 17 x 3 x 1 0 51 us which corresponds to 19 6078 kHz e For an ITC 18 the inter channel interval corresponds to 13 3333 clock ticks The ITC 18 delivers the closest available sampling rate by processing one output channel every 13 ticks This gives an effective sample interval of 13 x 3 x 1 25 48 75 us which corresponds to 20 5128 kHz 11 3 Timing of Input and Output Events In some situations it is important to know the precise timing of events on the active input and output channels When several input or output channels are active the timing of events will be slightly different for an ITC 16 compared with an I
21. name not number Also the names should not be too long because they will be used to label the axes in the data display window Dialogs are only presented for channels that have a cable connected see Section 2 2 The series of dialogs request signal names for the Analog Input channels the Analog Output channels the Digital Input channels and the Digital Output channels A dialog will not appear if there are no cables connected to a group of channels The dialogs for the Analog Input and Analog Output channels also request signal units Units should be given in the most convenient format For example if an instrument is measuring a small current then the most convenient units for the corresponding Analog Input signal may be nA If an Analog Output signal is controlling a fine mechanical positioner then most convenient units may be um All four dialogs have the same layout as shown at left for the Analog Inputs dialog signal names and units for ADC Inputs ADE Input Name Current Units ADC Input i Name Voltage Units cancer Cox 2 4 Signal Gains The Gain settings define the amplification between the signal at an external device measured in External Units e g p gm and the voltage signal at the analog input or output channel Gains can be specified in two different ways mY per External Unit d External Unit per Y cancer Cox 11 The Gains button brings up a series of di
22. on When the dialog is dismissed demo mode will be initiated and a Scope Window with a simulated trace will appear If the acquisition software is running in demo mode skip to section 2 2 10 Time ms 4 Connect electronic equipment to the digitizer After the digitizer has been installed and tested connect cables from the electronic equipment to the digitizer The digitizer has BNC connectors that are grouped and labeled as follows Connectors for Digidata 1320 Label ITC 16 and ITC 18 Label Analog input signals ANALOG IN ADC INPUT Analog output signals ANALOG OUT DAC OUTPUT Digital input signals DIGITAL IN TTL INPUT Digital output signals DIGITAL OUT TTL OUTPUT Triggering acquisition TRIGGER IN TRIG IN This manual will describe these connectors using the generic terms Analog Input Analog Output Digital Input Digital Output and Trigger Input Connect cables carrying voltage signals that are to be displayed and recorded to the Analog Input connectors Use the Analog Output connectors to deliver command voltages that will drive or trigger the electronic equipment The Digital Input connectors can be used to receive logic false true signals which are either 0 or 5 V The Digital Output connectors can be used to deliver logic signals which step between 0 and 5 V Digital Outputs are useful for triggering an instrument turning a signal on and off or for delivering timing signals to synchronize the equipment 5
23. opens a documentation window which describes keyboard shortcuts for controlling the digital oscilloscope Here 1s the list of the scope hot keys and their actions space bar halts the scope Automatically adjust Y axis range to the size of the signal Adjust Y axis to display the full Analog Input range Capture a trace for comparison with subsequent traces Erase the scope useful when the Erase between sweeps option 1s turned off O O mr p up arrow increase Y axis range zoom out down arrow decrease Y axis range zoom in right arrow increase sweep width left arrow decrease sweep width The following keys modify the Scope test pulse increase pulse amplitude decrease pulse amplitude 1 Invert pulse The following keys are active when using an internal trigger The test pulse in inactivated in this trigger mode increase trigger level decrease trigger level 3 4 Display The Display button in the Scope toolbar brings up the Scope Display Settings dialog scope Display Settings This asks whether to display information about the acquisition parameters in the scope window It also asks how sweeps are to be displayed AuUto Scale adjusts the scope display range to match the signal range every few sweeps bf Erase Between Sweeps Erase Between Sweeps When this check box is turned off each new O Subtract Baseline AC Couple sweep is overlaid on preceding sweeps This emulates the behavior of a
24. records the current and voltage responses on two input channels The series is repeated 4 times for a total of 24 episodes Spontaneous Events with Rate Acquires up to 100 episodes from an input channel but does not output any signal Searches each acquired episode for spontaneous events and monitors the event rate in a separate window Monitor Cm Acquires up to 500 episodes from an input channel while sending a square voltage pulse to an output channels Analyses each acquired episode and calculate the cell s membrane capacitance 74 10 3 Detailed Description of Each Protocol A Simple Pulse HE A Simple Pulse This protocol sends a 10 mV 10 ms voltage command pulse to Analog Output 0 The pulse is repeated up to 500 times and episodes are initiated as rapidly as possible The protocol acquires the signal at 20 kHz from the Analog Input channel named Current and displays it in the Acquisition Monitor window See Section 7 4 Channels for a description of how to add or remove channels from a protocol This protocol can be halted at any time by hitting the space bar It will halt automatically 10 when 500 episodes have been acquired anh Jk E JE i iL Time ms Pulse Average This protocol is identical to the previous protocol except that the 10 mV pulse is repeated only 50 times and the ensemble average of the acquired signal is calculated The running average is
25. signal the positions of the Lowpass Filter and Whole Cell Capacitance knobs The telegraphs may be read and the results stored with acquired data b4 Read Frequency Telegraph CO Read Capacitance Telegraph Frequency at ADC Input 5S o Capacitance at ADC Input la concer Cx The next dialog only appears if filter frequency or cell capacitance telegraphs are supported by the patch clamp amplifier Before each trace is acquired the signal on Analog Input 5 will be sampled and the voltage will be used to determine the amplifier s filter frequency setting This setting will be stored in the Notes section of the acquired data file 16 2 8 Clamp Mode The Clamp Mode feature is only relevant to electrophysiologists using a voltage clamp amplifier other than an AxoPatch 200B that is occasionally switched between current and voltage clamp modes The AxoPatch 200B has a mode telegraph output that signals a change of clamp mode When this telegraph is used the Clamp Mode program is not needed and is removed from the Configuration toolbar Most other voltage clamp amplifiers have only a single connection for an External Command signal The signal is interpreted as a voltage command when the amplifier is in voltage clamp mode and as a current command when the amplifier is in current clamp mode The gain and unit settings for the Analog Output channel that generates the External Command signal will change when the clamp mode is c
26. storage L Show Pulse Size oscilloscope Typing the letter e while the scope program is running erases the scope __ Show Sample Rate window When this check box is turned on the scope is erased before each new trace is O Auto Scale Scope displayed This emulates the behavior of a standard oscilloscope Sweeps Between Auto Scale a When this check box is turned on the signal Cancel l E ok amplitude 1s calculated over a short interval Subtract Baseline AC Couple 20 at the start of each sweep This baseline amplitude is subtracted from the signal before it is displayed This procedure emulates the AC couple option available on electronic oscilloscopes It is useful in situations where the signal of interest is superimposed on a larger drifting signal It stabilizes the signal at the center of the display range Show Pulse Size When this check box 1s turned on the amplitude of the test pulse is displayed in the scope window Show Sample Rate When this check box is turned on the acquisition sample rate is displayed in the scope window Auto Scale Scope When this check box is turned on the Y axis display range of the scope window is automatically adjusted to display the full amplitude of each signal auto scale The auto scale function is performed periodically The interval between each auto scale can be entered in the Sweeps Between Auto Scale field When the Auto Scale Scope check box is turned off the aut
27. tag hot key t depends on the sample rate and the size of the internal buffer in the digitizer If Chart is still running the buffer will not overflow in the next few seconds then a dialog appears asking for a YOu have 12 seconds to enter a comment comment describing the event Tag Comment The dialog indicates how long there is before the buffer overflows If there is 7 insufficient time to enter a comment then a ox tag is added without an associated comment Tags are displayed as vertical dashed lines in the chart window Tag comments are stored in the chart window s Notes together with the time that the event occurred These comments can be accessed by selecting Display gt Comments and Notes or by clicking the Note button in the vertical toolbar at the left of the chart window If the chart is halted and restarted the stop and start times will be included automatically in the list of event markers 30 4 6 Timebase The Timebase button in the Chart toolbar brings up the dialog shown at left This dialog controls the sample rate used by the Chart Settings Chart program to acquire signals When several input channels are active the signal on each channel will be sampled at the requested rate The sample rate should be at Sample Rate Hz 1088 least twice the highest frequency of interest in the signal The best results are obtained Record Length sec 1266 when the signal is low pass filtered at a
28. termination of the current protocol It also asks how long to pause Pause Between Protocols s before initiating the next protocol and whether to proceed if the current protocol is Run Protocol Named I U and Analysis interrupted by hitting the space bar on the keyboard t the termination of this protocol pause then run another protocol C Run Even If Acquisition Is Interrupted 59 7 15 P N Leak Subtraction The purpose of P N leak subtraction is to remove any linear component of the response to a stimulus and thereby to isolate the non linear response It is often used by electrophysiologists to study voltage activated currents It is termed leak subtraction because in a voltage clamp recording there is always a leak current present predominantly through voltage independent potassium channels This leak current has a linear current voltage relationship P N leak subtraction will remove the leak current from the response to a voltage step leaving only the voltage activated current The built in P N leak subtraction algorithm is relatively simple and does not permit the raw data to be recorded Only the leak subtracted data is displayed in the monitor window and recorded to disk A more sophisticated form of P N leak subtraction can be performed using online analysis programs It permits conditioning voltage steps to be delivered and raw data to be recorded An example protocol is supplied with AxoGraph that demonstra
29. the After Every Repetition field of the online analysis dialog LocalLanguage C Custom analysis program calculates and displays the standard deviation SD of the signal measured over the first 10 of each acquired episode void OnlineNoiseSD Declare local variables short window trace float yArray 0 float xMin xMax float noiseSD theScale string yUnits Get the first 10 of the frot trace GetFront window trace GetXRange window xMin xMax xMax 0 1 yArray yRange window trace xMin xMax Calculate the D noiseSD SD yArray Get the displayed Y axis units and scale factor DisplayedYUnits window trace yUnits DisplayedYScale window trace theScale Display the noise SD using the Y axis units DrawMove xMin Mean yArray 2 noisesSD DrawSetSize 12 DrawString concat noiseSD theScale 3 yUnits When the protocol is run the Acquisition Monitor window will display the baseline noise SD superimposed on each episode Note that the above program is exactly the same as the Scope custom analysis program presented at the end of Chapter 3 The program works in both contexts because it applies the analysis and directs the feedback to the front graph window When the Scope program is running this will be the Scope Window When a protocol is running this will be the Acquisition Monitor window The standard analysis programs AmplitudeSetup and AmplitudeM
30. time period The slope of the regression line is plotted in a separate monitor window The analysis program is a standard part of the data acquisition package see Chapter 8 Online Analysis A Simple 1 0 SSS p A Simple I V Episode 20 120 This protocol sends a series of 20 different voltage pulses to Analog Output 0 An episode is initiated every 0 5 seconds regular trigger mode The first pulse is 40 mV in amplitude and subsequent pulses are incremented by 10 mV per episode to a maximum of 150 mV This series of pulses is delivered twice for a total of 40 episodes The protocol 10 acquires signals from the Time ms Analog Input channels named Current and Voltage The acquired episodes describes the current voltage I V relationship of the preparation See Sections 7 6 and 7 7 for a description of how to add an incrementing series of pulses to a protocol lu dE 1E J 1S ee la JA E el E a T1 I V with Analysis This protocol is identical to the previous protocol except that an analysis program is run after the last episode is acquired This analysis program is applied to the recorded data file It only runs when the Record option is Data 665 0 5S pH used not with Preview The I V Episode 2i 2 analysis program generates a new graph window containing a plot of the current recorded on one channel versus the voltage recorded on the other I V plot The current
31. 2 The next dialog requests the number of pulses in the train and the onset time of the first pulse It also requests the pulse width and inter pulse interval If the options for incrementing width and interval were activated in the previous dialog then this dialog will also ask how much to increment the pulse width and inter pulse interval increment options are not shown at left The next two dialogs only appear if the protocol contains more than one waveform The protocol outputs 5 different waveforms Optionally increment pulse train onset pulse Width or interval with every waveform a increment Onset Time C Increment Pulse Width J Increment Inter pulse Inteval concer Cox Add or Edit a Pulse Train Increment pulse train onset pulse width oF inter pulse interval after every waveform Pulse Onset Increment ms i The first dialog asks whether the first pulse onset time the pulse width or the inter pulse interval are to be incremented following each waveform The next dialog asks how much to increment the first pulse onset time the pulse width and the inter pulse interval if these options were activated in the previous dialog If no increment options were activated this dialog does not appear The shape of the Pulse Train is now completely specified for every output waveform The waveforms in the protocol window are updated to display the new pulse trains 7 9 Edit Pulse One or more ou
32. 4 Test Seal 5 5 Test Cell 5 6 Test To Log 5 7 Test Setup 5 8 Test Pulse 5 9 Test Link 5 1 Introduction The Test Seal and Test Cell programs are designed to help electrophysiologists set up and perform patch clamp or voltage clamp experiments If the data acquisition software will not be used for electrophysiology experiments then review Section 1 5 Removing the Electrophysiology Features before skipping forward to Chapter 6 The Test Seal program monitors the input resistance of a patch recording electrode This is useful for checking the electrode is OK when it is first lowered into extracellular solution and for monitoring the formation of a GOhm seal The program delivers a series of small voltage clamp pulses typically 1 10 mV and monitors the clamp current in a scope window The electrode input resistance 1s calculated and displayed in the scope window overlaid on the trace The input resistance can also be directed to the log window The Test Cell program monitors three electrical parameters when recording in whole cell voltage clamp configuration These are the membrane capacitance and input resistance of the cell and the series resistance of the electrode The program delivers a series of small voltage clamp pulses and monitors the current in a scope window The three parameters are calculated from the shape of the response and displayed in the scope window overlaid on the trace The value of the three parameters
33. 42 86 Tea Lado 8 125 0 19 114 29 9 111 11 10 100 10 100 LS 88 889 11 90 909 12 3 80 IZ 03 333 13475 72 7127 This table assumes that only 1 Analog Input channel and 1 Analog Output channel are active The situation is more complicated when there are several active input or output channels The ITC can only process one input and one output channel every N clock ticks If there are 2 input channels or 2 output channels the effective sample interval is 2N ticks This places additional limits on the available sampling rates including the maximum sampling rate In general the maximum multi channel sampling rate is given by maximum multi channel rate maximum single channel rate maximum channels kHz where maximum channels is equal to the number of input channels or the number of output channels whichever is greater All Digital Output channels are processed simultaneously so any number of active Digital Output channels are counted as only 1 output channel when calculating maximum channels A similar argument applies for the Digital Input channels A case study demonstrating the restrictions on the available sampling rates due to hardware limitations An acquisition protocol samples data from 1 Analog Input channel while sending signals to 2 Analog Output channels and 2 Digital Output channels The 2 Digital Output channels are counted as a single output channel so there are 3 effective output channels There is only 1
34. 7 13 Custom Waveform for information on adding a custom waveform to a protocol Command mY 10 Time ms LTP Induction Pairing The phenomenon of long term potentiation LTP of a synaptic contact can be induced by pairing presynaptic stimulation with depolarization of the postsynaptic neuron This protocol delivers a train of 100 pulses at a rate of 100 Hz to Digital Output 0 to trigger a presynaptic stimulator Simultaneously a voltage command pulse of 50 mV is directed to Analog Output 0 to depolarize the postsynaptic neuron This paired stimulus is delivered every 10 seconds and is repeated 5 times The protocol acquires current and voltage data from the Analog Inputs named 0 5 Current and Voltage during the Time 5 paired stimulus See Section 7 6 Add Pulse for a description of how to add a train of pulses to a protocol LTP Induction Pairing Command mY Trigger Stim Toggle Digital Output 0 81 This protocol does not output any signal but it acquires up to 500 episodes each 20 ms second long from the Analog Input named Current Before the start of each episode the protocol runs an online program that toggles the setting of Digital Output 0 between low 0 Volts and high 5 Volts The purpose of this protocol is to demonstrate the command for setting the levels of Digital Output channels This command can be used to set the levels
35. Add an event marker tag to the log window and data file notes c Capture a trace for comparison with subsequent traces e Erase monitor useful when Erase between sweeps is turned off up arrow increase Y axis range zoom out down arrow decrease Y axis range zoom in right arrow increase time range zoom in left arrow decrease time range zoom out lt Scroll time range to the left gt Scroll time range to the right The following keys are active when using an internal trigger increase trigger level decrease trigger level 6 6 Protocol One or more Protocol windows are open Select the protocol to run 1 1 and Analysis Pulse Average cancer Cx 6 7 File Name 42 The Protocol button brings up a dialog asking which protocol should be used next time the Preview or Record programs are run If no protocols are open then a dialog will appear asking whether to open an existing protocol or create a new protocol see Section 6 2 Preview When the Record program is run a new data file is created to receive the acquired data The name of the new data file will be generated automatically from a root name and an incrementing sequence number The file name and sequence number are combined to generate a new data file name sequence number is incremented with each run File Name Glycine 36 9 98 Sequence Number concer Cx The File Name button initiates a series
36. After First Episode l Auto Scale After Each Episode Auto Scale After First Episode If this O Subtract Baseline AC Couple option is turned on the Y axis range of each monitor channel will be adjusted to display Mf Monitor Acquired Episodes the full amplitude of the signal auto scale after the first episode has been acquired LJ Only Monitor Never Record When this option is turned off the auto scale function can be performed at any time ox by hitting a hot key see Section 6 5 Auto Scale After Each Episode If this option is turned on the monitor will be auto scaled after every episode This option will slow the maximum episode repetition rate and at high repetition rates it will cause the axes to flash It is only recommended for slow episode repetition rates Subtract Baseline AC Couple When this option is turned on the amplitude of the signal on each monitor channel is calculated over a short interval at the start of each episode This baseline amplitude is subtracted from the signal before it is displayed This procedure emulates the AC couple option available on oscilloscopes It 1s useful in situations where the signal of interest is superimposed on a larger drifting signal It stabilizes the signal at the center of the display range This option only affects the signal displayed in the monitor It does not affect the signal recorded in the data file Monitor Acquired Episodes If this option is tur
37. AxoGraph X Data Acquisition Manual PLEASE NOTE e For the best figure quality when reading this document onscreen the zoom setting should be 147 e If the zoom setting has changed type 147 into the zoom field in the toolbar at bottom left e Search for a key word by clicking on the binoculars icon gin the toolbar at top right e Navigate this document using the bookmarks at left The bookmarks can be toggled in and out by clicking the toolbar icon at bottom left e Alternatively navigate using the table of contents on following pages Clicking on an item of interest will jump to the relevant page of the main text Copyright Copyright 1991 2003 by Dr John Clements All rights reserved Disclaimer All product names mentioned in the AxoGraph 4 9 Data Acquisition Manual are registered trademarks of their respective manufacturers Any mention of specific products should not be construed as an endorsement by Axon Instruments 11 Chapters pd p m SONDAAR WN j N Introduction Configuration Digital Oscilloscope Digital Chart and Tape Recorder Electrophysiology Test Pulse Protocol Driven Acquisition Create or Edit a Protocol Online Analysis The Protocol Launch List Example Protocols Precise Acquisition Timing Optimizing Performance Contents A RC 1 1 1 Explore the Acquisition Software Without a DIBITIZET cccccnnnononononancnonnncncnnnnnnnnnanononnnnnnnnnnccnnanananess l 1 2 Hardware Requirements
38. LOO IC VS o ack i ash a ares a ae casa in Sala are Sean auie ces gala xd ee seas a saucee aan E a een aca eaaee 29 LEEY MI ol hc ee ae En Oe a Re AEM RI ESI TiO E Seen OP OPT CET STR aS ee aE STENT Srey VE 29 AE ERA O 30 4 A E E A E E EAE 30 O AT a a N T A O Meets near ee 31 PATA A AA AATETTA II TAS ATS TTS 31 A II A A O 32 A E II 33 S Electrophysiology Test Pulse sciscvcasccdoseskciessoscececdelateteWaxedececccieseeucssuedoccdeceecavedecudebatesacedeucsatseesvevetedeascsasdeeases 34 Dich O O A A ee 34 Ie SO AA E EE Re Re E A O RON AAE 35 So Test Pube HORES 35 E PTT 36 SN E E EEE E E E ee Re E E PI E EE S ES E EE EE 36 BO LS E e A E O E EE EN O T AIEE IEE T E T AETA 37 TESSA AA TA 37 Sl AP A E EEE II II aa aaah ates e aco ceneES 38 A A A O SS neo ao RE OOM SR ean ORE TS SEER Po aE SITE SES 38 6 Protocol Driven ACQUISITION ep vzsieccgescsbeds desusscencececeasexaceasedesdeeceda Geevlasenaicedecacwaauenadecdestedetesstuads tesdedesieecedecteceiees 39 O TINE OCC IO Isto asc sce crane rane cals hen aalea feos Sus os iene a Sreceu ah oeaersaenaeeneae E E EE 39 A ca ohh oi ee ee A Fee ee SSeS 40 O E e o o A II IN 40 wr PRU INS ph cit are cee se ashe E E eames 41 A IO O ncauaa aime nunanaaumeanenasee 41 NS IN ae 42 O NA E o O O E O O A ORR RP RER 42 60 Delete Data Pes A odie A 43 TP Create or Edita Protocolar aisla A 44 e TO E Oaa A E 44 TE EE E en ieee mC re rt cree 45 E PP AO 45 E SRE ee nee envi Poe g PST ae eee eS Ea ee oe a 46 FD Me
39. N This protocol delivers a voltage clamp waveform consisting of two action potentials AP s separated by 10 ms test stimulus The paired action potentials are preceded by a series of 8 pairs of inverted scaled down action potentials pre pulses The output waveform is repeated 5 times The recorded response to each waveform is analysed using P N leak subtraction see description of previous protocol The linear or passive component of the current response is automatically subtracted to reveal non linear or voltage dependent components Command m tT a This protocol uses the online P N analysis programs PonNSetup and PonNMeasure see 100 206 Chapter 8 Online Analysis Time ms This protocol is relatively difficult to set up The online P N analysis programs expect the protocol to contain 4 pulses in the following order 1 Pre pulse Holding Level 2 Pre pulse Train 3 Test Holding Level 4 Test Pulse So the first step 1s to build a protocol in this format For example the protocol named I V with P N Leak Subtraction could be copied then edited so that it only contained one waveform but was repeated 5 times The second step is to replace the waveform containing rectangular pulses with a synthetic waveform containing pairs of AP s This can be done using AxoGraph s built in programming environment 79 Ramp Then Link to 1 1 gt HH Ramp Then Link to I V Ramp Then Link t
40. TC 18 The ITC 18 hardware separates the jobs of processing a channel moving a numerical value in or out and updating a channel changing the output voltage or sampling the input voltage Although only one input and one output channel can be processed at a time all channels can be updated simultaneously Consider a protocol with a sample rate of 10 kHz 2 Analog Input channels 1 Analog Output and 2 Digital Output channels The protocol specifies that a step output is applied to the Analog Output channel and both Digital Output channels at 1 ms after the start of the acquisition sweep At exactly 1 ms both input channels are sampled and all 3 output channels are stepped to a new voltage Even if a response occurs instantaneously it will not DAC Output be sampled on the Analog Input channels until the Command next sample point at 1 1 ms In general the first point of the response to a step command is not sampled ADC Input until 1 sample interval after the step is applied The Response figure at left shows the sampled Analog Input and A the Analog Output numerical values blue circles 09 1 41 42 and the actual voltage signals black lines This figure assumes that the response is instantaneous and Time ms that an ITC 18 is used 86 Both the ITC 16 and the Digidata 1320 series are less sophisticated than the ITC 18 These digitizers process and update a channel at the same instant When several channels are
41. Trigger The regular trigger mode can be used for both the digital oscilloscope and protocol driven data acquisition see Sections 3 7 and 7 12 In this mode acquisition of a sweep or episode is initiated at regular intervals The line trigger mode also initiates sweeps at a regular interval that is a multiple of the power line cycle time 1 50th or 1 60th second The precise timing of the sweep initiation is determined with reference to a microsecond clock on the computer motherboard The data acquisition software reads this clock at the start of the first sweep then calculates the time in microseconds at which to start each subsequent sweep The clock is read inside a tight loop and when it reaches the calculated start time the sweep 1s initiated The precision of this trigger scheme is limited by the accuracy and stability of the computer s clock and by the software s ability to read it in a tight loop without interruption The clock on a Macintosh motherboard was designed primarily for tracking the day and date It is reasonably accurate but may drift by a few seconds per day This corresponds to a few milliseconds per minute and is enough to prevent tight time lock to the power line cycle in line trigger mode over a minute or more The Macintosh operating system can interrupt a running program to perform low level system tasks such as monitoring a network connection If an interrupt occurs close to the sweep initiation time this coul
42. a window If the same name is given to two different channels AxoGraph will report an error and repeat the series of dialogs The signal units should be given in the most convenient form For example if an instrument is measuring a small current then the most convenient units for the corresponding Analog Input channel may be nA nanoAmps If an Analog Output is controlling a fine mechanical positioner then most convenient units for this channel may be um micrometers e The next two dialogs request the gains for the Analog Input and Analog Output channels The gains can be specified in either of two different ways The user can choose the most convenient format The options are a milliVolts at the digitizer per unit at the electronic equipment or b units at the electronic equipment per Volt at the digitizer For example An amplifier generates an output signal of 10 mV per nA The gain will be either 10 mV at the Analog Input per nA at the amplifier or 100 nA at the amplifier per Volt at the Analog Input A mechanical translator generates a movement of 0 4 um per mV of command signal The gain will be either 2 5 mV at the Analog Output per um at the translator or 400 um at the translator per Volt at the Analog Output e The next dialog requests the holding levels for the Analog Output channels Each Analog Output will be set to the holding level when AxoGraph is launched and returned to the holding level betwee
43. active they will be updated at different times within the sample interval Consider a protocol with a sample rate of 10 kHz 2 Analog Input channels Analog Output and 2 Digital Output channels The protocol specifies that a step output is applied to the Analog Output channel and both Digital Output channels at 1 ms after the start of the acquisition sweep At exactly 1 ms the first Analog Input is sampled and the Analog Output is stepped to a new voltage Then at 1 05 ms the second Analog Input is sampled and the two Digital Output channels are stepped to a new voltage Thus the second Analog Input channel and the Digital Output channels are offset in time by 1 2 a sample interval relative to the first Analog Input channel and the Analog Output channel ii DAC Output 1 12 sample point offset TTL Output 1 a ee es TTL Output 2 a i a i ADC Input 1 ADC Input 2 a A ooo mm 0 39 1 1 1 1 2 Time ms The figure at left shows the Analog Input the Analog Output and Digital Output values blue circles and the actual voltage signals black lines The figure assumes that the response to the Analog Output is instantaneous and that an ITC 16 is used Note that the 1 2 a sample interval offset arrows in this schematic is not shown in the protocol graph or in the acquired data graph To work out the precise sequence of events when using multiple channels of an ITC 16 or Digidata the channel processing sequence is requir
44. alogs that asks about the signal amplification or conversion factor for each of the electronic devices connected to the digitizer The first dialog ask how the gains are to be specified in the subsequent dialogs A choice is offered simply for convenience because different devices specify their gains in different ways The two subsequent dialogs request the signal gain amplification and conversion factor for each Analog Input and Analog Output channel that has a cable connected If the gain of an Analog Input or an Analog Output signal is specified via a telegraph then that channel will not appear in the gain dialog see Section 2 7 Two examples follow signal gains for DAC Outputs bain mm at digitizer per External Unit Piezo mY at DAC Tv um ok signal gains for DAC Outputs bain External Unit per at digitizer Piezo wm at DAC_1 o Analog Output Gain A piezoelectric mechanical positioner generates a movement of 40 um per Volt The positioner s command input is connected to Analog Output 0 and the signal name and units for this channel are Piezo and um see Section 2 3 Depending on the option chosen in the first dialog the gain will be specified either as 25 mV per um or as 40 um per V 12 Analog Input Gain A voltage clamp amplifier generates a signal of 10 mV per pA The amplifier s output is connected to the digitizer at Analog Input 0 and the signal nam
45. amplitude of a response that occurs within a specified time range during each episode The protocol should specify multiple repetition of a single output waveform a stimulus The AmplitudeSetup program name should be added to the Command dialog field After First Repetition It creates a new graph window titled Amplitudes that will monitor the response amplitude while the protocol is running It resizes the standard Acquisition Monitor Window so that both monitor windows are visible on the screen It then poses two dialogs that requests the time range over which to calculate the baseline signal and the time range within which the peak of the response will occur Peak amplitude is measured relative to the baseline signal If an Amplitudes monitor window already exists the amplitude measurements will be appended to this window The range dialog will not appear and the amplitude measurement range that was specified previously will be used To change the measurement range the monitor window must be closed before the protocol is run AmplitudeMeasure This program works in conjunction with the previous program It measures the response amplitude after each episode is acquired and adds it to the Amplitudes monitor window The AmplitudeMeasure program name should be added to the Command dialog field After Each Repetition The example protocol titled Pulse with Amplitudes demonstrates the use of these two programs 64 RsAndAmplitudeSetup
46. ave the same units e g mV Chart delivers an optional test pulse to one of the DAC or TTL Output channels send pulse to channel Command Piezo No Output Pulse cancer Cox 31 The digital chart recorder can optionally deliver a regular test pulse to one Analog Output or Digital Output channel The second dialog asks which channel if any is to receive the regular test pulse The frequency duration and amplitude of the test pulse can be edited via the Pulse button in the Chart toolbar see Section 4 9 below 4 8 Gains The Gains button in the Chart toolbar brings up two dialogs that request the signal gain amplification and conversion factor for each Analog Input and Analog Output channel These dialogs are described in detail in Section 2 4 Signal Gains The Gains button is also found in the Configuration toolbar but is duplicated in the Scope and Chart toolbars for convenience 4 9 Test Pulse Chart Settings Specify the amplitude of the test pulse Pulse Size mu The digital chart recorder can optionally deliver a regular test pulse to one Analog Output or Digital Output channel during continuous recording see Section 4 7 Channels If a test pulse channel has been selected then the Pulse button in the Scope toolbar brings up a series of three dialogs requesting the time between test pulses and their amplitude and duration The first dialog only appears if an A
47. aveform 1f the protocol 1s not repeated or to the dialog field After Last Repetition 1f the protocol is repeated see previous section This will guarantee that a complete data file is available for analysis The program name is followed by four numerical parameters in brackets separated by commas These specify two time ranges over which to measure the current and voltage amplitudes The time range parameters are given in milliseconds The average amplitude of each episode is calculated over the specified time range and the current amplitudes are plotted against the voltage amplitudes in a new graph window IV AnalysisRequestRange This is very similar to the previous program but does not require the four time parameters that define the current and voltage measurement regions Instead when the protocol is run the program poses a dialog requesting the measurement regions The example protocol titled I V with Analysis uses this program IV AnalysisNoVoltageRecord This is similar to the previous program but is designed for a protocol which does not record the voltage pulses on an Analog Input channel Instead the amplitudes of the voltage pulses are obtained from the acquisition protocol The voltage pulses are delivered to an Analog Output channel by the protocol When the protocol is run a dialog requests the time range for measuring the current amplitude AmplitudeSetup This program in cooperation with the following program monitors the
48. bsequent dialog is used to identify the that folder will be unlocked and can then be Proceed modified or deleted 44 7 Create or Edit a Protocol 7 1 Introduction 7 2 Create 7 3 Timebase 7 4 Channels 7 5 Repetitions 7 6 Add Pulse 7 7 Define Pulse or Ramp Shape 7 8 Define Pulse Train Shape 7 9 Edit Pulse 7 10 Delete Pulse 7 11 Display 7 12 Trigger 7 13 Custom Waveform 7 14 Add a Link 7 15 P N Leak Subtraction 7 16 Extract Protocol 7 17 Convert To Protocol 7 1 Introduction Data acquisition is controlled by a set of instructions contained in a protocol A protocol is a graph file that contains one or more Analog Output and Digital Output waveforms It also contains instructions about which Analog Input and Digital Input channels to record signals from how to trigger acquisition of each episode how many episodes to acquire etc A single protocol can output a complex series of waveforms When necessary more complex output sequences can be generated by linking several protocols together in a chain so that they run one after another This chapter contains detailed information about how to create and edit a protocol and how to link two or more protocols Example protocols are supplied in the Acquisition Protocols folder which demonstrate most of the available features see Chapter 10 The example protocols can by customized or extended using the commands described in this chapter AxoGraph presents a pop up menu in t
49. can also be directed to the log window AxoGraph presents a pop up menu in the toolbar at the bottom left of the screen When the Test Pulse item is selected in this pop up menu a new window titled Test Pulse Window will appear The following sections describe the function of each of the buttons in the Test Pulse toolbar 35 5 2 Test Channels The Test Channels button brings up two successive dialogs that define which Analog Input channel is connected to the voltage clamp current signal and which and Analog Output channel is connected to the voltage clamp command signals This must be done before using Test Seal or Test Cell The Test Seal and Test Cell programs analyse the voltage clamp current in response to a series of small voltage pulses Clamp current recorded on ADC Input channel Current O Voltage cancer Cx The Test programs deliver a voltage clamp command pulse to one of the DAC Output channels Voltage clamp command pulse sent to channel Command 0 Piezo concer Cx 5 3 Test Pulse Hot Keys Hot keys can be used to modify the scope display and the test pulse amplitude while the Test Seal or Test Cell program is running They are a subset of the scope hot keys described in Section 3 3 To display hot key information switch to the Scope toolbar and click on the Hot Key button space bar halts the test program a Automatically adjust Y axis range to the size of the signal
50. d again the data window will be closed before a new data file is created If the root data file name has not been specified a series of three dialogs will appear requesting the root name the sequence number and the location of the folder in which data files should be stored The root 41 name and destination folder can be changed via the File Name button which brings up the same three dialogs These dialogs are described in section 6 7 File Name 6 4 Resume If a protocol is interrupted by hitting the space bar then the Resume button continues running the protocol from the point at which it was interrupted The episodes acquired by the Resume program are appended to the data file window The Resume button will only work if a previous Record was interrupted and if the Can not Resume the protocol corresponding data file is still open If these conditions are not met the following dialog appears Resume only works when the previous Record was interrupted before the end of the protocol and the corresponding data file is still open 6 5 Hot Keys The Hot Keys button opens a documentation window with information about keyboard shortcuts for controlling the display format of the Acquisition Monitor window Here is a list of the monitor hot keys and their actions space bar terminates acquisition a Auto adjust Y axis range to the size of the signal f Adjust Y axis to display the full Analog Input range t
51. d prevent precise triggering For optimum performance it is recommended that the network connection is inactivated when acquiring data This can be done by restarting the computer while holding down the shift key The acquisition software always notes the time that a sweep is actually triggered and compares this with the requested trigger time If regular triggering is not achieved this will be reported when the acquisition is terminated 11 2 Hardware Limitations on the Sampling Rate Most digitizers place limitations on the available sampling rates because they are driven by an internal clock with a fixed tick rate The clock in the Digidata 1320 and 1321A tics every 0 1 us the ITC 16 clock ticks every 1 0 us and the ITC 18 every 1 25 us The sample interval must be a multiple of the clock interval and can not be less than 2 us 500 kHz for the Digidata 1321A 4 us 250 kHz for the Digidata 1321A or 5 us 200 kHz for the ITC digitizers A 5 us interval corresponds to 5 ticks on an ITC 16 or 4 ticks on an ITC 18 84 A table of the available sample intervals and sample rates Digidata 1320 Digidata 1321A Interval us Rate kHz Interval ps Rate kHz 4 250 2 500 4 1 243 90 Zel 476 19 4 2 238 10 a 454 55 4 3 ZA O O 434 78 4 4 221 27 2 4 416 67 4 5 222 22 PARE 400 4 6 217 39 2 6 384 62 4 7 212 71 2 7 370 37 ITC 16 ITC 18 Interval us Rate kHz Interval ps Rate kHz 5 200 g 200 6 166 67 6 29 160 7 1
52. d the whole cell membrane capacitance and input resistance A series of voltage clamp pulses is delivered and the current response is monitored The amplitude of the pulse and the passive electrical parameters are displayed at the bottom of the scope window Hitting the space bar on the keyboard halts 104 M0 the scope y 494 Current pA _ Test Pulse 5 m Series Resistance Membrane Capacitance Input Resistance The passive electrical parameters are calculated z by measuring the steady state current at the end of each pulse response then fitting an exponential to the first few milliseconds of the response The fitted exponential and steady state amplitude are shown as a red Time ms 37 line superimposed on the current response The amplitude is measured relative to the baseline current before the start of the response as indicated by the horizontal dashed green line Several factors will influence the reliability of the Test Cell parameter estimates It 1s important that the sampling rate and low pass filter frequency permit the current decay to be captured without distortion Also the test pulse should be long enough for the current response to decay to a steady state level Test Cell will provide a reliable estimate of series resistance and input resistance under most circumstances Test Cell only provides an accurate estimate of the total membrane capacitance when a cell is isopotential
53. de is complete If acquisition is interrupted part way through an episode the incomplete episode will be discarded A protocol may contain one or more waveforms per output channel As each waveform is output an episode is acquired When all waveforms in the protocol have been output the acquisition program loops back to the first waveform The protocol specifies how many times the series of output waveforms will be repeated The two programs that initiate data acquisition are Preview and Record Selecting Preview runs a protocol delivering output waveforms and monitoring the input signals but does not save the acquired signals Record runs a protocol and saves the acquired signals to a new data file A protocol window must be open before running either of these data acquisition programs AxoGraph presents a pop up menu in the toolbar at the bottom left of the screen When the Acquisition item is selected in this pop up menu the data acquisition programs are loaded into the toolbar AxoGraph assumes that data acquisition is about to commence and checks whether a protocol window is open If no protocols are open a dialog will appear asking whether to open an existing protocol or create a new protocol see Section 6 2 The most common response is to select Open Protocol then find and open a protocol file for example any file in the Acquisition Protocols folder Next the Acquisition Monitor window will appear This window monitors the input sig
54. display select Program gt Acquisition Options gt Performance and turn on the Interleave Acquisition and Display check box in the resulting dialog This option will not appear when a Digidata 1320 series digitizer is connected Scope and Data Acquisition Settings Faster sweep cycle times can be achieved by interleaving the acquisition af each sweep With the display of the previous sweep LJ Interleave Acquisition and Display concer Cx 88 12 3 Buffer Acquired Data to Memory By default protocol driven data acquisition displays each episode in the Acquisition Monitor window then saves it to a separate data file window as soon as it is acquired This second step involves the adjustment of the data window s display parameters and incurs a performance penalty Faster repetition rates can be achieved by buffering the acquired data to memory then moving it into the data window when the protocol terminates However it is recommended that buffering is turned off unless very fast episode repetition rates are required The performance gain is generally less than 15 and buffering can cause data to be lost if memory overflow occurs during acquisition To buffer acquired data to memory select Program gt Protocol gt Buffer and turn on the Buffer Acquired Data to Memory check box in the resulting dialog Faster episode repetition rates 7158 can be achieved by bufterring data to memory then building the data window on prot
55. down the Command key and typing 1 Cmd 1 The second protocol named A Simple I V Protocal 4 can be launched by selecting Launch 2 or by typing Cmd 2 se Similarly for the other protocols on the LK staunch ist 70 When a protocol is launched via a launch list command or keyboard shortcut the protocol window will be opened and data Run the protocol Pulse with Amplitudes acquisition will be initiated in either in the selected mode Preview or Record mode A dialog will Preview data not saved l appear asking which mode to use Record data saved to new file O Preview This dialog can be bypassed for faster launching Bypass requires that the launch mode for a given protocol be predefined as aah described in the next section 7 Record 9 3 Default Launch Mode Some protocols should always be launched in either Preview or Record mode A default Change the default launch mode launch mode can be predefined for each for the selected protocol protocol on the launch list Selecting Program gt Launch Protocol gt Default Launch Mode brings up a dialog which selects one A Simple 1 8 protocol on the launch list CO Pulse with Amplitudes concer Cx A Simple Pulse The subsequent dialog defines the default launch mode for the selected protocol When launching the protocol A Simple Pulse always Preview Cy Record y Ask cancer Cx 71 9 4 Ope
56. e Measure Amplitude This file is located in the folder Plug In Programs Acquisition Programs Protocol A description of the function of these programs and instructions for attaching them to a protocol are given in Section 8 3 above LocalLanguage C Online analysis program calculates and displays the peak amplitude of the signal measured over a specified time range of each sweep Define the monitor window name as a global variable because it is used by both the AmplitudeSetup and AmplitudeMeasure programs string AmplitudeFileName AmplitudeFileName Amplitudes void AmplitudeSetup boolean newWindow Check that this protocol is repeated at least twice The global variable prRepetitions defines the number of repetitions if prRepetitions lt 1 Exit Create a new monitor window using the NewAnalysisWindow utility routine NewAnalysisWindow AmplitudeFileName newWindow 1 68 SetFront acgAnalysisWindow 1 SetFront monitorWindow 1 if newWindow If a new monitor window was created request new baseline and peak amplitude measurement regions The parameters acqRefMin acgqRefMax acgAnalysisMin acqAnalysisMax are global variables available for use by any acquisition program GetRange monitorWindow Subtract Baseline acqRefMin acgRefMax GetRange monitorWindow Measure Peak Amplitude acgAnalysisMin acqAnalysisMax void AmplitudeMeasure
57. e a new protocol Mo protocol windows are open Either open a protocol file or create anew protocol window f 4 If Open Protocol is selected then a standard Open Protocol open file dialog appears Use it to open a Nei Protacai protocol file for example any file in the Acquisition Protocols folder Cancel ok f ox If New Protocol is selected a series of dialogs appears requesting the information needed to build a basic protocol These dialogs are described in detail in the next chapter In particular see section 7 2 Create a Protocol If several protocols windows are open the Preview program must decide which one to run If the front window is a protocol when the Preview button is pressed then that protocol will be selected Otherwise the protocol that was run most recently will be selected Any of the open protocol windows can be selected by pressing the Protocol button see Section 6 6 Protocol 6 3 Record The Record button runs a protocol and saves the acquired data to disk Hitting the space bar halts the protocol Record behaves exactly like Preview except that a new graph window is created to receive the acquired data The name of the new data file will be generated automatically from a root name and an incrementing sequence number At the completion of the protocol the new data file is saved to disk and locked to prevent accidental modification at a later date If the Record button is presse
58. e and units for this channel are Current and pA see Section 2 3 Signal gains for ADC Inputs Gain mW at digitizer per External Unit Current m at ADC_O pA Depending on the option chosen in the first dialog the gain will be specified either as Cancel i ox 10 mV per pA or as 100 pA per Volt Signal gains for ADC Inputs Gain External Unit per at digitizer Current pA at ADC O ok 2 5 Holding Levels The Holding Levels button brings up a dialog that requests the level at which to The DAC Output waveform is superimposed on a hold the signal on each of the active Analog A E ee Heer Output channels those that have a cable Holding level for connected Any command signals sent to an Analog Output will be superimposed on Command mu E the holding level For most purposes the holding levels will be set to zero as shown Piezo um at left The signals on the active Analog Output channels will be set to the requested holding levels immediately after clicking OK in this dialog All output channels will be set to their holding levels when AxoGraph is launched and at the termination of an acquisition protocol see Chapter 6 Protocol Driven Data Acquisition Scope and Chart test pulses will be superimposed on the holding level of the pulse output channel 13 2 6 Save and Load Configuration A mechanism is provided for switching quickly between different c
59. e top of the SCSIProbe window there is a popup menu labeled SCSI Buses Click on this popup menu In the list there should be an item labeled AdvanSys If this item does not appear then the AdvanSys PCI gt SCSI card has not been installed correctly or is not working correctly Select the item labeled AdvanSys A Digidata 1320 item should now appear in the list labeled SCSI Devices If this item does not appear then there is a problem with the connection to the Digidata or with the Digidata hardware If the Digidata 1320 item does appear then all the hardware is correctly installed and connected Problem AxoGraph data acquisition features are not available under the Program menu Most probable reason the AxoGraph data acquisition package is not loaded Open the AxoGraph 4 9 folder Open the folder named Plug In Programs and confirm that the Acquisition Programs folder is not present Search for this folder then move it into the Plug In Programs folder If the Acquisition Programs folder is already present then there may be more than one copy of AxoGraph installed on the computer Delete the other copies of AxoGraph Alternatively make sure that all AxoGraph aliases point to the latest version Launch AxoGraph 4 9 The acquisition programs should now appear under the Program menu Problem AxoGraph data acquisition programs only run in demo mode Most probable reason either the digitizer has not be
60. e voltage current clamp Mode knob This telegraph can automatically adjust the current voltage units and gain setting for the AxoPatch External Command channel wf Read Mode Telegraph Mode at ADC Input 6 ox 14 Next one or more dialogs appear asking for the channel that each of the telegraph signals is connected to The first dialog asks about the gain telegraph connection For Instrutech digitizers the telegraph signal must be connected to an ADC input channel as shown at left Before each trace is acquired the signal on Analog Input 7 will be sampled and the voltage will be used to adjust the gain for the signal on Analog Input 0 This is the Scaled Output signal from the patch clamp amplifier The gain telegraph dialog has a slightly different format when the digitizer is a Digidata 1320 series as shown at left This is because the Digidata has four dedicated telegraph input channels on the rear panel Telegraph signals must be connected to these channels Before each trace is acquired the signal on Rear Panel Telegraph 0 will be sampled and the voltage will be used to adjust the gain for the signal on Analog Input 0 This is the Scaled Output signal from the patch clamp amplifier The next dialog only appears if the amplifier is an AxoPatch 200 This amplifier supports a Mode telegraph which signals whether the AxoPatch is in current clamp or voltage clamp mode Both the Scaled Output and the External
61. eSetup This program in cooperation with the following program monitors the average rate of occurrence of spontaneous events with amplitudes larger than a specified threshold during each episode The protocol should specify multiple repetitions and have no output waveform The EventRateSetup program name should be added to the Command dialog field After First Repetition It creates a new graph window titled Event Rate that will monitor this parameter while the protocol is running It then poses a dialogs that requests the amplitude threshold for the events It also requests the minimum time between events so that an events with noise on its peak is not counted as two events and the maximum expected number of events per episode which is used to adjust the y axis range of the Event Rate monitor window EventRateMeasure This program works in conjunction with the previous program It measures the rate of occurrence of spontaneous events after each episode is acquired and adds the result to the Event Rate monitor window The EventRateMeasure program name should be added to the Command dialog field After Each Repetition The example protocol titled Spontaneous Events with Rate demonstrates the use of these two programs PonNSetup This program in cooperation with the following program performs P N leak subtraction The purpose of P N leak subtraction is to remove any linear component of the response to a stimulus and thereby to isolate the
62. earch measure seal resistance during patch recordings measure electrode series resistance membrane capacitance and membrane resistance during whole cell recordings 2 Configuration 2 1 Introduction 2 2 Connections 2 3 Channel Names 2 4 Signal Gains 2 5 Holding Levels 2 6 Save and Load Configuration 2 7 Telegraphs 2 8 Clamp Mode 2 1 Introduction AxoGraph needs to know which input and output channels of the digitizer receive connections It also needs to know the gain amplification and the units for the signal carried by each connection AxoGraph uses this information to scale the input and output signals and to label the axes in the data display windows There are five separate programs that can be used to describe the connections and signals Connections Channel Names Gains Holding Levels and Telegraphs The program Full Configuration runs each of these five programs one after the other Running all five programs fully describes the connections to the digitizer If the connections and the signal gains remain constant configuration will only need to be performed once when the data acquisition software is first installed Signal amplification is the configuration parameter that is most likely to be altered When ever the gain of a signal is adjusted run the Gains program to inform AxoGraph of the change AxoGraph presents a pop up menu in the toolbar at the bottom left of the screen When the Configuration item is selected
63. easure will be presented below But first several global variables and a utility procedure are introduced The standard online analysis programs take advantage of several global parameters defined in a file named Custom Analysis Globals Here is a brief description of each of these global variables acqRefMin acqRefMax eee baseline time range acqAnalysisMin acqAnalysisMakx the peak measurement time range acqRsMin acqRsMak 000eeeeees time range for calculating series resistance ACGRSPUISESIZE wiadecccarincdnesascrsasedveatsondenes series resistance test pulse amplitude acqAnalysis Window 00esesseeeees destination window for the analysis results acqAnalysisEpisodes 00eeseseeees a counter for number of episodes analysed acqAnalysisData ooooocccccccncccnonnnnnnnnnns an array that accumulates the analysis results ac RSDAta cusadacisa alot ias an array that accumulates the Rs results acqAnalysisAVerage ccccccccnnnnnnnnnnnanononos an array for ensemble average calculation 66 67 These variables are defined as global for two reasons 1 so that they can be accessed by all the online analysis programs and 2 so that the time range values will default to their most recent settings in the range dialogs The standard online analysis programs also use several global variables that are created and maintained by the data acquisition programs monitorWindow cocoocooocccnccc
64. ed All active Analog Input and Analog Output channels are processed and updated in ascending numerical order followed by the active Digital Output channels 87 12 Optimizing Performance 12 1 Memory and Network Settings 12 2 Interleave Acquisition and Display 12 3 Buffer Acquired Data to Memory 12 4 Disable Acquisition Monitor 12 1 Memory and Network Settings AxoGraph acquires data to memory so the maximum recording time is limited by the available memory This can be increased simply by quitting all other programs before launching AxoGraph It can also be increased by installing additional memory a minimum system configuration of 32 MByte is recommended The amount of available memory can also be increased by activating virtual memory however this incurs a performance penalty and is not recommended The maximum sustained sampling rate and the maximum sweep rate will both be reduced by a few percent initially However the performance penalty increases dramatically as the amount of acquired data approaches the limits of physical memory Once this limit is reached the acquisition program will come to a grinding halt and may freeze causing the loss of all the acquired data This is a flaw of the current implementation of virtual memory It is therefore recommended that virtual memory is switched off during data acquisition This can be done via the Memory control panel It can also be done by restarting the computer while holding down t
65. en correctly installed and connected see above or the demo version of the data acquisition package is loaded To check whether the demo acquisition package is loaded open the AxoGraph 4 9 folder Open the folder named Plug In Programs and confirm that the Acquisition Programs Demo folder is present Drag this folder out of the Plug In Programs folder Search for the folder named Acquisition Programs then move it into the Plug In Programs folder Launch AxoGraph 4 9 A text box will appear saying that the programs are loading If no dialog box appears as the programs are loading then the acquisition package has found and initialized the digitizer Problem data acquisition programs run but do not display the correct signal Most probable reason the digitizer is not correctly connected to the electronic equipment or the acquisition package is not correctly configured or the digitizer 1s not working correctly It is important that the electronic equipment and the digitizer reference their electrical signals to the same ground This is not usually a problem as most equipment uses the BNC cable outer shield as the signal ground However some amplifiers output a signal that is not referenced to the BNC cable shield but to a separate signal ground They have an external connector usually labeled signal ground that must be connected to the same ground as the digitizer Instrutech digitizers have an external connector labeled signal gro
66. er for the chart data file A new chart window will then appear with the selected name The following sections describe the function of each of the buttons in the Chart toolbar E analyze Chart Name Name Hot keys Keys ACE 28 29 4 2 Run Chart The Run Chart button starts the digital chart recorder The signal on one or more channels will be displayed in the scrolling chart window Hitting the space bar on the keyboard stops the chart Clicking the Run Chart button restarts the chart 4 3 New Chart The New Chart button first closes the old chart window if one is open then opens a new chart window The user is given the option of saving or discarding the old chart data The new chart file name is generated by incrementing the sequence number at the end of the file name 4 4 Hot Keys The Hot Keys button opens a documentation window with information about keyboard shortcuts for controlling the digital chart recorder Here is a list of the chart hot keys and their actions space bar halts the chart recorder a Auto adjust Y axis range to the size of the signal f Adjust Y axis to display the full Analog Input range t Add an event marker tag and a comment to the chart up arrow increase Y axis range zoom out down arrow decrease Y axis range zoom in left arrow increase the time range zoom out right arrow decrease the time range zoom in 4 5 Event Markers The action of the event marker
67. f Adjust Y axis to display the full Analog Input range up arrow increase Y axis range zoom out down arrow decrease Y axis range zoom in right arrow increase sweep width left arrow decrease sweep width The following keys modify the test pulse size increase pulse amplitude decrease pulse amplitude 1 Invert pulse 36 5 4 Test Seal Test Pulse Window SS Hp E The Test Seal button activates the scope window that monitors the recording electrode input resistance or seal resistance A series of voltage clamp pulses is delivered and the current response is monitored The amplitude of the voltage clamp pulse and the seal resistance are displayed at the bottom of the scope ee T m nj T J pa estPulse 5 mY window Hitting the space 4007 Seal Resistance 10 1 MO bar on the keyboard halts the scope 5 Time ms The electrode input resistance 1s calculated from the amplitude of the response to each voltage clamp pulse The amplitude is measured by averaging the current over the last half of the pulse response as indicated by the horizontal red line superimposed on the current response The amplitude is measured relative to the baseline current before the start of the response as indicated by the horizontal dashed green line 5 5 Test Cell Test Pulse Window SF E The Test Cell button Episode 2 2 activates the scope window that monitors the electrode series resistance an
68. f time Double Exponential Waveform Waveform rises and decays exponentially If the protocol already contains a custom waveform a third option appears which permits the removal of all custom waveforms The format of the next dialog depends on the response to the previous dialog If the General Waveform option was selected the dialog shown at left appears This dialog requests the peak amplitude of the custom waveform when to initiate the waveform time zero and what function of time the waveform should follow In this example the output waveform is defined as a chirp function an exponentially modulated sinusoid The chirp function starts at 10 ms time zero and the preceding 10 ms of the output waveform is zeroed If the protocol contains a series of waveforms this dialog will appear once for each waveform 58 If the Double Exponential Waveform option was Selected the dialog shown at left Set waveform 1 toa double exponential appears This dialog requests the peak amplitude the rise and decay time constants f t expl t Rise exp t Decay of the double exponential waveform and when to initiate the waveform time zero Peak Amplitude mu In this example the waveform starts at 10 ms time zero and the preceding 10 ms is Rise ms zeroed If the protocol contains a series of waveforms this dialog will appear once for Decay msp waveform When a protocol contains a custom Time Zero ms
69. for Data Acquisition occccccnnnnnoonoooncnononcnnnnnnnnnnononnnnnnnnnnnnnnnnnnonnnnnnnnncnninnnnnnss 1 1 3 Install Acquisition Hardware and Software oooooooocccncccnocnnoononononnnnnnnnnnnnononn noo nnnnnnncnnnnnnnnnn non nn nn nnncnnnnonnnnss 2 1 4 Trouble Shoot Hardware and Software Installation cccccnnnnnononononnnnncnnonnnnnonannnonnnnnnncncnnnannananonoss 5 1 5 Optionally Remove the Electrophysiology Features oocccccnnnnoononononnnononcncnonnnnnnnon ono nononnnnncnnnnnnnnnnnnoss 16 Summa OBESA AA E A T 7 2 CONSUL ON td 9 PA EE EE EEE A A ET I EAERI IE T ET E E A ETE A OE 9 22 COMEMOS AA AR 9 ON o A ER 10 DA A A 11 A LEVE A A AA 12 2 6 Save a d Load Conti AA 13 A A II 13 O aca a e sca d onset cesme geen tec voce saccades A E E A E 16 3 DistalOsculos COPE iran A iia 18 Ss ea a sa he sod ecnteaetum EEA E E AE EE 18 BZ ARUN SC OMG IE E E A TA ET E E E teenies 18 EPOCE A PE O E sna dona E E AE E E E E E a 19 Die UN 19 A E OR 20 O OO COn a E E E wgacecanaces 20 IAA O A A 21 3S Channels ena aa sncsesdscnssas A duclusesuspeuiov ess acoeunacase ceca a ea AAA AEA 23 S E A EEIEIE I A A E E E EE E E TAE E heme ESE E E TTT 24 A A Rul E E E A E E oe A A E E E E 24 E A EA TE E P EET DET AS P A A A E E AETA 293 IIZ MOUS OMANI SA A 26 111 4 Dieital Chart and Tape Recorded 28 AL MINEO C LOIN oea A a E noe AA A EEE A A E nem ednoeeaenenss 28 A anaa A A eae ae 29 Ci FING NI WN IM AI th eR a O Ron TO 29 PE SE
70. ft blank The protocol will now run the program AmplitudeSetup immediately after the first repetition of the output waveform or waveform series This program restricts the time region over which to perform subsequent amplitude measurements The program AmplitudeMeasure will run after each repetition and performs the actual amplitude measurement This program only operates on the most recently acquired episode It adds each amplitude measurement to a second monitor window that plots the response amplitude versus time 63 8 3 Standard Analysis Programs Several online analysis programs are provided as part of the data acquisition package These programs are used by some of the example protocols described in Chapter 10 They can be added to any suitable protocol These programs can also be extended or modified to provide new online analysis functions Here is a brief descriptions of each of the standard analysis programs IV Analysis CurrentMin CurrentMax VoltageMin VoltageMax This program produces a current voltage 1 V plot from a data file consisting of a series of current transient recorded on one Analog Input channel in responses to a series of voltage pulses recorded on a second Analog Input channel The names of the two input channels should contain the words Current and Voltage so that the program can determine which is which The IV Analysis program name should be added to the Command dialog field After Last W
71. gitizer connector labeled Trigger Input This permits the start of each episode to be synchronized with an external event Keyboard When this mode is selected an episode is initiated each time the tab key is pressed This permits the start of each episode to be determined manually Regardless of which trigger mode is selected a following dialog will provide the option to Save Trigger Times to Notes If this option is turned on the time at which each episode is triggered will be added to the Notes field of the data file when the protocol terminates 7 13 Custom Waveform 57 Waveforms on the Analog Output channels are not limited to pulses and ramps They can be set to arbitrary functions of time via the Custom Waveform button This button initiates a series of two or three dialogs If the front protocol has two or more active Analog Output channels then the first dialog asks which channel is to receive the custom waveform dialog not shown Define a custom waveform on DAC Output O Command Select the type of waveform General Waveform 3 Double Exponential Waveform y Remove Custom Waveform cancel ox set waveform 1 to an arbitrary function of time For example fiti expi 1710 Peak Amplitude pm Time Zero ms f t expl t 48 sin t 66 The next dialog asks what kind of custom waveform to add to the Analog Output channel The options are General Waveform Any mathematical function o
72. graph outputs that signal the gain and other settings from one or more 4xoPatch Dagan Warner or NPI amplifiers How many amplifiers are present with telegraph outputs connected button brings up the dialog shown at left Number of Amplifiers i cancel ox The next two dialogs request the manufacturer of the patch clamp amplifier shown at left and the amplifier s model number not shown Describe the connections to the amplifier telegraph outputs Telegraphs from 4xoPatch Dagan Warner and NFI amplifiers are supported The amplifier is an AxoPatch Dagan G Warner 3 MPI concer ox Gain Telegraph AxoPatch amplifiers have a Gain Telegraph output It signals the position of the Output Gain knob This telegraph can automatically adjust the gain setting for the ADC Input channel that receives the AxoPatch Scaled Output signal ba Use Gain Telegraph Gain Telegraph at ADC Input Scaled Output at ADC Input a ss cancel Cox AxoPatch amplifiers have a Gain Telegraph output lf this signal is connected to a Digidata telegraph Input channel rear panel it can automatically adjust the gain setting for the Analog Input channel that receives the amp 4xoaPatch Scaled Output signal ba Use Gain Telegraph Rear Panel Telegraph Channel jo Scaled Output at Analog Input jo concer Cox Mode Telegraph Axo0Patch amplifiers have a Mode Telegraph output It signals the position of th
73. hanged In addition some amplifiers have a Scaled Output signal that reports the holding current when the amplifier is in voltage clamp mode and the membrane potential when the amplifier is in current clamp mode The gain and unit settings for the Analog Input channel that receives the Scaled Output signal will change when the clamp mode is changed The Clamp Mode button can be used to inform the acquisition program that the clamp mode has changed AxoGraph then makes all the necessary changes to the configuration settings for the External Command and Scaled Output channels The dialog shown at left indicates that the amplifier is now operating in voltage clamp mode When the recording mode is changed between voltage and current clamp adjust the gain and Units of the voltage and current channels The Clamp Mode program will only work if Change the recording mode to the configuration programs Connections and Channel Names have already been run and 2 Current Clamp the External Command signal has been Edit Parameters Used When Changing Mode cancel ox The first time the Clamp Mode program is run it is necessary to enter the gain and unit settings for the External Command signal in current and voltage clamp modes Selecting the Edit Clamp Mode Parameters option in the previous dialog then clicking OK brings up a series of dialogs that request the mode switch parameters The first dialog asks which Analog Output chan
74. he shift key which has the additional benefit of inactivating all system extensions and freeing up several MBytes of system memory When an acquisition program is running AxoGraph does not share CPU time with any other program running in the background However the Mac operating system can interrupt the acquisition program to perform low level tasks Network tasks are of particular concern For optimum performance it 1s recommended that the network connection is inactivated This can be done via network control panels by physically unplugging the connection or by restarting the computer while holding down the shift key 12 2 Interleave Acquisition and Display By default the digital oscilloscope and the protocol driven data acquisition programs work in a sub optimal manner Acquisition of a sweep can not begin until the display of the previous sweep is complete For Instrutech ITC series digitizers faster repetition rates can be achieved by initiating the acquisition of a new sweep while the previous sweep is being displayed However it is recommended that interleave is turned off unless very fast sweep repetition rates are required regular trigger interval is set to zero The performance gain is generally less than 25 and at lower repetition rates regular trigger interval gt sweep width 200 ms interleave introduces a lag between data acquisition and screen display that may be annoying To interleave acquisition and screen
75. he toolbar at the bottom left of the screen When the Protocol item is selected in this pop up menu the protocol editing commands are loaded into the toolbar and the most recently used protocol window is brought to the front If no protocols are open a dialog will appear asking whether to open an existing protocol for example one of the protocols supplied in the Acquisition Protocols folder or create a new protocol see Section 7 2 Create Commands for adding more advanced features to a protocol are loaded into the toolbar by selecting the More Protocol item in the toolbar popup menu The commands in both these tool bars are always applied to the front protocol window The following sections describe the function of each of the buttons in the Protocol and the More Protocol toolbars Protocol createl Add Pulse Edit Pulse Delete Pulse Display Trigger Repetitions Timebase Channels l More Protocol Custom Waveform Add 4 Link F on N commands Extract Protocol Edit Launch List 45 7 2 Create The Create button creates a new protocol window and initiates a series of up to five dialogs which request the basic information needed to build the protocol They request the acquisition sample rate and episode width the input and output channels the number of waveforms in the output series and how many times to repeat the output series These dialogs are described in sections 7 3 7 4 and 7 5 The information p
76. ia a standard file dialog then selected and run via buttons in the Acquisition toolbar see Chapter 6 An alternative mechanism is provided that can open and run a protocol with a single keystroke This is termed launching the protocol The launch mechanism requires that the protocol file reside in a specific folder and the file name must be a member of a predefined launch list Setting up the launch list takes a little work but the benefit 1s faster and more convenient access to protocols that are used most frequently The three programs for setting up and editing the launch list are listed under the Launch Protocol submenu of the Program menu Each of the programs is described in the following sections 9 2 Edit Launch List Selecting Program gt Launch Protocol gt Edit Launch List brings up the launch list dialog containing 8 fields only 4 are shown here A protocol file name can be entered in each field The name is case sensitive and must include any trailing or leading blanks Each of the protocols on the launch list should be saved in the Acquisition Create a list of protocols to launch Protocols sub folder of Acquisition Package Via the Cmd Keys or Launch Protocol menu In this example the first protocol on the list name is case sensitive is named A Simple Pulse and it can be Protocol 1 A Simple Pulse launched by selecting Program gt Launch Protocol gt Launch 1 Protocol 2 or by holding
77. imes in a single recording session The gain and sampling rate can be changed during a recording session The chart can be annotated at any time during the recording with comments and event markers In addition to the standard features of a chart recorder outlined above AxoGraph s digital chart recorder can send a regular test or stimulus pulse to an Analog Output during continuous recording Chart data is acquired to memory and is not automatically written to disk To write the acquired data to disk the chart file must be saved manually This can be done at any time during a recording session The digital chart recorder will halt with an error message when all available system memory is exhausted The maximum chart length can be estimated as follows Switch to the Finder and select About This Computer under the Apple menu Note the size of the Largest Unused Block of memory If Chart is recording from N channels then AxoGraph requires 4 x N 1 bytes per sample point and 4 x N 1 x SampleRate bytes per second For example if there is 36 MBytes of memory available and Chart is recording 2 channels at 1 kHz then Maximum Chart Length Available Memory 4 x N 1 x SampleRate 36 000 000 4 x 2 1 x 1 000 3 000 seconds or 50 minutes AxoGraph presents a pop up menu in the toolbar at the bottom left of the screen When the Chart item is selected in this pop up menu a dialog will appear asking for the file name and destination fold
78. in this pop up menu a new row of buttons appears in the toolbar The following sections describe the function of each of the buttons in the Configuration toolbar Configuration Configuration Connections nnections Channel Names nel Na mes Gains ins Holding Levels ing Levels Telegraphs eqraphs 2 2 Connections The Connections button initiates a series of dialogs that ask which digitizer channels have a cable connected The first dialog shown at left requests an overview of the connections This helps to simplify the Overview af connections to the Instrutech Which types of input and output channel have one or more cables connected subsequent series of dialogs Wi DAC Outputs Wi ADC Inputs f TTL Outputs C TTL Inputs k C Rear Panel TIL Outputs conca Coe 10 Next a series of dialogs request the connections for the Analog Input channels DAC Output channels the Analog Output channels etc Each of that have a cable connected these dialogs has the same layout as shown at left for the Analog Outputs dialog f DAC Output 6 ef DAC Output 1 I DAC Output 2 I DAC Output 3 cancer Cx 2 3 Channel Names The Channel Names button initiates a series of dialogs that request signal names for the input and output channels that have a cable connected The names should clearly distinguish between the various channels because all subsequent dialogs request channels by
79. isode width is changed the widths of the stimulus pulses if any can be handled in one of two ways Pulse widths can be left unchanged or they can be scaled to match the change in the episode width Turing on the Scale Protocol Pulse Widths check box will select the second option 46 Some stimulus paradigms require a conditioning stimulus to be delivered well before the test stimulus Often the response to the conditioning stimulus and the intervening period are of little interest so only the response to the test stimulus needs to be recorded Turning on the Only Record a Sub section of Episode check box in the Timebase Range Selector dialog then clicking OK will Only record data present a standard range selector Fram To Mig o dialog that asks which sub section of the protocol to record A second dialog asks whether the acquisition monitor window should display the entire width of the protocol or only the sub section The acquisition monitor can optionally that is being acquired display data during the entire protocol or only during the period when data is acquired Wf Only Monitor Acquisition Period cancel Cox 7 4 Channels The Channels button initiates a series of two dialogs that request the input and output channels to be used by the front protocol Only channels that have a cable connected are listed in the dialogs see Section 2 2 Connections Channels are listed by name not by number see Sec
80. ivate the program bring up the Analyse dialog and enter the name ScopeSpectrumSetup in the Before First Sweep field and ScopeSpectrum in the After Every Sweep field The source code for the CustomNoiseSD analysis program is presented on the next page This code can also be found in the file named Custom Analysis which is located in the Acquisition sub folder of the Acquisition Programs folder It should be a useful starting point for writing simple custom analysis programs The CustomNoiseSD program is automatically loaded when AxoGraph is launched because it is located in a sub folder of the Plug In Programs folder When a new custom analysis program is written it can be loaded and tested as follows First save the new custom analysis program in the Plug In Programs folder or sub folder Next select the AxoGraph menu item Program gt Reload Plug Ins Finally enter the custom analysis program name in the After Every Sweep field of the online analysis dialog as shown above 21 Source code listing for the program CustomNoiseSD The first line of the source file must be LocalLanguage C to inform AxoGraph that the following code is written in the C programming language localLanguage C Custom analysis program calculates and displays the standard deviation SD of the signal measured over the first 10 of each sweep void CustomNoiseSD short window trace float yAr
81. log asks which channel if any is to receive the Trigger Stim synch pulse No Pulse cancer Cx 24 3 9 Gains The Gains button in the Scope toolbar brings up two dialogs that request the signal gain amplification and conversion factor for each Analog Input and Analog Output channel These dialogs are described in detail in Section 2 4 Signal Gains The Gains button is also found in the Configuration toolbar but is duplicated in the Scope and Chart toolbars for convenience 3 10 Test Pulse The digital scope can optionally deliver a test pulse to one Analog Output or Digital Output channel during each sweep see Section 3 8 Channels If a test pulse channel has been selected then the Pulse button in the Scope toolbar brings up a series of three dialogs requesting the amplitude onset time and duration of the test pulse The first dialog only appears if an Analog Output channel has been selected It asks for the pulse amplitude Specify the amplitude of the test pulse The pulse amplitude can also be adjusted using Hot Keys see Section 3 3 Scope Settings Pulse Size mu The second dialog asks whether the onset and duration are to be specified in fixed time units ms or as a fraction of the sweep Specify the test pulse onset and width in fixed time width Units or relative to the sweep width Cunas If fixed time units are specified then the third dialog requests the pulse o
82. mode The slope is calculated over a 0 5 ms interval that is slid along the input signal The 5 ms period preceding E the trigger is also acquired See Section 7 12 Trigger for a description of how to specify internal trigger mode for a Current nA Time ms protocol This figure shows the first 10 ms of a data file produced by the Trigger on Event protocol Two episodes were triggered when the signal slope exceeded 1 nA ms The protocol specifies a 5 ms pre trigger interval and a 0 5 ms slope calculation region so the region that triggered the acquisition is displayed from 5 0 to 5 5 ms 80 Keyboard Trigger This protocol sends a 10 mV 10 ms voltage command pulse to Analog Output 0 The pulse is repeated up to 100 times Each episode is initiated via the keyboard by hitting the tab key keyboard trigger mode The protocol acquires the signal from Analog Input 0 The time at which each episode is triggered is saved in the Notes field of the data file See Section 7 12 Trigger for a description of how to specify the keyboard trigger mode for a protocol Chirp This protocol sends a chirp waveform to Analog Output 0 The custom waveform consists of a 1 kHz sinusoid with an amplitude that decays exponentially The initial amplitude of the sinusoid is 50 mV and it decays with a 4 ms time constant The waveform is repeated up to 100 times See Section
83. n Listed Protocols Selecting Program gt Launch Protocol Open Listed Protocols opens each of the protocols on the launch list This is a convenient way to open all the protocols required for a given experiment with a single menu selection 9 5 Remove the Launch List Features If the launch list programs described above are not required they can be removed to simplify the Program menu Open the Acquisition Programs folder inside the Plug In Programs folder Drag the Launch Protocol folder to Trash or store it in the AxoGraph folder Quit and re run AxoGraph 12 10 Example Protocols 10 1 Introduction 10 2 Brief Overview of Each Protocol 10 3 Detailed Description of Each Protocol 10 1 Introduction Twenty one example protocols can be found in the Acquisition Protocols sub folder of the Acquisition Package folder They demonstrate many of the advanced features of the data acquisition package These protocols can be extended or customized using the protocol editing tools described in Chapters 7 and 8 The example protocols assume a minimal configuration of connections to the digitizer They assume that Analog Outputs 0 and 1 both have a connection and that Analog Input channels named Current and Voltage are connected A few of the example protocols also assume that Digital Output 0 is connected A brief outline of the example protocols is given in the next section This is followed by a more detailed description of each pr
84. n be run during a protocol each at a specified time point A program can only be run before or after an episode not while an episode is being acquired A program that is run before the first episode can set up global parameters that control subsequent analysis e g restrict the analysis time range A program that is launched after each episode can provide graphical feedback about some feature of the acquired data e g the response amplitude A program that is run after the last episode can analyse the entire data file that has just been acquired and generate a summary graph e g create an amplitude histogram for all the responses Several standard online analysis programs are provided as part of the data acquisition package Some of the example protocols found in the Acquisition Protocols folder incorporate these standard programs see Chapter 10 The following sections show how to add online data analysis programs to a protocol describe the standard online analysis programs and provide some guidelines and tools for writing new online analysis programs For general information about writing analysis programs see the chapter on programming in the AxoGraph Online Manual 62 8 2 Add Analysis Programs to a Protocol Online analysis programs are added to a protocol via the Commands button in the More Protocol toolbar If several protocol windows are open the analysis programs are added to the front protocol window Runa custom command or
85. n periods of data acquisition In general all holding levels should be set to zero e The next dialog asks whether the user will ever switch a patch clamp amplifier between voltage clamp and current clamp modes If the answer is yes then a series of dialogs is present that simplifies the task of switching the amplifier clamp mode If the answer is no then the configuration procedure is complete The connections to the digitizer are now fully described Some additional configuration may be required before running the data acquisition programs sampling rate trigger mode etc Chapters 3 to 9 contain a detailed description and guide to all the data acquisition programs Section 1 5 provides a brief overview of the features available in these programs 1 4 Trouble Shoot Hardware and Software Installation This section presents some common problems and possible solution Problem At start up AxoGraph reports that it can not find the digitizer Most probable reason digitizer has not been correctly installed and connected Shut down the computer Check that the cable between the digitizer and computer is securely plugged in at both ends Check that the digitizer power cable is securely connected Make sure the digitizer is switched on and its front power light is on Reboot the computer If a Digidata 1320 series digitizer is connected Launch the SCSI monitoring application SCSIProbe that was installed from the AdvanSys CD At th
86. nalog Output channel has been selected It asks for the pulse amplitude Chart settings Specify the test pulse parameters Time Between Pulses sec Pulse Onset ms Pulse Width ms 4 10 Analyse Chart settings Runa custom analysis program every pulse cycle Before First Pulse Run After Each Pulse Run Ka 32 The second dialog asks for the onset time to the first pulse the time between subsequent pulses and the duration width of each pulse The Analyse button in the Chart toolbar brings up the online analysis dialog The chart recorder optionally runs a custom analysis program after each test pulse cycle see Section 4 9 Test Pulse A custom analysis programs can also be run before the first pulse and would typically set up global parameters that control subsequent analysis For information about writing a custom analysis program see the chapter on programming in the AxoGraph Online Manual 33 4 11 Custom Analysis The source code for the ChartNoiseSD online analysis program follows The code for this program can also be found in the file named Custom Analysis which is located in the Acquisition sub folder of the Acquisition Programs folder The ChartNoiseSD program is automatically loaded when AxoGraph is launched because it is located in a sub folder of the Plug In Programs folder When a new custom analysis program is written 1t can be loaded and tested a
87. nals when a protocol is running 40 The following sections describe the function of each of the buttons in the Acquisition toolbar Acquisition Acquisition Resume Resume Protocol File Name Name i st Sea 6 2 Preview The Preview button initiates data acquisition under the control of a protocol The waveforms in the protocol window are delivered to the Analog Output and Digital Output channels and signals are acquired from the Analog Input and Digital Input channels specified in the protocol The input signals are displayed in the Acquisition Monitor window Acquisition will terminate after the number of repetitions specified in the protocol Alternatively hitting the space bar on the keyboard halts acquisition at any time The Preview program is intended for monitoring the response to a protocol without saving the data It tests whether a protocol is working correctly before committing data to disk The name of the protocol that is being run is shown at the top left of the Acquisition Monitor window and the episode number is shown at the top right of the window The display gain and time range of the monitor window can be changed using the keyboard while the protocol is running see Section 6 5 Hot Keys A protocol window should already be open before pressing the Preview button If no protocols are open then a dialog will appear asking whether to open an existing protocol or creat
88. nd a trigger pulse during each sweep custom analysis program can be applied to every sweep scope sweeps can be triggered in several different ways at regular user defined intervals locked to the line frequency from an external logic signal from a signal on an analog input channel trigger on amplitude or first derivative threshold crossings display a pre trigger period e Digital chart recorder scrolling display mark events with tags and comments optional output of a regular timer or test pulse measure and display event frequency custom analysis applied during continuous acquisition e Protocol driven data acquisition regular external or keyboard trigger for episodic data acquisition unlimited episode width deliver complex waveforms to the analog and digital outputs square pulses trains of pulses and ramps incrementing pulse amplitudes and onset times arbitrary series of pulse amplitudes from a table arbitrary waveform output enter a function exponential sine wave etc convert a recorded signal to an output waveform hot keys for initiating predefined protocols real time data analysis during acquisition monitor event amplitude or rate versus time real time analysis programs can be customized or extended all 16 rear panel digital input and output channels can be accessed between episodes using an online analysis command e Patch clamp test pulse for electrophysiology res
89. ned off no feedback will be provided during data acquisition The only reason for turning it off would be to increase performance see Section 12 4 Disable Acquisition Monitor Only Monitor Never Record If this option is turned on the acquisition monitor will display the acquired data but will not record it to a data file even if the Record button is pressed A non recording protocol could be used to deliver a conditioning stimulus as part of a series of linked protocols or it could be used to create a custom monitoring feature similar to Test Cell 55 The second Display dialog asks what online analysis feedback to display in the Acquisition Monitor window Acquisition Monitor Settings analyze each episode and display results f y j Fo The first three options activate standard Display programs that analyses each episode immediately after it is acquired The programs identify the largest event in each episode and measure three parameters describing that event Peak Amplitude C Rise Time L Half Width The last two options simply report parameters describing important features of Lj Pulse Size the acquisition protocol U Sample Rate Peak Amplitude The peak amplitude of the event is displayed Rise Time ecccionicnosiono The 20 80 rise time of the event 1s displayed Half Width The width of the event at 50 of peak amplitude 1s displayed PUISS SIZE sosse The amplitude of the fi
90. nel is connected to the External Command of the voltage clamp amplifier The channels are listed by name When the recording mode is changed from current to voltage clamp the External Command channel switches from a current to a voltage command Select the External Command channel Command Cd Piezo cancel ox Clamp mode switch options Units for External Command channel Yoltage Command Units my Current Command Units anei Cor Mode switch options Gains for voltage current command channel mU per Volt PA per Volt 2600 concer Cx Voltage Gain Current Gain When the recording mode is changed from current to voltage clamp the Scaled Output channel switches from a voltage to a current signal Select the Scaled Output channel Current 3 Yoltage cancel ox When the recording mode is changed from current to voltage clamp one of the clamp output channels switches from a voltage to a current signal Specify the signal name for this channel in voltage clamp and current clamp modes Yoltage Clamp Mode Current Current Clamp Mode oltage cancer Cox 17 The second dialog requests the units for the voltage and current clamp commands These will generally be mV for the voltage command and either pA or nA for the current command The third dialog requests the gains for the voltage and current clamp commands
91. non linear response It is often used by electrophysiologists to study voltage activated currents It is termed leak subtraction because in a voltage clamp recording there is always a leak current present predominantly through voltage independent potassium channels This leak current has a linear current voltage relationship P N leak subtraction will remove the leak current from the response to a voltage step leaving only the voltage activated current P N leak subtraction works as follows Each output waveform in the protocol is delivered several times at a reduced amplitude before being delivered at full amplitude The responses to the reduced amplitude waveforms are ensemble averaged scaled up then subtracted from the response to the full amplitude waveform Any component of the input signal that scales linearly with the output waveform amplitude will be subtracted away by the P N procedure This eliminates any passive or leak component of the response Only the active voltage dependent component of the response will remain The resulting leak subtracted signal is displayed in a separate P N analysis window 65 The reduced amplitude waveforms may be superimposed on a long step to a different holding level A hyperpolarized holding level is typically used to inactivate any voltage dependent channels that may be open at the resting level The P N protocol must be set up to contain 4 pulses in the following order 1
92. nonanonancnonononons the window number of the acquisition monitor data Window cccccccceeseeeeccceeeeeesseeeeees the window number of the data file Gf running Record prRepetitions sccuscaceniedessadeccnensiensaneccsnnedss the number of times the active protocol is repeated prEpisodes cooocoooocococcccnononocncnnnnonananannnnnnos the number of episodes in the data file prPulseOnset O oconnnnnnncconooococcncnnnnnnos the onset time of the first pulse in ms prPulseWidth O ooonnnnnnnicucononocccnncnnnos the width of the first pulse in ms prPulseAmplitude O the amplitude of the first pulse in output channel units Several of the standard online analysis programs take advantage of a utility procedure NewAnalysisWindow This procedure is passed a window name a boolean variable set to true if a new analysis window is created and the number of traces in the analysis window the number of different analysis results to monitor This procedure creates a new monitor window in which to plot analysis results during data acquisition It resizes both the new window and the Acquisition Monitor window so that both are visible on the screen If the monitor window is already open it updates its X axis range so that it can receive new measurements Two of the standard online analysis programs described in Section 8 3 are presented below The original source code for these programs can be found in the fil
93. nset time and pulse width in milliseconds not shown Scope Settings The test pulse duration will remain constant even 1f the scope sampling rate and sweep Specify the test pulse onset and width width settings are changed Pulse Onset of sweep If relative timing is selected the test pulse will change in duration when the sweep Pulse Width of sweep width is changed The pulse will always occupy the same fraction of the sweep and the third dialog asks which fraction that will be 3 11 Analyse Analyze each scope sweep by running the three standard analysis programs or custom analysis programs names are case sensitive Analysis is only applied to the front channel Wf Peak Amplitude Rise Time O Half Width Before First Sweep Run After Every Sweep Run CustomNoiseso Scope Window EE PB bee x Current pA Time ms ele lelels lt 23 The Analyse button in the Scope toolbar presents the online analysis dialog The digital scope optionally analyses each sweep immediately after it is acquired and displays the results overlaid with the sweep Three standard analysis programs identify the largest event in each episode and analyse that event They optionally measure the peak amplitude the 20 80 rise time and the half width of the event Custom analysis programs can also be run before the first sweep and after every sweep For example the program CustomNoiseSD calcula
94. number of times to PA repeat the series TJ If a protocol has no active output channels then the Waveforms field will not appear in this dialog If the specified number of Repetitions 1s greater than one then a second dialog will Optionally average the response to a repeated appear asking whether the acquired episodes protocol and display the running average should be averaged and whether to display the running average in the Acquisition O Average Responses Monitor window O Display Running Average If Average Responses is selected then the ensemble average of the response to each waveform will be calculated when the protocol terminates For example 1f the protocol contains a series of 3 output waveforms then there will be 5 ensemble average traces per Analog Input channel A new window is created to receive the average traces This is separate from the data window which receives the raw traces cancer Cx If Display Running Average is selected then the ensemble average 1s calculated after every repetition of the waveform series The running average trace is superimposed on the raw trace in the Acquisition Monitor window If more than one output waveform was specified then the Display Running Average option will not be available 48 7 6 Add Pulse When a new protocol is created all output waveforms are set initially to a flat line at the holding level usually zero on each active output channel The outpu
95. o 1 4 This protocol outputs a voltage command ramp superimposed on a square pulse to Analog Output 0 The pulse is 100 mV in amplitude and the ramp is 200 mV in amplitude Both the pulse and the ramp start at 10 ms and are 180 ms wide Their superimposition results in a voltage command waveform that sweeps over the range from 100 to 100 mV gt JE a E J JE JE 70 100 The waveform is Time ms repeated 10 times The protocol acquires data from the Analog Inputs named Current and Voltage The acquired data estimates the current voltage I V relationship See Section 7 6 for a description of how to add a ramp to a protocol An important feature of this protocol is that it contains a link to another protocol When all 10 ramps have been delivered and the data saved to a new file the acquisition program pauses for 5 seconds then runs a second protocol named I V with Analysis Two or more protocols can be linked in this way to form a chain or loop and thereby generate output sequences of arbitrary complexity See Section 7 14 Add a Link fora description of how to add a link to a protocol Data 003 EE Trigger on Event Episode 2 2 This protocol does not output any signal but it acquires up to 500 episodes from the Analog Input named Current Each episode is triggered when the slope of the signal on the input channel exceeds a threshold of 1 nA ms internal trigger
96. o nnnnnnccncnonnnnnos 83 11 3 Timing of Input and Output Events 0 cccccccasccsseecsossecceesesaeedecscedccsaacseeesactecnncadeccssadeosssnccneessaaedeaece 85 12 Op mizine Performan E sannin a E AE T E AANIKA EEE LEEDO TENTE OTEP ESEE rSn 87 121 Memory and NetWork Seine S einden a O sees 87 12 2 Interleave Acquisition and Display ccccsssssssssccccceeescsessnnsseeeceecesceseessseeeeeeeeeseseesaaeaeecessessaaaees 87 123 Butter Acquired Data to Mem dit ini 88 12 4 Disable Acquisition Monitor Sa A A A 88 1 Introduction 1 1 Explore the Acquisition Software Without a Digitizer 1 2 Hardware Requirements for Data Acquisition 1 3 Install Acquisition Hardware and Software 1 4 Trouble Shoot Hardware and Software Installation 1 5 Optionally Remove the Electrophysiology Features 1 6 Summary of Features 1 1 Explore the Acquisition Software Without a Digitizer The AxoGraph data acquisition package can run in demo mode and generate simulated electrical signals This permits the various acquisition programs and options to be explored The installer named Install AxoGraph 4 9 Demo installs and preloads the acquisition software package The acquisition programs can only run in demo mode The installer named Install AxoGraph 4 9 does not preload the acquisition software package It needs to be loaded manually as follows Open the AxoGraph 4 9 folder then the Data Acquisition Package folder Move the Acquisition Prog
97. o scale function can be performed at any time by typing the letter a while the scope program is running see Section 3 3 Hot Keys 3 5 Average The Average button brings up the dialog shown at left Scope can optionally average the sweeps When the Average Sweeps check box is on and display the running average a running average of successive sweeps is calculated and displayed in the scope window This is useful for improving the Mf Average Sweeps signal to noise characteristics of the displayed signal Number of Sweeps to Average 3 6 Timebase The Timebase button in the Scope toolbar brings up the dialog shown at left This dialog controls the sample rate used by the Scope program to acquire signals When several input channels are active the signal on each channel will be sampled at the requested rate This dialog also controls the Sample Rate KHz width of each sweep in milliseconds Sweep Width ms 5S o lt The sample rate should be at least twice the highest frequency of interest in the signal The best results are obtained when the Cancel signal is low pass filtered at a frequency equal to half the sample rate scope Settings 21 Because of limitations in the digitizer hardware it is not always possible to sample data points exactly at the requested sample rate See Section 11 2 Hardware Limitations on the Sampling Rate for a discussion of these limitations 3 7 Trigger The T
98. ocol termination When recording data with this protocol C Buffer Acquired Data to Memory cancel ox _ 12 4 Disable Acquisition Monitor The maximum episode repetition rate is limited by the time it takes to acquire an episode plus the time it takes to de multiplex the acquired episode and copy it to the data window plus the time it takes to display the episode in the monitor window Under many circumstances the display step is the slowest The episodes repetition rate can be increased by disabling the display of each episode during acquisition Acquired episodes will still be displayed but only when acquisition terminates To disable the acquisition monitor select the Protocol item in the toolbar pop up menu This will bring the current protocol window to the front or request a protocol filename if no protocol windows are open Press the Display button in the Protocol toolbar to bring up the Acquisition Monitor Settings dialog see Section 7 11 Turn off the Monitor Acquired Episodes check box
99. of three dialogs requesting the root file name the sequence number and the folder in which data files should be stored In this example the next three data files will be given the names Glycine 30 9 98 019 Glycine 30 9 98 020 Glycine 30 9 98 021 43 After specifying the root data file name and sequence number two more dialogs appear requesting the destination folder to receive the data files Save data files in the selected folder Select Data oh GigaDrive Desktop Cancel 6 8 Deleting Data Files Data files are locked so that they can not be accidentally modified or deleted Two mechanisms are available for deleting data files that have been backed up or are no longer required The first mechanism takes advantage of an hidden feature of the Mac OS The data files are dragged to Trash as usual but then the option key is held down while selecting the menu item Special gt Empty Trash The option key overrides the error message that normally appears when trying to delete locked files The second mechanism uses an AxoGraph program to unlock the data files before dragging them to Trash The files to be deleted should be gathered in a single folder Selecting the AxoGraph menu item Program gt Acquisition gt Unlock Data Data files are locked for their protection Files brings up the dialog shown at left Only unlock them if they have been backed up and you now wish to delete them A su
100. of any of these channels including the channels that can only be accessed at the rear panel of the digitizer However it can only be run between episodes It can not be used to change a Digital Output setting while an episode is being acquired Command mY Acquisition Stimulus Preceding Episode This delivers two rectangular pulses to Analog Output 0 and records the response on Analog Input 0 Only the response to the second stimulus pulse is recorded The first pulse is the conditioning stimulus and it is delivered 1 sec before the start of the recorded episode The second pulse is the test stimulus and the response is only recorded over the last 0 5 sec of this protocol Data acquisition can be restricted to a sub section of a protocol via the Timebase button see Section 7 3 In this protocol the monitor window is set to display the entire 1 5 second time range The monitor range can be restricted to display only the acquisition sub section via the Timebase button see Section 7 3 82 Multi Channel Complex HH Multi Channel Complex codo e dE This protocol demonstrates how pulses pulse trains and ramps can be concatenated to build complex waveforms see Sections 7 6 to 7 9 It delivers a series of 6 waveforms to 3 output channels while acquiring 6 episodes from the Analog Inputs named Current and Voltage The first pulse on the Command channel has an amplitude of 20 mV on the fir
101. of the data acquisition package and 1t can be modified to monitor any parameter of the response see Chapter 8 Online Analysis Monitor Cm This protocol sends a 5 mV 10 ms voltage command pulse to Analog Output 0 The output pulse is repeated up to 1000 times and episodes are initiated as rapidly as possible The protocol acquires the signal from the Analog Input channel named Current An online analysis program is run after each episode The program assumes that the recorded current is from a voltage clamped spherical cell and that whole cell capacitance neutralization is not active The program analyses the amplitude and decay of the current transient recorded and estimates the total membrane capacitance Cm of the cell The measured Cm values are plotted in a separate monitor window that is created when the protocol is first run The online Cm measurement program is a standard part of the data acquisition package see Chapter 8 Online Analysis To add this online analysis feature to a protocol click the Commands button then enter MonitorCmSetup in the After First Episode field and MonitorCm in the After Each Episode field These programs assume the first pulse in the protocol is a rectangular test pulse 83 11 Precise Acquisition Timing 11 1 Stability and Accuracy of Internal Trigger 11 2 Hardware Limitations on the Sampling Rate 11 3 Timing of Input and Output Events 11 1 Stability and Accuracy of Regular
102. onfigurations of the digitizer connections This feature is useful when the instruments connected to the digitizer are changed between two or more standard configurations on a regular basis A set of connection parameters can be saved to a file by selecting Program gt Acquisition Options gt Save Configuration The file is saved in the same folder as the AxoGraph application A set of parameters can be loaded by selecting Program gt Acquisition Options gt Load Configuration 2 7 Telegraphs The telegraphs feature is relevant to electrophysiologists using a patch clamp amplifier manufactured by Axon Instruments Dagan Warner or NPI If a patch clamp amplifier is not being used then review Section 1 5 Removing the Electrophysiology Features before skipping forward to Chapter 3 AxoGraph can read and interpret the telegraph signals gain setting etc that are output by most patch clamp amplifiers Most importantly it can adjust the gain of the specified input signal according to the voltage on the gain telegraph channel Other supported signals include the AxoPatch voltage clamp mode telegraph filter frequency telegraph and whole cell capacitance compensation telegraph For experiments involving simultaneous presynaptic and postsynaptic recording there may be two or more amplifiers connected to the digitizer AxoGraph can support telegraphs from up to four amplifiers simultaneously The Telegraphs AxoGraph can read the tele
103. otocol 10 2 Brief Overview of Each Protocol A Simple Pulse Acquires up to 500 episodes from an input channel while sending a square voltage pulse to an output channel Pulse Average Acquires and averages 50 episodes from an input channel while sending a square voltage pulse to an output channel Pulse Amplitudes Acquires up to 500 episodes from an input channel while sending a voltage pulse to an output channel Analyses each acquired episode and monitors the response amplitude in a separate window Pulse Stimulus and Rs Acquires up to 500 episodes from an input channel while sending voltage pulses to two output channels Analyses each acquired episode and monitors the response amplitude and the series resistance Pulse Slope Acquires up to 500 episodes from an input channel while sending a voltage pulse to an output channel Analyses each acquired episode and monitors the slope of the response over a selected time region Pulse Two Amplitudes Acquires up to 500 episodes from an input channel while sending a voltage pulse to an output channel Analyses each acquired episode and monitors the response amplitude at two selected time points A Simple I V Delivers a series of 20 square voltage pulses covering a range of amplitudes from 40 to 150 mV to an output channel Records the current and voltage responses on two input channels I V with Analysis Delivers a series of 20 square voltage pulses covering a range of amplitudes to an out
104. package is preloaded The acquisition programs can be run by clicking toolbar buttons at the bottom of the screen or from submenus of the Program menu e Install AxoGraph 4 9 installs only a demo version of the acquisition software and does not preload the acquisition package The acquisition package will need to be loaded manually as described below If the acquisition package is not preloaded it can be manually loaded as follows Open the AxoGraph 4 9 folder then the Data Acquisition Package folder Move the Acquisition Programs Demo folder into the Plug In Programs folder Launch the AxoGraph 4 9 application 3 Test the software installation This section assumes that the AxoGraph data acquisition software has just been loaded for the first time If a digitizer has not been installed skip to step c a Make sure the digitizer is plugged in and the power is on b Connect a cable from Analog Input 0 to Analog Output 0 c Launch AxoGraph A toolbar will appear at the bottom of the screen d Change the toolbar popup menu setting to Scope e Click on the Run Scope button in the toolbar A Scope Window will appear with a live updating trace containing a square Current pA pulse Scope Window If AxoGraph can not communicate with the digitizer or if initialization of the digitizer hardware fails an alert will appear asking you to check that a digitizer is connected to the computer and that it is switched
105. pe is running Slope Calculation Range ms Trigger Level pA ms Pre Trigger Interval ms 5 The next dialog asks whether to initiate a sweep when the signal amplitude crosses a threshold level or when the slope of the signal exceeds a threshold level If Amplitude Threshold is selected the next dialog requests the direction of threshold crossing low to high or high to low that initiates the sweep It also requests the threshold level that must be crossed The signal can optionally be displayed both before and after the threshold crossing event that triggers the sweep This is achieved by setting the Pre Trigger Interval to a value greater than zero If Slope Threshold is selected the next dialog requests the range over which to calculate the slope and the slope threshold level that initiates the sweep The slope of the signal is calculated by fitting a line over the specified calculation range This calculation is continuously updated as the signal is acquired until the slope exceeds the specified threshold If the Trigger Level is set to a negative value then a sweep will be triggered when the slope of the signal is more negative than the threshold This permits triggering on negative going events 23 3 8 Channels The Channels button in the Scope toolbar initiates a series of three dialogs The first dialog asks which Analog Input and Digital Input signals to display in the scope window Only channels tha
106. program at selected times during each waveform series Command Before First Waveform f After First Waveform Ps Before Each Waveform fe After Each Waveform fo After Last Waveform concer Cx kun a custom command or program at selected times during 4 repeated acquisiton protocol Command Before First Repetition f After First Repetition Before Each Repetition fo After Each Repetition After Last Repetition cancer Cx The Commands button brings up one or two dialogs depending on the number of waveforms and repetitions specified in the protocol If a protocol contains more than one output waveform then the dialog at left will appear requesting the names of programs or procedures to execute at various points during the series of waveforms Program names are not case sensitive and any field can be left blank This protocol will now run the current voltage analysis program IV Analysis after the last waveform in the series has been output and the last episode has been acquired The program is passed 4 parameters that define the time intervals in milliseconds over which to measure the current and voltage The program will analyse the entire data file that has just been acquired If a protocol repeats a waveform series then the dialog at left appears requesting the names of programs or functions to execute at various points during the protocol Program names are not case sensitive and any field can be le
107. ptions were activated in the previous dialog then this dialog also asks how much to increment the pulse onset and width increment options are not shown at left The shape of the Pulse or Ramp is now completely specified for every output waveform The waveforms in the protocol window are updated The following series of dialogs will appear if a Pulse Train has been specified 4dd or Edit a Pulse Train Specify the amplitudes of pulses in the train First Pulse Amplitude mU Increment per Pulse mu Increment per Waveform mW Add or Edit a Pulse Train Optionally increment pulse width or inter pulse Interval With every pulse in the train C Increment Pulse Width C Increment Inter pulse Interval concer Cox The first dialog asks for the amplitude of the first pulse in the train and how much to increment the amplitude for each subsequent pulse in the train If more than one output waveform has been specified a third item also appears in this dialog It asks how much to increment the amplitude of every pulse in the train after each waveform The following two dialogs define the pulse width and inter pulse interval The first dialog asks whether the width or the interval are to be incremented for each pulse in the train 4dd or Edit a Pulse Train Define timing of pulse train in first waveform Pulses in Train la First Pulse Onset ms Pulse Width ms Inter pulse Interval ms 5S 5
108. put channel Records the current and voltage responses on two input channels then analyses them to generate an I V plot I V using Command Table Delivers a series of 10 square voltage pulses to an output channel and records the current and voltage responses on two input channels An arbitrary series of pulse amplitudes 1s specified in a table I V with P N Leak Subtraction Delivers a series of 10 voltage pulses to an output channel and records the current responses The linear or passive component of the current response is automatically subtracted to reveal any non linear or voltage dependent component Paired Pulse AP with P N Delivers a voltage clamp waveform consisting of two action potentials separated by 10 ms The paired action potentials are preceded by a series of 8 pairs of inverted scaled down 73 action potentials The output waveform is repeated up to 5 times The recorded response to each waveform is analysed online using P N leak subtraction The linear or passive component of the current and voltage responses is automatically subtracted to reveal non linear or voltage dependent components Ramp Then Link to I V Delivers a voltage command waveform that ramps over the range from 100 to 100 mV Repeats the waveform 10 times and records the current and voltage responses on two input channels When complete this protocol automatically runs another protocol Keyboard Trigger Acquires up to 100 episodes from an in
109. put channel while sending a square pulse to an output channel Each episode is triggered via the keyboard by hitting the tab key Trigger on Event Acquires up to 500 episodes from an input channel but does not output any signal Each episode is triggered when the slope of the signal on the input channel exceeds a threshold Chirp Acquires up to 100 episodes from an input channel while sending a chirp waveform to an output channel This custom waveform consists of a 1 kHz sinusoid with an amplitude that decays exponentially LTP Induction Pairing Delivers a train of 100 pulses at a rate of 100 Hz to trigger a presynaptic stimulator Simultaneously delivers a voltage command pulse of 50 mV to depolarize the postsynaptic neuron This paired stimulus is delivered every 10 seconds and is repeated 5 times Toggle Digital Output 0 Record a series of up to 500 episodes Before the start of each episode toggle the setting of Digital Output 0 This is achieved using online analysis commands Stimulus Preceding Episode Delivers two rectangular pulses to an output channel Only the response to the second stimulus is recorded The first pulse is the conditioning stimulus and it is delivered 1 sec before the start of the recorded episode The second pulse is the test stimulus and the response is recorded over the last 0 5 sec of the protocol Multi Channel Complex Delivers a series of 6 complex waveforms to 3 output channels and
110. ral of the features of the data acquisition package were developed specifically for electrophysiology research These include the patch clamp test pulse and support for telegraph signals from patch clamp amplifiers If these features are not required they can be removed to simplify the user interface Open the Acquisition Programs folder in the Plug In Programs folder Drag the Electrophysiology folder out of the Plug In Programs folder Quit and re launch AxoGraph to reset the Program menu and toolbars 1 6 Summary of Features e Full support of Axon Digidata 1320 series digitizers and Instrutech ITC 16 and ITC 18 digitizers Axon Digidata 1320 series 16 analog and 4 digital inputs 2 analog and 8 digital outputs 4 dedicated telegraph inputs maximum sample rate of 500 kHz Digidata 1321A or 250 kHz Digidata 1320 Instrutech ITC series 8 analog and 4 digital inputs gain control on analog inputs ITC 18 only 4 analog and 4 digital outputs maximum sample rate of 200 kHz All digitizers external trigger can initiate acquisition all 16 rear panel digital input and output channels can be accessed between episodes using an online analysis command e Digital storage scope measure and display peak amplitude rise time half width hot keys for changing the display range settings on the fly AC couple via baseline subtraction average multiple sweeps Sweep capture and overlay optional output of a test pulse a
111. rams Demo folder into the Plug In Programs folder Launch the AxoGraph 4 9 application The installer named Install AxoGraph 4 9 Acq installs and preloads a fully functional acquisition software package If no digitizer 1s connected the acquisition programs revert to demo mode Skip to section 2 2 for more detailed information on the acquisition programs 1 2 Hardware Requirements for Data Acquisition Data is acquired to memory so a minimum of 32 MByte RAM is recommended for episodic data acquisition and 64 MByte for continuous acquisition at a high sampling rate see Section 4 1 for additional information on memory requirements AxoGraph runs on all Power Macintosh computers and on older Macs with a 680x0 CPU and an FPU Data acquisition requires a Digidata 1320 series or an Instrutech ITC 16 or ITC 18 digitizer The Digidata 1320 consists of an external unit that connects to the Mac via a SCSI cable Newer PowerMacs that do not have a built in SCSI bus G4 and newer will also require the installation of a SCSI card into a PCI bus slot inside the computer The Digidata ships with a SCSI card The ITC 16 and ITC 18 digitizers consist of an external rack mounting data acquisition unit and a bus interface card which plugs into a slot inside the computer For the ITC 16 interface cards are available for both NuBus and PCI bus computers The ITC 18 is only available with a PCI bus card For more information on digitizer hardware visit
112. ray 0 float xMin xMax float noiseSD theScale string yUnits Get the front window and trace number GetFront window trace GetXRange window xMin xMax Get the baseline region before the start of the test pulse xMax startPulsePnt samplelnterval yArray yRange window trace 0 xMax Make sure we got at least 3 points if ArraySize yArray gt 2 Calculate the SD noisesD SD yArray Get the displayed Y axis units and scale factor DisplayedYUnits window trace yUnits DisplayedYScale window trace theScale Move to a point just below the baseline DrawMove xMin Mean yArray 2 noisesSD DrawPixelMove 5 12 DrawSetSize 12 Display the noise SD in Y axis units DrawString concat noiseSD theScale 3 yUnits 4 Digital Chart and Tape Recorder 4 1 Introduction 4 2 Run Chart 4 3 New Chart 4 4 Hot Keys 4 5 Event Markers 4 6 Timebase 4 7 Channels 4 8 Gains 4 9 Test Pulse 4 10 Analyse 4 11 Custom Analysis 4 1 Introduction The Chart program implements all the main features of a chart recorder It can continuously acquire data at audio rates up to 50 kHz on some computers so it could also be used as a digital tape recorder The program is designed for continuously monitoring and recording electrical signals The signals are plotted versus time in a scrolling chart window The digital chart recorder can be stopped and restarted many t
113. rigger button in the Scope toolbar brings up the Scope Trigger Mode dialog scope Trigger Mode This dialog controls how sweeps are Regular Initiate sweep at regular intervals ende l ane gt Initiate sweep at signal on ADC Input Ta ated MIE Eroa ree at noun ODORS External Initiate sweep at signal on TRIG IN Line Initiate sweep locked to line frequency Regular When this mode is selected sweeps are Regular initiated at a regular interval that is specified Internal in a subsequent dialog not shown If the sweep Start to start time is set to zero sweeps will be initiated as rapidly as 2 Line possible If Scope can not keep up with the requested sweep start to start time a ok warning dialog will appear when the program is halted External Internal When this mode is selected sweeps are initiated whenever a signal on one of the Analog Input channels crosses a specified threshold The procedure for setting the threshold is described below External When this mode is selected sweeps are initiated whenever a 5 Volt signal appears on the digitizer connector labeled Trigger Input This permits the start of the scope sweep to by synchronized with an external event Line When this mode is selected sweeps will be synchronized with the power line voltage cycle The Axon Digidata 1320 series incorporates a hardware line trigger The Instrutech ITC series need to be triggered from software so a second dialog appears
114. rm The first dialog asks about the pulse amplitude The protocol outputs 10 different waveforms Method tor calculating pulse amplitudes G Same Amplitude for all Waveforms Constant Amplitude Increment CH Enter a Table of Amplitudes cancel Cox The options are Same Amplitude the pulse has the same amplitude on all waveforms Constant Increment the pulse amplitude is incremented after each waveform Table of Amplitudes the pulse amplitude is different on each waveform and the individual amplitudes are entered into a table The next dialog requests the amplitudes of the pulses The format of the dialog depends on which option is selected in the previous dialog The pulse amplitude dialog is self explanatory and is not shown The next two dialogs request the pulse onset and width for each waveform The first dialog asks whether the pulse onset time or width are to be incremented These The protocol outputs 10 different waveforms parameters can be incremented after every waveform or after every few waveforms Optionally increment pulse onset or width after every one or more waveforms LJ Increment Pulse Onset Time Cl Increment Pulse Width Increment After Every fo 4dd or Edit a Pulse Define timing of the pulse Pulse Onset ms Pulse Width ms 7 8 Define Pulse Train Shape 51 The next dialog requests the pulse onset and width If the onset and width increment o
115. rovided via these dialogs is used to build a preliminary protocol in which the output waveforms are set to a flat line at the holding level typically zero on each channel see Section 2 5 Holding Levels Additional information about the shape of the output waveforms and the method used to trigger each episode should be added to the preliminary protocol using buttons in the Protocol toolbar These commands are described in sections 7 6 through to 7 12 7 3 Timebase The Timebase button brings up a dialog requesting the data acquisition sample rate and episode width for the front protocol AWS LN as This dialog controls the sample rate used by the data acquisition programs to acquire signals When several input channels are sampling Rate KHz active the signal on each channel will be sampled at the requested rate This dialog Episode Width ms also controls the width of each episode in milliseconds bd Scale Protocol Pulse Widths The sample rate should be at least twice the highest frequency of interest in the signal The best results are obtained when the signal is low pass filtered at a frequency equal to half the sample rate U Only Record a Sub section of Episode Because of limitations in the digitizer hardware it is not always possible to sample data points exactly at the requested sampling rate See Section 11 2 Hardware Limitations on the Sampling Rate for a discussion of these limitations When the ep
116. rst pulse in the protocol is displayed Sample Rate The data acquisition sample rate is displayed in kHz 56 7 12 Trigger The Trigger button brings up the Acquisition Scope Trigger Mode Trigger Mode dialog This dialog controls Regular Initiate sweep at regular intervals how to initiate trigger the acquisition of Internal Initiate sweep at signal on ADC Input every episode There are four options External Initiate sweep at signal on TRIG IN Line Initiate sweep locked to line frequency Regular When this mode is selected episodes are initiated at a regular interval that is specified 2 Internal in a subsequent dialog not shown If the External episode start to start time is set to zero episodes will be initiated as rapidly as Y Line possible If the Acquisition program can not keep up with the requested episode start to ok ae e a adi dialog will appear when the protocol is halted Regular Internal When this mode is selected episodes are initiated whenever a signal on one of the Analog Input channels crosses a specified threshold When an Internal trigger is selected a series of three dialogs appears that request additional information about the trigger threshold and pretrigger interval These dialogs were described in a previous chapter see Section 3 7 Trigger External When this mode is selected episodes are initiated whenever a 5 Volt signal appears on the di
117. s follows First save the new custom analysis program in the Plug In Programs folder or sub folder Next select the AxoGraph menu item Program gt Reload Plug Ins Finally enter the custom analysis program name in the After Each Pulse field of the online analysis dialog as shown above Source code listing for the program ChartNoiseSD The first line of the source file must be LocalLanguage C to inform AxoGraph that the following code is written in the C programming language LocalLanguage C Custom analysis program calculates and displays the standard deviation SD of the signal measured over the last 10 of each test pulse cycle void ChartNoiseSD short window trace float yArray 0 float xMin xMax float noiseSD theScale string yUnits Get the last 10 of the most recent test pulse cycle GetFront window trace xMax ChartCumulativePoints samplelnterval xMin xMax 0 1 PulseCycleTime yArray yRange window trace xMin xMax Calenlate the 8D Y noiseSD SD yArray Get the displayed Y axis units and scale factor DisplayedYUnits window trace yUnits DisplayedYScale window trace theScale Display the noise SD using the Y axis units DrawMove xMax Mean yArray 6 noisesSD DrawSetSize 12 DrawString concat noiseSD theScale 3 yUnits 34 5 Electrophysiology Test Pulse 5 1 Introduction 5 2 Test Channels 5 3 Test Pulse Hot Keys 5
118. sode field see Section 8 3 Standard Analysis Programs for additional details Pulse Two Amplitudes This protocol is similar to the previous protocol but it measures the amplitude at two different points in the response It sends a5 mV 10 ms voltage command pulse to Analog Output 0 and analyses the current response recorded on the Current channel The analysis program measures average amplitude over two separate time periods relative to the average current in a baseline period These time periods are specified manually The amplitude at both time points is plotted in a separate monitor window The analysis program is a Standard part of the data acquisition package see Chapter 8 Online Analysis 75 Pulse Stim cc p E Pulse Stimulus and Rs This protocol sends a 5 mV 10 ms voltage command pulse to Analog Output 0 and after a delay sends a 1 ms Digital Output pulse 5 V to Digital Output 0 This pair of output pulses is repeated up to 500 times and an episode is initiated every 1 second regular trigger mode The protocol acquires the signal from the Analog Input channel named Current See Section 7 4 Channels for a description of how to add or remove channels from a protocol a if E r T y E E o s Trigger Stim 40 Time ms amplitudes and Rs BB The Pulse Stimulus and Rs protocol also runs an online analysis program after each episode The program anal
119. st episode The pulse amplitude increments by 20 mV with each episode On every second episode the onset time 1s incremented by 10 ms and the pulse width is reduced by 10 ms This pulse is followed by a train of 5 pulses The amplitude of the first pulse in the train 1s 20 mV and the amplitude is incremented by 5 mV for each pulse in the train The amplitude of all pulses in the train are incremented by 10 mV with each episode The pulse train is followed by two successive ramps The amplitude of both ramps increments with each episode The Piezo channel delivers a pulse train which consists of 5 pulses each 20 mV in amplitude The width of the first pulse is 10 ms and the width is incremented by 10 ms for each pulse in the train The interpulse interval is constant at 10 ms A Digital Output pulse is delivered at the start of each episode to the Trigger Stim channel Command mY Piezo um E 17m pa dy de a sp jm 100 Time ms Spontaneous Events with Rate This protocol does not output any signal but 1t acquires up to 100 episodes each 1 second long from the Analog Input named Current It runs an analysis program after every episode that scans along the acquired signal searching for spontaneous events The value of spontaneous event rate 1s plotted in a separate monitor window that is created when the protocol is first run The analysis program that measures the event rate 1s a standard part
120. superimposed on the individual acquired signals in the Acquisition Monitor window and the final ensemble average is saved in a separate data file when the acquisition program is halted See Section 7 5 Repetitions for a description of how to activate online averaging Pulse Amplitudes This protocol sends a 5 mV 10 ms voltage command pulse to Analog Output 0 This output pulse is repeated up to 500 times and an episode is initiated every 0 4 seconds regular trigger mode The protocol acquires the signal from the Analog Input channel named Current The Pulse Amplitudes protocol runs an online analysis program after each episode The program analyses the response recorded on the input channel and measures the amplitude relative to the average current in a baseline period preceding the response Both the baseline and the amplitude measurement periods are defined by placing cursors in the monitor window after the first episode has been acquired The measured amplitudes are plotted in a separate monitor window that is created when the protocol is first run The online amplitude measurement program is a standard part of the data acquisition package and it can be modified to monitor any parameter of the response see Chapter 8 Online Analysis To add this online analysis feature to a protocol click the Commands button then enter AmplitudeSetup in the After First Episode field and AmplitudeMeasure in the After Each Epi
121. t have a cable connected are listed see Section 2 2 Connections Channels are listed by name not by number see Section 2 3 Channel Names Scope displays one or more of the ADC and TTL Input channels Scope channels Current O Voltage When two or more input channels are selected the signals will be displayed by default in separate groups within the scope window The signals from multiple channels can be overlaid by selecting either Combine or Group under the Trace menu Channels should only be combined if their signals have the same units e g mV The digital scope can optionally deliver a test pulse to one Analog Output or Digital Output channel during each sweep The second dialog asks which channel if any is to receive the test pulse Scope delivers an optional test pulse to one of the DAC or TTL Output channels Test pulse to channel Command O Piezo No Output Pulse cancer Cox The duration and amplitude of the test pulse can be edited via the Pulse button in the Scope toolbar see Section 3 10 below The scope can optionally deliver a brief synchronizing pulse to a Digital Output channel every time a sweep is triggered This pulse could be used to trigger a waveform generator or an electronic Scope delivers an optional 1 ms pulse to one of the TTL Output channels at the start of each sweep Sweep start pulse to channel oscilloscope for example The third dia
122. t waveform can be edited in two different ways The first is by adding square pulses pulse trains or ramps as described in this section The second is by adding a custom waveform which is an arbitrary function of time as described in Section 7 13 Custom Waveform The Add Pulse button initiates a series of dialogs that request the output channel that the pulse should be added to the shape and size of the pulse and the onset time and width of the pulse Add a pulse or ramp to the waveform on the selected output channel Add a pulse to r The first dialog only appears if two or more Clamp Command output channels are active It asks which Piezo output channel the new pulse should be added to Only channels that have a cable za connected are listed see Section 2 2 ox Conmnections Channels are listed by name not by number see Section 2 3 Channel Names The next dialog asks what type of pulse to add Add a new pulse to The options are DAC Output 0 Command mv PUSS ciao a single rectangular pulse Select the t f pulse to add eet ere es ee Pulse Train several rectangular pulses Pulse at regular intervals Ramp a continuous change in the d Pulse Train output signal y Ramp concer Cx 49 Each type of waveform Pulse Pulse Train and Ramp is illustrated in the following protocol window Neu Protocol Pulse mY E E E Ramp mY
123. tes the standard deviation of the signal in the baseline region before the start of the test pulse The code for this program can be found in the file named Custom Analysis which is located in the Acquisition sub folder of the Acquisition Programs folder When the digital scope is run with online analysis active the scope window will display the results superimposed on each sweep The figure at left shows a single Scope sweep with the peak amplitude the rise time and the noise SD analysis results superimposed The noise SD was calculated and displayed by a custom analysis program which is described below 26 3 12 Custom Analysis Custom analysis programs can be run before the first sweep and after every sweep A program run before the first sweep would typically set up global parameters that control subsequent analysis For information about writing a programs in AxoGraph see the chapter on programming in the AxoGraph User Manual Two example online Scope analysis programs are supplied with AxoGraph They are CustomNoiseSD Calculates and displays the standard deviation SD of the signal over the first 10 of each sweep To activate the program bring up the Analyse dialog and enter the name CustomNoiseSD in the After Every Sweep field ScopeSpectrumSetup ScopeSpectrum calculates the power spectrum of the signal in each sweep and displays the result in a separate window on log log axes To act
124. tes this feature The protocol is named I V with P N Leak Subtraction and is described briefly in a later section see Section 10 1 The example protocol does not use the built in P N feature described here The P over N button brings up a dialog that asks whether P N leak subtraction should be performed on the acquired data When this feature is activated each output waveform in the protocol is automatically delivered several times at a reduced amplitude before being delivered at full amplitude In this example each output waveform is scaled to 1 10th of its original amplitude It P N Leak Subtraction is output 8 times at reduced amplitude before being delivered at full amplitude 4 scaled down waveform is delivered several The responses to the reduced amplitude times The response is averaged and subtracted waveforms are ensemble averaged scaled from the response to the full scale waveform up 10 times then subtracted from the Perform P N Leak Subtraction response to the full amplitude waveform The resulting signal is displayed in the P N Repetitions monitor window and saved to the data file Any component of the input signal that P N Scale scales linearly with the output waveform amplitude will be subtracted away by the P N procedure This eliminates any passive or leak component of the response Only the active voltage dependent component of the response will remain 60 7 16 Extract Protocol When
125. tion 2 3 Channel Names This protocol acquires data from one or more of the ADC and TTL Input channels Acquisition channels kf Current L Voltage When the protocol is initiated via the Preview or Record programs see Section o 6 2 and 6 3 data will be acquired from the specified Analog Input and Digital Input channels The active Analog Output and Digital Output channels are specified in the second dialog This determines which channels will This protocol delivers an output waveform be included in the protocol window to the DAC channels during data acquistion Waveforms can be constructed in the active Output waveform to output channels by adding pulses and ramps Lf Command see Section 7 6 Add Pulse or by adding custom waveforms that can be any function Mm Piezo of time see Section 7 13 Custom Waveform cancer Cox 47 7 5 Repetitions If a protocol has active output channels then 1t may contain a single output waveform or a series of different waveforms on each channel When the protocol is run the series of waveforms may be repeated any number of times The 4 protocol outputs a series of different waveforms The series may be repeated any number of times n episode is acquired for each Waveform and each repetition Waveforms Repetitions button brings up a dialog that requests the number of different waveforms Repetitions 168 in the series and the
126. tion will improve the reliability of the parameter estimates but will cause the scope window to update more slowly The response to several pulses is averaged before being used in computations by the programs Test seal and Test Cell specify the number of pulses to average Pulses per Computation cancer Cox 38 A single exponential fit is only suitable for a spherical cell or a cell with very short fine Test Cell fits a single or double exponential process dendrites axon etc For most to the current transient during the test pulse 4 amp short interval at the peak is skipped because it may be corrupted by electrode artefact neurons the current response will decay with a multi exponential time course and a double exponential fit will provide a more accurate estimate of membrane capacitance if Fit Double Exponential Even if the Fit Double Exponential option is on AxoGraph will automatically revert to a After Pulse Onset Skip ms single exponential fit when appropriate 5 8 Test Pulse The Test Pulse button brings up a series of three dialogs requesting the amplitude onset time and duration of the test pulse These dialogs are described in detail in Section 3 10 Test Pulse The pulse amplitude can also be adjusted using Hot Keys see Section 3 3 The first dialog requests the pulse amplitude The second dialog asks whether the onset and duration are to be specified in fixed time uni
127. tput pulses have been defined Select the pulse to edit Pulse 1 Command Cd Pulse 2 Command cancel ox 7 10 Delete Pulse One or more output pulses have been defined Select the pulse to delete Command Ramp 1 CG Command Pulse 2 cancer Cx 53 The Edit Pulse button brings up a dialog asking which pulse pulse train or ramp to modify The pulses are listed by name It is not possible to alter the waveform type pulse pulse train or ramp but the amplitude and timing parameters can all be changed These parameters are edited via a series of dialogs that are described in the previous two section see Sections 7 7 and 7 8 The Delete Pulse button brings up a dialog that asks which pulse pulse train or ramp to delete The pulses are listed by name The output waveforms are updated to reflect the change 54 7 11 Display The Display button brings up a series of two dialogs that controls the behaviour of the Acquisition Monitor Settings Acquisition Monitor window AuUto Scale adjusts the monitor y axis range to match the signal range Turn off monitor for increased speed Erase Before Each Episode If this option is turned on the monitor will be erased immediately before each episode is displayed If it is turned off episodes will be overlaid The monitor can be erased at any time by hitting the e hot key see Section 6 5 _ Erase Before Each Episode Ul Auto Scale
128. ts milliseconds or as a fraction of the sweep width If fixed time units are specified then the third dialog requests the pulse onset time and pulse width in milliseconds The test pulse duration will remain constant even if the scope sampling rate and sweep width settings are changed If relative timing is selected the test pulse will change in duration when the sweep width is changed The pulse will always occupy the same fraction of the sweep and the third dialog asks which fraction that will be 5 9 Test Link The Test Link feature provides a mechanism for noting the test pulse results to the log window then immediately running a data acquisition protocol The Test Link button brings up a dialog that requests the file name of an acquisition protocol see Chapter 6 for an explanation of protocol files When the Test To Log program is halted by hitting the space bar the test results will be directed to the log window the selected protocol will be activated and data acquisition will commence See Section 5 6 above for a description of the Test To Log program 39 6 Protocol Driven Acquisition 6 1 Introduction 6 2 Preview 6 3 Record 6 4 Resume 6 5 Hot Keys 6 6 Protocol 6 7 File Name 6 8 Deleting Data Files 6 1 Introduction The Scope and Chart programs described in Chapters 3 and 4 were designed to emulate familiar laboratory instruments and so have limited waveform output capabilities Both programs can only send a simple
129. und for this purpose The Axon Digidata uses the BNC cable earth as its signal ground To check the configuration of the AxoGraph data acquisition package Select the Configuration popup menu in the toolbar Click on the Full Configuration button Describe each of the cables that are connected from the electronic equipment to the digitizer Make sure that all these cables are actually connected as described To check that the digitizer is working correctly Disconnect all BNC cables from the digitizer Connect a cable directly between Analog Output 0 and Analog Input 0 Launch AxoGraph and switch the toolbar popup menu to Scope Click on the Run Scope button then the space bar to halt the Scope Click on Channels button Acquire data from Analog Input 0 and direct the test pulse to Analog Output 0 Click on the Pulse button and enter a large pulse amplitude Select Relative time and specify an Onset of 10 and Width of 50 Click on the Run Scope button Hit the a key to autoscale the sweeps A square pulse should be visible If a square pulse is NOT displayed this would indicate a serious problem with the digitizer hardware If a square pulse is displayed this would indicate that everything is working correctly with the AxoGraph acquisition package and the digitizer hardware The problem must lie with the electronic equipment or its connection to the digitizer 1 5 Optionally Remove the Electrophysiology Features Seve
130. ut channels For an Instrutech ITC series digitizer the group numbers should be between 0 and 3 Selecting the item Program gt Protocol gt Convert To Protocol from the AxoGraph menu brings up a dialog that asks whether the front window contains suitable data for conversion If Proceed is selected the program performs the conversion by adding protocol information to the Notes field of the window and by changing the names of the groups if necessary to conform with the Analog Output channel names All protocol parameters will be set to default values It will be necessary to adjust some of these parameters using the buttons in the Protocol toolbar For example the Trigger Repetition and Channel parameters may need to be changed For technical reasons it is not possible to edit the sample rate or the active Analog Output channels of the converted protocol and pulses can not be added 61 8 Online Analysis 8 1 Introduction 8 2 Add Analysis Programs to a Protocol 8 3 Standard Analysis Programs 8 4 Writing New Analysis Programs 8 1 Introduction An acquisition protocol can periodically launch a data analysis program while the protocol is running online analysis Analysis programs can operate on the last acquired episode or on the entire data file Online analysis can provide immediate graphical feedback about any feature of the acquired data and can automate routine data analysis tasks Several different online analysis programs ca
131. yses the response recorded on the Current channel to the 5 mV voltage command pulse It calculates the series resistance Rs from the size of the applied voltage step Vcmd and the peak amplitude of the resulting current transient Iresp using the formula Rs Iresp Vemd The program also measures the peak amplitude of the current Ipeak over a selected time period for example following the Trigger pulse on Digital Output 0 It calculates the amplitude relative to the 100 average current in a baseline period Time s Both the baseline and the peak time periods are defined by placing cursors in the monitor window after the first episode has been acquired The value of Rs and Ipeak are plotted in a separate monitor window that is created when the protocol is first run The analysis program that measures Rs and Ipeak is a standard part of the data acquisition package and it can be modified to monitor any parameter of the response See Chapter 8 Online Analysis for information on adding analysis programs to a protocol and on writing or editing custom analysis programs 76 Pulse Slope This protocol is similar to the previous protocol but it measures the slope of the response over a selected time regions It sends a5 mV 10 ms voltage command pulse to Analog Output 0 and analyses the current response recorded on the Current channel The analysis program fits a regression line over a selected

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