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AxoGraph X User Manual

1. M 14 5 1 Opena Graph WindOwW 5 irt eheu OD e e ERREUR U ERE 14 52 Zoom and Unzoom ecu temo n e ERR SEHE ae t Ye UE eren 14 5 3 SCO icit RR EUR EURO EORUM REIR ER EE E EO UR 14 5 4 Review Multi Episode Data ie ec esee enne nennen enitn tenete treten teen nne nenne 15 M Dis play Options E M 6 1 Axis Range and Title 6 2 Comment Title and Notes Options eterne eene neret retener nennen nennen 17 6 3 Symbol and Line Display Options esses nennen ene nrenet reete eene nenne 17 6 4 Symbol Legend eese tee ee ER ERE HH He EEE ere ette nempe recens 18 6 5 Automatic AXIS Units pe o RO CCP EORR REC EE EU CHO TES ITE RE SO E EO E Eth 18 6 6 Font Size up M 18 6 7 Axis and Scale Bar Display Options eee eseeseeeeseeeeseceecseecseeeeesceecseecsaesseaesecsesaeeesasseeesseeaees 19 6 8 Axis Tick Length and Line Thickness Options ceeeeeceessesesseeeesceeesseeecseeeseecseeacseeesseeesseeseeersees 19 6 9 Interval Batts eter Oo ERG EC ER UE I pP EP RR bo Rc tO EE RE 19 6 10 Set the X and Y Axis and Error Bar Data Columns esee 20 6T Error Bar Options seeevouse ote tenen eU CUR ENUR DDR RERT USE RI 20 6 12 Zero Crossing AX6eS iuis serere Ie AO SU RETRO WR HO Ee ERE eo eee Re eene 21 6313 Log ARES ed RO DRIBERRUTO ORO DORO RT EORR IRIS
2. 29 9 1 Scale and Offset X and Y Data nete tecocmeeeerettiUErHe hp et SE TENNESSE SESSE RERE Ses 29 9 2 Change X Axis ZetO eie eee toca dee ir tee e OE ee RE e Eee ere ettet 29 9 3 Normalize Peak Amplitude neto eo neu ee eode oen dg oe tb 30 9 4 Subtract iti S 30 9 5 Digital FIET eee rtt er RECUERDE EE RT ER UE Deu 30 9 6 Mouse Measurements sessi nene aE EEE EE teens treten eene entente ene nnene 31 9 7 Cursors Measurements toro ue Ono RRU Ro ER ERREUR UR 32 9 8 Peak Amplitude Measurement sees teen nenne tetnetrennetetne nenne tremens nne 33 9 9 Peak Shape Meas rement en eoitome opere n RO DU OO DE Hep CHO e ERU 33 9 10 Peak Det ction eee teet eee e Pe HORE ER ER dete Re re etre e 34 9 11 Convertto Histogram 5 nne no epe D Rd ORO RE ert 35 9 12 Power Spectrum m 35 I0 Data Analysis TEXN siscicccssickicsccsccsscusscocscosscosenccvadastacsudseadensvesevscussonssseunncvssacesssasd ssssbesssoesssnsussvssusssosesessonsssosy 36 10 1 Create a Graph From the Text Editor essit re ree Rte tec eb Hed 36 10 2 Simple Statistics in the Text Editor essere eene nennen emen enne 37 10 3 Tidy Selected Text eoo eoe rette er e Re ete debo pede 37 10 4 Convert Vertical Lists into a Tab Delimited Table esee 38 10 5 Transpose a Tab Delimited Table nee een Or RE REPRE OE HERR MIR He ign
3. 18 1 18 2 18 3 18 4 18 5 18 6 18 7 18 8 18 9 18 10 18 11 Math Functions Trigonometric String Manipulation Byte and Bit Manipulation Array Functions Advanced Array Functions General Purpose Procedures Create Array and String Variables User Dialog Array Procedures Advanced Procedures 18 1 Math Functions abs x sqrt x sqr x exp x In x exp10 x log10 x trunc x round x odd i Absolute value Square root Square Exponential e raised to the power x Natural Log to the base e 10 raised to the power x Log to the base 10 Next lowest integer Nearest integer Returns TRUE if i is an odd number 18 2 Trigonometric pi Sin x Cos x Tan x Sinh x Cosh x Tanh x ArcSin x ArcCos x ArcTan x Returns the value of pi 3 1415926 Sin Cos Tan Hyperbolic Sin Hyperbolic Cos Hyperbolic Tan Inverse Sin Inverse Cos Inverse Tan 79 80 18 3 String Manipulation NewString variableName StringToNum aStr NumToString aNum PtoCString aStr CtoPString anArr Length aStr Pos subStr aStr Concat aStr x bStr Copy aStr start len Delete aStr start len Insert iStr aStr start This procedure creates a new 255 character string It provides an alternative to formally declaring a string variable at the start of a program String variables can also be created on the fly For example aString abc123 Function that c
4. Close window Close the specified window If the content have changed the user will be asked whether to save the changes ForceClose window Close the specified window If the content have changed the changes will be discarded DeleteFile fileName Delete the specified file from the current folder Lock fileName Lock the specified file It can not be modified or deleted when locked Unlock fileName Unlock the specified file nWindows Returns the number of open windows 19 3 Edit Text Select window start end Select a range of text in the specified window extending from the character start to the character end GetSelection window start end Returns the current text selection range in the specified window The range extends from the character start to the character end ShowSelection window Scroll the selected text into view in the specified window ClearText window Clear the selected text in the specified window CutText window Cut the selected text from the specified window 90 CopyText window Copy the selected text from the specified window PasteText window Paste any text on the clipboard into the specified window 19 4 Read and Write Text Files ReadFrom window offset The next Read or ReadLn will start from the specified window and character offset If the offset parameter is omitted the end of the current selection is used GetReadFrom window o
5. is the sample interval FitExpPlus dx yArr amplitude timeConstant addedConstant Fits a single exponential with added constant to the data in yArr and returns the result in the parameters amplitude timeConstant and addedConstant The parameter dx is the sample interval FitDoubleExp dx yArr al tcl a2 tc2 addedConstant Fits a double exponential with added constant to the data in yArr and returns the result in the parameters al tcl a2 tc2 and addedConstant The parameter dx is the sample interval FitMultiExp dx yArr al tcl a2 tc2 a3 tc3 addedConstant Fits a triple exponential with added constant to the data in yArr and returns the result in the parameters al tcl a2 tc2 a3 tc3 and addedConstant The parameters al a2 and a3 are the amplitudes of the three exponentials and the parameters tcl tc2 and tc3 are the corresponding time constants The parameter dx is the sample interval If any of the four amplitude parameters al a2 a3 and addedConstant are set to zero before calling FitMultiExp they will be constrained to zero during the fit procedure FFT arr Performs a fast Fourier transform on the contents of arr and returns the 82 result in the same array ComplexFFT realArr imaginaryArr Performs a complex
6. 84 Real 4 Scaling Factor 88 Integer 2 Ist Data point 90 Integer 2 2nd Data point etc 20 2 Floating Point Data AxoGraph uses three different formats to store floating point data depending on the context The formats are 1 IEEE 4 byte 32 bit format real in Pascal and float in C 2 IEEE 8 byte 64 bit format double in Pascal and C 3 IEEE 10 byte 80 bit format extended in Pascal and C AxoGraph stores all graph data points using the 32 bit floating point format on both PPC and 68K machines The 32 bit format contains 24 bits of precision Thus a 32 bit floating point number can be used to average approximately 4 000 integer data points acquired by a 12 bit A D converter without loss of information All intermediate values generated during arithmetic calculations and data manipulations are performed using a higher precision format On 68K machines the 80 bit extended floating point format is used and on PPC machines the 64 bit double format is used All of AxoGraph s floating point parameters and all floating point variables in its built in languages are stored and processed internally using the higher precision floating point format Elements of array variables are stored in 32 bit format Higher precision results are rounded into the 32 bit format when they are passed into an array or a graph 95 20 3 Formulas and Algorithms Most of the formulas and algorithms used by AxoGraph fo
7. Groups start at the first element If average False then arr i is replaced by arr first increment i 1 If average True then arr i is replaced by the average of the array elements from first increment i 1 to first ncrement i 1 e Copy Array theArray from to Returns a copy of a subrange of theArray The parameters from and to specify the subrange to copy If these parameters are omitted the entire array is copied e CombineArrays arrl arr2 A function for combining two arrays The arrays arr1 and arr2 are combined into a new array and returned 83 18 7 General Purpose Procedures Beep Generates the alert or beep sound Random Returns a random number between 0 and 1 Wait sec Halt execution for the specified period in seconds Also Pause or Sleep CurrentTime Return the time in seconds since Jan 1st 1904 PauseUntil sec Halt execution until the specified time is reached GetDate Return a string containing today s date GetTime Return a string containing the time hours min sec FreeMemory Return the currently available memory in bytes For example check memory before creating an array 4 bytes per element nWindows Return the number of open windows ListBuiltIns List all built in procedures and functions ListPrograms List all loaded programs procedures and functions ListGlobals List all global variables and their current values GetSigFigs sf Get the default number of significant
8. Position the position where the mouse is clicked Distance the distance between two mouse clicks Amplitude the trace amplitude at the mouse click position Amplitude Difference the difference in trace amplitudes between two mouse click positions Measure Mouse Click Position Select the Position radio button then click OK in the 1 168 s 0 690 nR Mouse Measurement dialog The mouse cursor changes to a cross hair and the Measure Mouse Click Position dialog appears This window shows the x and y coordinates of the mouse and these values constantly update as the mouse is moved around in the graph window Click the mouse button with the cross hair cursor positioned anywhere in the graph The position of the click is marked on the graph and its x and y coordinates are written to the log More than one position measurement can be made and the measurements accumulate in the log The appearance of the mouse cursor and the behavior of the measurement feedback window will vary slightly depending on the style of mouse measurement selected Position Distance Amplitude or Amplitude Difference When a series of files is to be analysed the repeated presentation of the Mouse Measurement dialog will slow the measurement process To bypass this dialog turn on the Shift Cmd M bypasses dialog check box and initiate mouse measurements using the Shift Cmd M key combination When dialog bypass is active the Mouse Measurement
9. D k N4j42 Q j D d j 1 Q 3 N D Increasing the index by 1 j gt j 1 gives d j k 2 j 1 d j 1 N j k N4j 3 2 j 1 d j 2 2 j 5 N j Thus d j is now expressed in terms of the two higher coefficients d j 1 and d j 2 as well as k This expression has the property that if a reasonably large value of j is selected say 64 then for any defined k and arbitrary starting values for d 64 and d 65 1 0 and 0 0 the expression can be recursed inward to j 0 getting an estimate of d j at each step The resulting coefficients are extremely accurate values of the coefficients that would be obtained by Chebyshev transforming the function exp t tau The method is much faster than performing the transform At the end of the inward recursion the values are exact apart from a normalization factor It turns out that the sum of the coefficients must be 1 0 Thus summing all the coefficients together during the inward recursion gives a factor at the end of the calculation that can be divided into each coefficient This last step gives the Chebyshev coefficients of the exponential exp t tau Having obtained the Chebyshev coefficients for taul and tau2 and calling the two arrays containing the newly calculated coefficients d taul j and d tau2 j Then another regression equation is formed d j al d_taul j a2 d tau2 j 103 Since d j d tau1 j and d_tau2 j are known then al and a2 can be calculated by simp
10. Display gt Interval Bars To add an interval bar click on the Add Interval s Offset button then enter the Bar Title and Bar Title From To 0 1 specify the interval From gt To in o TUE Any number of bars can be added to a graph By default bars are drawn at the bottom of the lowest trace or A group They can be positioned aiia ia higher by setting Offset to a value in the range 0 to 1 If Offset is set to 1 the bar is drawn at the top of the lowest trace or group By default the interval bar title is drawn above the bar Turn on the Title Below Bar check box to display the title underneath the bar Interval bar thickness is the same as scale bar thickness see section 6 8 Interval Bars To hide all interval bars turn off the Display Interval Bars check box To delete an interval bar highlight its Bar Title and click the Delete button 20 6 10 Set the X and Y Axis and Error Bar Data Columns Re assign X and data columns Ada Derete _Column Number _ Column Name Trace X Y ErrBar Display gt Error Bars A dialog appears for specifying which data columns contain the X Y and Err Bar data for each trace In the example shown here 6 data columns left hand panel have been assigned to 2 traces right hand panel 1 Membrane Potential V 2 Current A 3 Error A 4 Membrane Potential V 5 Current A 6 Error A The first trace will plot column 2 Curr
11. FFT FitLine etc are also supported The programming languages are interpreted which means that code can be run instantly it does not need to be compiled first This provides many advantages For example code can be executed one line at a time as it is written intermediate results can be checked at any point and subroutines can be executed and tested before being integrated into a larger program The Language Implementations are Not Standard AxoGraph s implementations of Pascal Basic Fortran and C are designed for writing simple data analysis programs Some advanced language features are not implemented In particular AxoGraph C is a greatly simplified version of this large complex language None of the languages is case sensitive Normally C is 68 case sensitive Even if you are very familiar with one of the above languages please take a few moments to check the documentation for that language to see what features are available 16 2 A Programming Example in Basic Here is an example of how to create a simple program using the Basic language First select Program _ Preferences and turn on the Basic radio button then click OK A program always begins with the word program This word is followed by a string enclosed in quotes The string is appended to the Program menu when the program is loaded If the second last character of the string is a slash then the last character becomes a command key equivalent for ru
12. NewGraph fileName Create a new graph file Returns the window number OpenGraph fileName Open the graph file with the specified name It must be in the current folder If no fileName parameter is given a standard file dialog is presented OpenGraph returns the window number of the newly opened file If the file is already open its window number is returned If the file is not found return 1 Save window Save the contents of the specified window to disk Close window Close the specified window If the content have changed the user will be asked whether to save the changes 17 5 Get a Location Range or Point via a Dialog The variable aStr passes a message or prompt that is added to the dialog GetLocation w aStr xLocation Pose a dialog and returns the user selected x axis location GetRange w aStr xMin xMax Pose a dialog and returns the user selected x axis range GetPoint w aStr t xPoint yPoint Pose a dialog and returns the user selected x y point and the selected trace t Baseline w t xMin xMax Subtract a baseline calculated over the region xMin to x Max Note The window w is brought to the front if necessary 17 6 Set the Axis Title and Range 74 Xaxis w is an x axis title and displayed range Yaxis w t is a y axis title and displayed range These commands are used in a similar fashion to Xdata and Ydata For example the following 2 lines copy the X and Y axes from an old window to a ne
13. Program gt Copy and Paste gt Copy Style Select clipboard 1 File gt Close to close the front window File gt Open Graph turn on the Default Display Settings check box then select the same graph file It will open and display the graph data using the default style settings Program gt Copy and Paste gt Paste Graph Style Select clipboard 1 then click OK Turn on the Paste Axis Titles check box and the Paste Trace Grouping check box and turn off the other two check boxes then click OK The graph style information from clipboard 1 is applied to the front graph window Program gt Copy and Paste gt Edit Style Names Rename clipboard 1 to Alternate Blue and Red Traces 14 6 Copy and Paste Graph Data Program gt Copy and Paste gt Copy Data A dialog appears asking which subset of the data in the front graph window to copy to an internal clipboard If Copy All X and Y Data is selected then all the graph data will be copied If Copy Y Data Traces from Front Group is selected then only the traces in the front group will be copied This data cannot be pasted into another program it can only be pasted back into a graph window within AxoGraph Program gt Copy and Paste gt Paste Data A dialog appears asking which subset of the data on the internal clipboard to paste into the front graph window 54 Program gt Copy and Paste gt Graph Copied Data A new graph window is created from the data on the internal clipboard
14. SFY SF SY N Co Auto and Cross Correlation The cross correlation between two traces is calculated as follows Let Yi be the ith point of one trace and Zi be the ith point of the second trace Both traces contain N data points Let SY Sum over i Yi SYY Sum over i Yi Yi SZ Sum over i Zi SZZ Sum over i Zi Zi Co Square Root SYY SY SY N SZZ SZ SZ N Let CCi be the ith point of the cross correlation trace It has 2 N 1 points To calculate the ith point first define ko j N M N Abs ko where Abs is the absolute value function ka ko kb ko if ka lt 0 then ka 0 if kb lt 0 then kb 0 SYi Sum over k Yka k fork 1 to M SZi Sum over k Zkb k fork 1 to M SYZi Sum over k Yka k Zkb k fork 1 to M CCi SYZi SY1 SZ M Co The autocorrelation of a single trace is calculated using the same algorithm as above but with Yi substituted for Zi throughout 98 20 4 Simplex Optimization Algorithm AxoGraph uses a simplex algorithm to fit general equations and to fit exponentials when the faster Chebyshev algorithm is not selected It finds the combination of parameters that give the best fit between the fitted function and the data This is usually done by minimizing the sum of squared errors SSE between the data and the fitted function The simplex method is a standard algorithm that can be found in many numerical recipe books It should
15. against the same y axis Combine Traces into Numbered Groups Trace Group x 1 Normalized Binding 2 Normalized Peak 3 Normalized Separate All Combine All Combine Groups or 1 vl Number groups from zero As an example of arbitrary grouping assign trace 1 to Group 1 and traces 2 and 3 to Group 2 as shown below then click OK C Other buttons facilitate grouping when large numbers of traces are present Combine All merges all traces into a single group 1 Separate All separates groups by assigning each trace to a different group Combine Groups Of assigns sequences of N consecutive traces to the same group where N is entered in the adjacent text edit box Combine Every assigns every Nth trace to the same group where N is entered in the adjacent text edit box Number groups from zero begins the automatic group numbering from zero 25 7 3 Pile Tile or Offset Groups Before proceeding Trace gt Separate to separate all groups of traces This will aid the following descriptions Trace Pile All active not hidden groups are expanded to fill the graph window Click on the tag of the front group top left It is sent to the back and the next group is brought to the front Trace gt Tile All active groups are rearranged into equal sized non overlapping sub windows If the shift key is held down when this item is selected the groups are not repositioned but are resized into non overlappin
16. then select and open the file AxoTapeTags DAT in the Example Data folder This is an AxoTape file that includes tag markers Tags in Chart files can also be displayed Display Show Tags turns on the display of AxoTape tags To hide the tags select Display Hide Tags If no tags are present in a file the Show Tags menu item will be dimmed 24 7 Traces Groups and Windows 7 1 Separate or Combine All Traces 7 2 Combine Traces into Groups 7 3 Pile Tile or Offset Groups 7 4 Next Group 7 5 Show All Traces 7 6 Hide Traces 7 7 Delete Traces 7 8 Manipulating Graph Windows 7 1 Separate or Combine All Traces All sections in this chapter assume that a multiple trace graph window is open and that it is the front window File gt Open then select a multiple trace graph file Dose Response in the Example Data folder for example Trace gt Separate Split up any groups into individual traces Each trace is displayed in a separate sub window Trace Combine All traces in a graph are combined into a single group The combined traces are overlaid and plotted against the same axes When several traces are grouped they are treated as episodes and can be browsed and analyzed using tools described in Chapter 12 7 2 Combine Traces into Groups Trace gt Group This dialog permits arbitrary grouping of traces When two or more traces are placed in the same group i e given the same group number they will be overlaid and plotted
17. two exponential curves are to be fitted Select a region and click OK The optimization procedure begins and will take some time to converge over a minute on slower machines The slower performance is the price paid for the flexibility of the fitting procedure When the search converges the fitted exponentials are appended to the graph window and the optimal parameters are sent to the log window 46 12 Data Analysis Multi Episode 12 1 Remove Corrupt Episodes from Analysis and Display 12 2 Average Multi Episode Data All sections in this chapter assume that a multi episode graph window is open and that it is the front window File gt Open Digitized then select a multi episode graph file NMDAStep DAT in the Example Data folder for example this is a Clampex file with 16 episodes 12 1 Remove Corrupt Episodes from Analysis and Display Display a single episode for example episode 9 in the NMDAStep DAT file See section 5 4 for information on how to display selected episodes The decay phase of this episode contains a small recording artifact and should probably be removed from the display and from subsequent analysis This can be done as follows Display gt Mask Displayed The currently displayed episode disappears and is replaced by the next episode If another episode is masked it is added to a cumulative list of masked episodes If more than one episode is displayed when Display gt Mask Displayed is selected all the displa
18. xMin writeln An The minimum of the parabola was found at x x Min 87 19 File Management Functions 19 1 Change Folders 19 2 Create and Open Files 19 3 Edit Text 19 4 Read and Write Text Files 19 5 Read and Write Binary Data Files 19 1 Change Folders ChangeFolder folderName Set the current working folder If no parameter is given a standard file dialog is presented If the folder name is a string literal the brackets are optional The command cd is equivalent to ChangeFolder GetFolder refNumber Get the reference number of the current working folder SetFolder refNumber Make the folder with the specified reference number the current working folder 19 2 Create and Open Files Files List all files in the current folder dir and ls are equivalent to Files If followed by t or t only text files are listed If followed by g or g only graph files are listed If followed by d or d only digitized files are listed If followed by f or f only folders are listed FileExists fileName Returns true if the named file exists in the current directory IsATextFile fileName Returns True when the named file exists in the current directory and is a text file IsAGraphFile fileName Returns True when the named file exists in the current directory and is a graph file IsADigitizedFile fileName Returns True when the named file exists in
19. 2 Round 4 Sigma 1 bz 1 2 Sigma 2 1 2 N 2 1 Fj exp b G i 2 forj 1toN The data is then smoothed by sliding the Gaussian envelope represented by the filter coefficients along the data trace Each data point Yk is replaced by a filtered data point Y k as follows 96 Y k Sum over j Fj Yk j i Ensemble Average The ensemble average of a selected list of episodes in a digitized data file is calculated as follows Let Yij be the ith point of the jth episode Assume the ensemble average is calculated over all N episodes in the file Let SYi Sum over j Yij Now the ith point of the ensemble average Ai is Ai SYi N Ensemble Variance The ensemble variance of a selected list of episodes in a digitized data file is calculated as follows In addition to the symbols defined above let SYYi Sum over j Yij Yij Now the ith point of the ensemble variance Vi is Vi SYYi SYi SYi N N Residual Variance The residual variance of a selected list of episodes in a digitized data file is calculated as follows First the ensemble average of the N episodes Ai is calculated see above Then the ensemble average is optimally scaled to fit each episode The optimal scale factor for the jth episode Cj is calculated as follows SYAj Sum over i Yij Ai SAAj Sum over i Ai Ai Cj SYAj SAAj The optimally scaled average is subtracted from each trace Y ij Yij Cj Ai The r
20. Chebyshev transform to obtain d j d k Sum over j f G TkG Rk Define db j N j 2 d j 1 2 j 3 d j N j 1 d j 11 2 1 If db j is calculated from the d j and f t is an exact sum of exponentials then d j al d_taul j a2 d tau2 j where d taul j is the Chebyshev transform of a single exponential exp t taul and d tau2 j is the transform of exp t tau2 Calculating db j yields db j al db taul j a2 db tau2 j However each db taul j is related to the d taul j via d taul j k1 db taul j where k1 1 k1 1 expC l tau1 1 exp l taul Thus db j a1 d tau1 j k1 a2 d tau2 j k2 101 Performing the operation dbb j N j 2 db j 1 2 j 3 db j N j D db j 1 2 j 1 and employing the same identity dbb j al d taul j K1 2 a2 d tau2 j K2 2 There are now three equations d j al d taul j a2 d tau2 j db j al d taul j k1 a2 d tau2 j K2 dbb j al d taul j K1 2 a2 d_tau2 j k2 2 The left hand side coefficients are calculated from the d j Now there must exist a pair of parameters x1 and x2 such that d j x1 db j x2 dbb j al d_taul j 1 x1 k1 x2 k1 2 a2_d_tau2 j 1 x 1 k2 x2 k242 zero for all j The only way this is possible is if x1 k1 k2 and x2 k1 k2 Substituting x1 and x2 in the expression 14 xl kl x2 k1 2 gives 1 k1 k2 k1 k1 K2 k1 2 gt 1 1 k2 k1 k2 k1 gt 0 Thus if x1
21. Display Symbols and Lines Turn on both the Line and the Symbol check boxes Click on the open triangle symbol set Symbol Size to 4 and Skip By to zero then click OK The current voltage graph is now complete It can be saved or closed and discarded 49 13 3 Calculate an Amplitude Histogram and Open Time Histogram from Single Channel Data AxoGraph s general purpose analysis features can be applied to the analysis and display of single channel data Several dedicated single channel analysis programs are available for more specialized analysis for example Fetchan and pStat on the IBM PC or TAC on the Macintosh File gt Open Digitized Select the file SingleChannel DAT This is a Fetchex binary data file containing 80 seconds of simulated single channel data The data is free of offset drift and only a single well resolved channel is present Analyse gt Convert to Histogram Turn on the Simple Histogram radio button and set Bin Width to 0 03 pA Zero Centered then click OK A range selector dialog appears Select all the data and click OK A new graph window appears containing a point by point amplitude histogram of the single channel data The two peaks correspond to the closed and open states Window gt SingleChannel DAT Copy Bring the SingleChannel DAT Copy window to the front again Display Type of Axes Normal Change to normal axes since peak detection and measurement does not work with raster display Analyse gt Pea
22. E ERO RERO EE ER ERE 21 G14 Raster Displays P M 22 6 15 Split Clock SUpport terrine seese seeen rrea esset e o OU TOR RR Re ER PERDU E eed 23 6 16 Display Tags oe teet tdt etre eet EUER RH RH e e trea E re 23 7 Traces Groups and Windows eere A tos ens tos ens R T 24 4 1 Separate or Combine AII Traces eben teinte rre Pe Ue het 24 7 2 Combine Traces into Groups sss etenim tnene treni tenete ene treten tenete eterne 24 To Pie Tile or Offset Groups et EO EROR te EO TE tO RO Or ERE ERE 25 TA DECIDE 25 1 5 Show AILTr ces adopt pO OTRO A 25 7 6 Hide Traces p E 25 T4 Delete Traces cR ORO eo E HE E ER et eet te a ease 25 7 8 Manipulating Graph Windows esses nee nete nennen trennen nne nenne nenne 26 LM Print r Exp rt a Grap e 8 1 Use a Page Setup Scaling of 25 for Optimal Output Quality 8 2 Page Layout Print a Full Page Graph eese eene nennen tenente nennen 8 3 Page Layout Print a Half Page Graph essere nenne nennen 8 4 Copy and Paste a Graph to a Drawing Program sese enne nrenenee 8 5 Copy and Paste a Graph to a Word Processor sese eene nennen nennen 8 6 Export to a PICT File terere eret tee te REIR sseceseusedescucnesscevtanseceaescssecbeys iv 9 Data Analysis Cen
23. FE petto 72 17 4 Graph File Management eccessi nennen enne enitn ene nente E EE ette tn ener enne 73 17 5 Get a Location Range or Point via a Dialog essere ementi 73 17 6 Set the Axis Title and Range sess eene teen trenenn ener vvs torrone 74 LET Maniptilate Graph Windows nodu eret breed pt etr pe I ib Posi 75 17 8 Manipulate Traces and Groups ennemi n nenne tenne trennen nenne 76 17 9 Temporary Analysis Feedback oomen t o re e HERO ERE ETE 78 18 Math String and Array Functions 18 1 Math Functions teen re ee re e Re E REEL E RET e e HE sg 18 2 Irigonometric iR ERIReneoethes tos eedem tg ep ne nes 18 3 String Manipulation 5 oett teeth dederant teh ERR ERE EEEE EEEE EEEE EEEE ES SESK 18 4 Byt and Bit Manipulation teme rte teo RUD nee pergerent 18 5 Array Functions cete Re HU EE UR ERERUR UE SEXE TREE XE EIER E ERE Eee Edd 18 6 Advanced Array Functions hene o e De e RR HE EO e EHE HERE r bna etg Pee 18 7 General Purpose Procedures essent nennen eene eene tr inen nenne nenne 18 8 Create Array and String Variables sees nennen tenente nennen 83 18 9 User Dialogs eee eed ere e REPRE Hd euer ee aee eade 84 18 10 Array Procedures eo Re RUTRUM Phe CRUS 85 18 11 Advanced Procedures ueste deitate ete ette eee REIR HERR ERE E EESE EEEE ESk 86 19 File Management Functions 4 eere eee eee reete eene eee tn stas tn staat
24. Graph Utilities Interpolate Front Graph The number of data points in the front open graph is increased by interpolating between data points in the x and y axis columns This can be used to facilitate the combination of data sets acquired at different sample rates or binned with different bin widths 56 Program gt Graph Utilities gt Tile Graph Windows Tile all currently open graph windows in a square pattern that fills the screen A dialog appears asking how many horizontal and vertical tiles to use 14 9 More Utilities The commands in the More Utilities sub menu perform some common types of manipulation on traces in a graph window Program gt More Utilities gt Blank Artifact A dialog appears which is used to select a region over which to perform blanking Select a range that covers the artifact but precedes the rise of the response then click OK An average is calculated over a small region immediately before the selected region and then all points in the selected region is set to this value effectively blanking out the artifact Program gt More Utilities gt Interpolate Artifact A dialog appears which is used to select a region over which to perform interpolation Select a range that covers the last part of the baseline and the first part of the rise of the response then click OK Data points in the selected region are adjusted so that they form a line between the first and last points of the region This can be used to blank
25. Number of Exps group turn on the One radio button turn off the Added constant check box then click OK The Range Selector dialog appears Select the range from 0 to 700 ms then click OK The exponential time constant suggests a mean open time of 150 ms The Save Exponential dialog appears Turn on the Append to current file radio button and click OK 14 The Program Menu 50 14 1 Overview of the Program Menu 14 2 Customize the Program Menu 14 3 Commands Under the Program Menu 14 4 The Default Plug In Programs 14 5 Copy and Paste Display Styles 14 6 Copy and Paste Graph Data 14 7 Document Utilities 14 8 Graph Utilities 14 9 More Utilities 14 10 Select Events 14 11 Trace Appearance 14 12 Trace Manipulations 14 13 Trace Transforms 14 14 Electrophysiology Tools 14 15 Statistics 14 16 Online Help 14 17 Add Custom Programs to the Menu Load or Evaluate enter List Programming Prefs Reload Drop Ins gt Copy and Paste Document Utilities Graph Utilities More Utilities Select Events Trace Appearance Trace Manipulations Trace Transforms Electrophys Tools Statistics vwvrowevrvrvvrvvy Programming Help Online User Manual 36 14 1 Overview of the Program Menu Items in the Program menu are divided into six groups The first group contains the single command Load or Evaluate This command evaluates numeric expressions or executes program fragments The se
26. Programs 15 2 Case Studies Using Analysis Programs 15 1 The Analysis Programs The programs described in this section are found in files and folders inside the Event Detection Package folder the Electrophysiology Analysis folder and the Models and Utilities folder These programs need to be loaded before they can be used Instructions are provided in the online documentation on how to load each program After an analysis program is loaded it can be run via a menu item or sub menu item under the Program menu The Event Detection Package folder contains documentation and programs for detecting capturing sorting and analysing asynchronous events in continuous or episodic data files Detection algorithms include amplitude threshold first derivative threshold and template matching techniques The Electrophysiology Analysis folder contains the following analysis packages Current Voltage I V Analysis Population Spike Analysis Quantal Analysis A brief overview of these packages is supplied below Additional information is supplied in online documentation that can be accessed via the Program menu For example after loading the Current Voltage Analysis package select the menu item Program gt Current Voltage Analysis gt About I V Analysis The Models and Utilities folder contains the following analysis packages Chemical Kinetic Model Electrophysiology Models Foreign File Import Export More Analysis Programs Utility Programs The Ch
27. Set the Vertical and Horizontal fractions to 100 to specify a full page Graph Size Fraction of Page graph then select File gt Print Vertical 100 75 Horizontal o 28 8 3 Page Layout Print a Half Page Graph File gt Page Layout Set Vertical to 50 The graph will now be output to the top half of a page CN The orientation of the output page should be changed to vertical otherwise ae a a very short wide graph will be produced File gt Page Setup and set Orientation to vertical as shown File gt Print The graph is printed in the top half of a vertical page 8 4 Copy and Paste a Graph to a Drawing Program File gt Page Setup Set Reduce or Enlarge to 25 and Orientation to horizontal see section 8 1 File gt Page Layout For the highest resolution and output to a full page set both Vertical and Horizontal to 100 Edit gt Copy Copies the graph to the clipboard Switch to a drawing program and paste the graph into a new document In the drawing program again change the Page Setup settings to Reduce or Enlarge 2546 and set Orientation to horizontal see section 8 1 The pasted graph will occupy the top half of one page It can be edited and then printed at full resolution Note that when the Page Setup scaling factor is set to 2596 AxoGraph increases the text point size by 400 If the text was set to 12 point in AxoGraph it will appear set to 48 point in the drawing program 8 5 Copy and Pa
28. TRO RR OD PRO EERE E ERE UE SEO DOR aeons 4 Open Import Save and Export Files eere eee eere esee esee e tenens tns tns tns ense tos ens tns tn stone tests enses ens enaeo 4 1 Opena File easi esee eR e ER e PH e ae ee erre tette tet 4 2 Create a New Text Editor WIndOW teer ERR ERU ER REPE A EAE 4 3 Open a Text File for Editing sess nennen EEEE EEEE aoa A KESKSEST E 44 Open an AxoGraph Data File derer rette ie Per EAS OSE RESES iii 4 5 Default Display Settings in the Open File Dialog sese 9 4 6 Import a KaleidaGraph or CricketGraph Data File sees ener 9 4 7 X Axis Data in Imported Files sees enne nenne treten nne teene tenerent 10 48 Open a Text Data File asioi Roe eneq meteo genauere e RE 10 4 9 Append a Graph File to an Open Graph Window eese nennen eene 11 4 10 Open an AxoData or pClamp Digitized Data File esee 11 4 11 Open a Foreign Digitized File sesesseesesee eene nennen nennen trennen trennen 11 4 12 Save a E18 iu doses Reoo OH OR Rete e VOCE ORDER HEU t DOS EOS ERE 12 4 13 Export a Graph Window to a KaleidaGraph File eene nenne 12 4 14 Export a Graph Window to a Text Data File sees rennen 13 4 15 Export Only Part of a Graph Window essesseseeeeeee eene enne eene neene trennt etenn eene 13 S Browse Graph Dataene nennu
29. The display style of the original graph data is preserved The commands for copying and pasting graph data are also available via the Copy and Paste toolbar at the bottom of the screen An alternative way to combine data from separate graph windows is by selecting Program gt Graph Utilities gt Combine Selected Graphs This feature is described in section 4 8 A Worked Example File gt Open Graph then select a graph file 2 Pulses in the Example Data folder for example Program gt Copy and Paste gt Copy Data Select Copy Y Data Traces from Front Group then click OK Program gt Analyse gt Scale and Offset Y Enter a scaling factor of 2 then click OK Program gt Copy and Paste gt Paste Data Select Paste Y Data then click OK The two copied traces are added to the front window The display style of the original traces is preserved 14 7 Document Utilities The commands in the Document Utilities sub menu can be used to open and close multiple graph digitized or text files with a single menu selection Program Document Utilities List Documents This command lists the names of documents in a selected folder A dialog appears asking which class of documents to list Graph Digitized Text or All documents This is followed by a standard dialog that indicates the folder containing the files to be listed For example choose the Graph radio button in the first dialog then select the Example Data folder The following file names
30. across the full width of the page Long column titles may require a larger BED separation When writing source code for an AxoGraph program a setting of 4 spaces is useful for indenting code blocks Text gt Text Width The editor can work with text of any width A text edit region wider than a single page is often Width of text ___Slpages convenient For example analysis results consisting of 7 or O Print full width of text more tab delimited columns will be wider than a single page Enter the width of the text edit region into the Width of text ok box A setting of 5 pages is adequate for most situations A width setting of 1 page combined with automatic line wrap is useful for simple word processing Turn on the Print full width of text check box to print the full width of the text editor region If this check box is off only a single page width is printed 41 11 Data Analysis Curve Fit 11 1 Fita Line 11 2 Fit an Exponential 11 3 Fita General Equation 11 4 Fit a User Defined Function All sections in this chapter assume that a graph window open and that it is the front window File gt Open then select a graph file 2 Pulses in the Example Data folder for example 11 1 Fit a Line Linear regression can be performed on the front trace or group of traces Range Selector Analyse 2 Fit Line Use the Range Selector dialog to select the range of data to fit The range From To s can be entered by typi
31. and the result is displayed in a new graph window Program gt More Utilities gt Average Aligned Traces A dialog appears asking for a range of windows and or traces over which to average The program assumes that all the chosen traces contain an event of variable latency It presents each trace in turn together with a dialog for manually identifying the onset of the event The program aligns the responses by discarding some of the baseline from traces with delayed responses then constructs an average of the aligned events in a new graph window Program gt More Utilities gt Maximum Slope A dialog appears asking for the number of data points over which to calculate the local slope Select a value typically 2 to 20 then click OK A second dialog appears which is used to select a region to search for the point of maximum local slope Select a range then click OK For each trace in the front group the point of maximum local slope is found and a red line is drawn indicating the point and the slope Detailed numerical results are sent to the log window 57 14 10 Select Events The commands in the Select Events sub menu are used to restrict automatically mask or unmask selected events in an episodic data file Traces can be masked and excluded from subsequent analysis based on the amplitude or shape of the event they contain Program Select Events gt Mask Large Events All episodes in the front group are examined Episodes that contain an ev
32. appearance of the line when printed Line Thickness Dashed Line va 18 The symbol used for each trace is selected from a palette The symbol size is entered as the radius of the symbol in pixels For example the default symbol size is 3 which generates a symbol 6 or 7 pixels in diameter By default a symbol is displayed for every data point To improve the graph s appearance some symbols can be skipped When the Skip by distance radio button is selected a symbol is displayed only when a data point is more than N pixels away from the previously displayed data point The value of N is entered in the box labeled Number of pixels between each symbol This style will display symbols where they are needed most on steeply sloping regions of a signal However it can produce undesirable results when the signal is noisy preferentially displaying the points with Skip by distance points the greatest deviation from the underlying signal Number of pixels between each symbol When the Skip by points radio button is selected a gap of N data points is left between each displayed symbol The value of N is entered in the box labeled Number of points between each symbol This style displays data points in a more regular manner but may leave steeply sloping regions of a signal looking bare Histograms are displayed as filled or outlined columns or as oft off of a cityscape A gap can be left between neighboring columns The width of the
33. are included for reading Igor binary data files experiments CED Neurocom and C Lab data acquisition files AxoBASIC users can create Clampex and Fetchex style data files ABF format using routines supplied with AxoB ASIC and these files can be read directly into AxoGraph AxoGraph s own file format has been kept very simple The format is described in the Technical Information section Programmers can use public domain routines for reading and writing AxoGraph files which are provided in the folder AxoGraph Data File Format in the Documentation folder 1 4 Axon s Data Acquisition Software for MS Windows and MS DOS AxoGraph can read data files generated by all of Axon s data acquisition software for PC and Windows AxoTape continuous acquisition for PC AxoBASIC continuous and episodic acquisition for PC pClamp 6 CLAMPEX episodic acquisition for PC pClamp 6 FETCHEX continuous acquisition for PC AxoScope continuous acquisition for PC and Windows pClamp 7 continuous and episodic acquisition for Windows ABF file format pClamp 8 continuous and episodic acquisition for Windows With the exception of AxoBASIC all these programs record data in the ABF file format The MS Windows programs use a ABF filename extension and the MS DOS programs use a DAT filename extension 1 5 System Requirements AxoGraph will run on any Macintosh equipped with at least 8 MByte of RAM and a 68020 030 040 or PowerPC CPU It will ru
34. axis values in the currently visible units Individual data points should generally be manipulated by loading a trace into a temporary array then accessing elements of the array For example the following short Pascal program calculates the square root of each non negative data point in the 1st trace of the 2nd window localLanguage Pascal Square root example Pass the trace to a temporary work array tempArr Ydata 2 1 Process each element of the array e FOR i 1 to ArraySize tempArr do begin if tempArr i gt 0 then tempArr i sqrt tempArr i else tempArr i 0 end Pass the array back to the trace Ydata 2 1 tempArr Throw away the work array Unload tempArr 73 Access a Single Point on a Data Trace Xpoint w xLocation Ypoint w t xLocation Epoint w g e xLocation REpoint w g r e xLocation where xLocation defines the x axis location in user units The data point nearest to xLocation is accessed NOTE When more than one data point per trace is to be processed do not use Xpoint Ypoint etc Instead use Xdata Ydata etc to create a temporary working array then process elements of the array For a worked example see the square root example in the preceding section Access a Section of a Data Trace 17 4 Graph File Management Additional commands are described in File Management Functions access this document via Program gt Programming Help
35. b This procedure requests parameter values from the user via a standard dialog Similar to PoseDialog but only a subset of the parameters will be displayed The parameter list must have the following format The 1st parameter is a string containing general information The 2nd parameter is a string describing the first requested variable The 3rd parameter is a numeric boolean or string variable which 85 will receive the requested value The 4th parameter is a boolean expression which determines whether this item will be displayed in the dialog Subsequent parameters are optional but if present they must repeat the sequence text string then numeric boolean or string variable then boolean expression After executing this procedure the values entered into the dialog by the user are returned in the variables a b e VariableRadioDialog Introduction Describe A a display a Describe B B display b This procedure the user to select one item from a list via a standard radio button dialog Similar to RadioDialog but only a subset of the parameters will be displayed The parameter list must have the following format The 1st parameter is a string containing general information The 2nd parameter is a string describing the first requested variable The 3rd parameter is a boolean variable The 4th parameter is a boolean expression which determines whether this item will be
36. etd00233 01662236 v0023103 00001520 59 14 12 Statistics The commands in the Statistics sub menu perform some basic statistical tests on traces in a graph window There are commands for assessing the quality of a theoretical fit to a data set using the Chi Squared statistic and for comparing two data sets using the Kolmogorov Smirnov statistic Program gt Statistics gt Assess Quality of Fit Calculate the Chi Squared statistic and probability level for an optimized fit to a data trace The front graph should contain at least a data trace and a fitted trace If the graph is not a histogram then either a standard error trace or error bars on the data trace are also required A dialog appears asking for the trace number of the data and of the fitted curve Cancel then select Trace gt Group to help determine the correct trace numbers to enter Enter the trace numbers and the number of free parameters in the equation used to fit the data If the trace is a binned histogram then turn on the Fitted Data is a Histogram check box Click OK A second dialog appears requesting the range over which to calculate the Chi Squared statistic If the data trace is not a binned histogram then the standard error of each data point is required A third dialog appears asking for the trace number containing the standard errors If the data trace has error bars then it is assumed that these represent standard error The results of the Chi Squared analysis
37. example of how to create a simple program using the Pascal language First select Program gt Preferences and turn on the Pascal radio button then click OK A program always begins with the word program This word is followed by a string enclosed in quotes The string is appended to the Program menu when the program is loaded If the second last character of the string is a slash then the last character becomes a command key equivalent for running the program Load the program by selecting highlighting it in the text editor window then hitting the enter key program Example Program 0 begin writeln Calculate factorial numbers from 1 to 10 f 1 for i 1 to 10 do begin f f i writeln Factorial 1 f end end If the program loads successfully Example Program should now be the last item under the Program menu To run the program select Program gt Example Program 16 5 A Programming Example in C Here is an example of how to create a simple program using the C language First select Program gt Preferences and turn on the C radio button then click OK A program always begins with the word program This word is followed by a string enclosed in quotes The string is appended to the Program menu when the program is loaded If the second last character of the string is a slash then the last character becomes a command key equivalent for running the program Load the program by selectin
38. fast Fourier transform on the contents of realArr and imaginaryArir and returns the result in the same arrays Both arrays should be the same size Array Manipulation NewArray variableName arrSize This procedure creates a new array with arrSize real elements and sets all elements to zero It provides an alternative to formally declaring an array variable at the start of a program SetArraySize variableName arrSize This procedure changes the size of an existing array to arrSize elements and does not alter the value of existing elements SetArray arr value from to A procedure to fill an array with a single numerical value The from and to parameters are optional For all i in the selected range arr i value FillArray arr first increment from to A procedure to fill an array with regularly spaced numerical values The from and to parameters are optional For all i in the selected range arr i first increment 1 1 e ConcatArrays arrl arr2 A procedure for combining two arrays The arrays arr1 and arr2 are combined into arr1 DecimateArray arr first increment average A procedure to decimate reduce the number of points in the array The size of the array is reduced by approximately 1 increment Groups of increment points are replaced with either the last point in the group or with the average of the group depending on the setting of average
39. for Optimal Output Quality 8 2 Page Layout Print a Full Page Graph 8 3 Page Layout Print a Half Page Graph 8 4 Copy and Paste a Graph to a Drawing Program 8 5 Copy and Paste a Graph to a Word Processor 8 6 Export to a MacDraw II File 8 1 Use a Page Setup Scaling of 25 for Optimal Output Quality A graph can be output from AxoGraph in three different ways It can be printed exported to a MacDraw PICT file or it can be copied and pasted to any program that supports graphics all drawing programs and most word processors LaserWriter 8 Page Setup For the best graph output Page Attributes sj quality it is important to select File gt Page Setup Format for and set the Scale factor to 25 as shown at left pet For a full page graph the X ae Orientation should ieu generally be set to horizontal as shown Scale s 8 2 Page Layout Print a Full Page Graph File gt Page Layout Determines how large the output graph will be in when a graph is printed or copied to the clipboard relation to a printed page This not only its size is adjusted to a fraction of one printed page applies to printed graphs but also to graphs that are copied to the clipboard Use Page Setup to specify page size and orientation or exported to a PICT format file 4 scaling factor of 25 gives the best resolution Use the following settings to define the size of the graph relative to one printed page
40. from one another by Offset between sweeps Raster display mode was designed to aid figure production It significantly complicates the issues of mouse interaction and screen feedback For this reason some types of analysis cannot work properly when a graph is displayed in raster mode These analysis features are disabled when in raster display mode Normal axes are recommended for data analysis 23 6 15 Split Clock Support File gt Open Digitized then select and open the file SplitClock DAT in the Example Data folder This is a Clampex file that was acquired at two sample clock rates The first half of each trace was sampled at 50 kHz and the second half was sampled at 66 6 Hz When the file is opened only the first half of the acquisition period is shown and the first sample rate is used Display gt Multiple Clocks gt 2nd Clock switches the display to the second half of the acquisition period and changes to the second sample rate To view and analyse split clock files with the entire width of the data trace displayed correctly it is necessary to export the digitized data to a graph file Display gt Save a Copy then click on the Options button and turn on the All Data radio button and the Open Copied File check box Click OK then Save This operation will take a few seconds Trace Combine overlays the traces Zoom on the left end of the x axis to view the data sampled at the faster rate 6 16 Display Tags File Open Digitized
41. gap in pixels is entered in the Sap DE Pe eons o jJ Gap between columns text edit box Symbol Size 6 4 Symbol Legend Display gt Show Symbol Legend displays a movable symbol legend It lists the y axis titles for all displayed traces and indicates the symbol used to display each trace Drag the small black square to move the legend 6 5 Automatic Axis Units AxoGraph automatically handles Standard International SI units if they are present in the axis title For example if the initial units are pA and the axis range is adjusted to 1500 pA to 2000 pA then the displayed axis range will automatically switch to 1 5 nA to 2 0 nA This feature can be switched off Edit gt Preferences then turn off the Automatic SI unit conversion check box see section 3 5 Axis Title Units To raise an SI unit to a power use the caret symbol For example Area millimeters squared would be indicated as mm 2 in the Units field of the axis title as shown here 6 6 Font Size and Style The font size and style of all text in a graph window can be changed using the Font Size and Style sub menus under the Text menu To modify the font size and style of individual text elements copy and paste the graph to a drawing or page layout program See section 8 4 for more information about copy and paste 19 6 7 Axis and Scale Bar Display Options All sections in this chapter assume that a graph window is open File gt Open then
42. negative going population spike e g from 11 to 25 ms Next select a point close to the peak of the population spike 15 ms The point of maximum slope on the rising phase is located using the selected number of regression points and is indicated by a red line The two positive going peaks and the negative going population spike are detected and marked with black crosses A line is interpolated between these two peaks and the population spike amplitude is measured from the population spike minimum negative peak to the interpolated line The interpolated line and the population spike amplitude are drawn in green All results are output to the log window 16 Writing Programs 16 1 Introduction to Programming AxoGraph 16 2 A Programming Example in Basic 16 3 A Programming Example in Fortran 16 4 A Programming Example in Pascal 16 5 A Programming Example in C 16 1 Introduction to Programming AxoGraph All the information in this chapter additional information about the programming languages and simple example programs are available via AxoGraph s online help Program gt Programming Help AxoGraph contains a built in programming environment Programs can be written in Pascal Fortran Basic or C and executed from any text window or via the Program menu The default language is specified using the menu command Program gt Programming Prefs but can be overridden if the first line of the source file is of the form LocalLanguage Lan
43. not be confused with another type of simplex algorithm used in constrained optimization problems e g airline bookings The following description of the simplex algorithm used in AxoGraph is adapted from Chapter 2 6 of Methods for Unconstrained Optimization by J Kowalic and M R Osborne Elsevier New York 2068 A simplex is a set of n 1 points in n dimensional space In the case n 2 the corresponding figure is a triangle and in the case n 3 it is a tetrahedron The optimization search proceeds as a series of spatial operations on the simplex that tend to move it towards the function minimum The three basic operations used in this method are reflection expansion and contraction which are defined below First define three important vertices of the simplex Xmax is the vertex where f Xmax Maximum f Xi fori 1 to n 1 X2nd is the vertex where f X2nd Maximum f X1 for i 1 to n 1 i1 lt gt max Xmin is the vertex where f Xmin Minimum f Xi fori 1 to n 1 also define the centroid of all vertices except Xmax Xo 1 n Sum over i Xi fori 1 to n 1 1 lt gt max The three operations used in the simplex method are defined as follows Reflection the maximum vertex is reflected in the centroid of the remaining vertices and Xmax is replaced by Xreflect 2 Xo Xmax Expansion Xreflect is expanded in the direction along which a further improvement of the function value is expected The distance from Xo to Xre
44. parameters or via a mouse click Trace Appearance automatically set trace colors and symbols Trace Manipulations add subtract or duplicate traces subtract sloping baseline Trace Transforms integrate differentiate and custom filter data traces Electrophys Tools accurately measure and analyse synaptic amplitudes Statistics test the quality of a theoretical fit or whether two data sets are related The programs under each of these hierarchical menus are described in the following sections 53 14 5 Copy and Paste Display Styles Program gt Copy and Paste gt Copy Style A dialog appears asking which internal style clipboard is to receive the display style from the front graph window This style information includes for each trace the symbol type and size line type and thickness the color the grouping of traces group position x and y axis ranges the data columns assigned to each trace etc This style information can be pasted back into another graph window within AxoGraph it cannot be pasted into another program Up to 8 graph styles can be stored Style clipboards are preserved when AxoGraph is quit and re run Program gt Copy and Paste gt Paste Style A dialog appears asking from which internal style clipboard the display style is to be pasted A second dialog then appears asking which aspects of the display style are to be pasted The line and symbol styles for each trace are always pasted Turning on the Paste Axis Titles check box
45. pastes the title for each group Turning on the Paste Axis Ranges check box pastes the axis range for each group Turning on the Paste Trace Grouping check box groups the traces in the front graph window in the same way that they were grouped in the original window Turning on the Paste Column Assignment check box assigns the data columns to the traces and error bars in the front graph window in the same pattern that was used in the source window This check box generally should be switched off unless error bars are present It can produce confusing results See section 6 10 for information on how to assign columns to traces and error bars If there were N traces in the copy graph but there are gt N traces in the paste graph then the style of trace 1 will be pasted to trace N 1 the style of trace 2 will be pasted to trace N 2 etc Program gt Copy and Paste gt Edit Style Names A dialog appears that permits the names of the 8 style clipboards to be changed These names will appear in the dialog when the Copy Style or Paste Style commands are selected Informative style names can be defined such as Log Linear Axes or Thick Lines for All Traces The commands for copying and pasting graph styles are also available via the Copy and Paste toolbar at the bottom of the screen A Worked Example File gt Open Graph then select a graph file that has a customized display style 2 Pulses in the Example Data folder for example
46. procedure the values entered into the dialog by the user are returned in the variables a b e ChoiceDialog Introduction Alternative Default userChoseDefault This procedure offers the user an either or choice via a standard dialog The parameter list must have the following format The 1st parameter is a string containing general information The 2nd parameter is a short string describing the alternative option The 3rd parameter is a short string describing the default option The 4th parameter is a variable that returns True if the default option is selected or False if the alternative option is selected e RadioDialog Introduction Describe A a Describe B b This procedure requests the user to select one item from a list via a standard radio button dialog The parameter list must have the following format The 1st parameter is a string containing general information The 2nd parameter is a string describing the first requested variable The 3rd parameter is a boolean variable Subsequent parameters are optional but if present they must alternate text string then a boolean variable After executing this procedure the boolean variable a b corresponding to the item selected by the user will be set to true and all others will be set to false e VariableDialog Introduction Describe A a display a Describe B B display
47. sample interval for a window GetScreenSize xSize ySize Returns the size of the main screen in pixels The menu bar at the top of the screen is not included GetWindowSize w xSize ySize SetWindowSize w xSize ySize Get or set the size of a window in pixels GetWindowLocation w xLoc yLoc SetWindowLocation w xLoc yLoc Get or set the location of a window in pixels The point xLoc yLoc defines the top left corner of the window The y coordinates increase down the page a counter intuitive Macintosh standard IsATextWindow window Returns True when the specified window is a text file IsAGraphWindow w Returns True when the specified window is a graph file IsADigitizedWindow w Returns True when the specified window is a digitized file 17 8 Manipulate Traces and Groups Pile w Pile all the visible groups in a window Tile w Tile all the visible groups in a window Stack w Stack or Offset all the visible groups in a window Show w t Reveal a hidden trace Hide w t Hide a visible trace DeleteTrace w t Delete a trace TraceIsShown w t Returns TRUE if a trace is not hidden GetGroup w t g Returns the group number a trace belongs to SetGroup w t g Assign a trace to a group T NthGroupNumber w n Returns the number of the nth group in a window GetFrontOfGroup w g t Returns the number of the front trace in the group GetEpisodes w e1 e2 Return the first and last display
48. select a graph file 2 Pulses in the Example Data folder for example fnis ond scale Bar Options Display gt Scale Bars and Axes To display labeled axes turn on the Axes check box To display y axis titles turn on the Y Titles check box By default the y axis title is displayed horizontally at the top of the y axis To rotate the title to the vertical and display it beside the y axis turn on the 90 Rotated Y Titles check box To display scale bars turn on the Scale Bars check box By default the x and y scale bars are locked together so that when one is dragged both move To move Show C Axes L H Asis for Every Trace O Titles O 902 Rotated Titles EJ Scale Bars PJH Scale Bar Locked to V the x and y scale bars independently turn off the X Scale Bar Locked to Y check box 6 8 Axis Tick Length and Line Thickness Options Display gt Scale Bars and Axes then click the Extras button The dialog expands and options for controlling the axis tick length and axis and scale bar line thickness are presented Ticks may be located on the inside or outside of the axis line Selecting both inside and outside causes the tick to stroke through the axis line Line thickness is specified in pixels 6 9 Interval Bars Interval bars are similar to scale bars except they are NMDA 20 uM tied to a specific region of the x axis They are useful for indicating a period of drug application or other experimental condition
49. style information is not documented Graph File Format Header Byte Type Contents 0 OSType AxoGraph file header identifier AxGr 4 Integer AxoGraph file format version number 1 6 Integer Number of columns to follow Each column Byte Type Contents 0 Longint Number of points in the column columnPoints 4 String 79 Column title units should be in brackets Current pA 84 Real 4 1st Data point 88 Real 4 2nd Data point etc Note OSType is a 4 byte character identifier Pascal Integer type is equivalent to C short or 2 byte integer Pascal Longint type is equivalent to C long or 4 byte long integer Pascal Real 4 type is equivalent to C float or 5 byte floating point Pascal String type has no direct C equivalent It is a character string with the string length stored in the first byte 94 Digitized File Format supported by version 4 1 and later Header Byte Type Contents 0 OSType AxoGraph file header identifier AxGr 4 Integer AxoGraph file format version number 2 6 Integer Number of columns to follow First column X data Byte Type Contents 0 Longint Number of points in the column columnPoints 4 String 79 Column title units should be in brackets Current pA 84 Real 4 Sample Interval Each subsequent column Byte Type Contents 0 Longint Number of points in the column columnPoints 4 String 79 Column title units should be in brackets Current pA
50. text The average value the standard error of the mean S E M and the number of points in each column are output below the previously selected values For example Amplitude mV Area mV ms 2 5294 2 5294 2 615 2 615 2 22 2 22 2 5144 2 5144 2 6094 2 6094 2 68 2 68 2 7919 2 7919 2 8494 2 8494 2 2644 2 2644 2 5638 2 5638 Average 0 07103 0 07103 S E M 9 9 Number Of Points 10 3 Tidy Selected Text The following text is in a format typical of the analysis results that accumulate in AxoGraph s log window TrialA measure position X s Y nA 0 1584 0 03036 0 2988 2 309 2 745 1 209 TrialB measure position X s Y nA 0 01 2152 1 87 570 4 A common task is to tidy up analysis output of this kind by deleting superfluous information and concatenating the relevant measurements into a single list The list of measurements can then be plotted and 38 subject to further analysis The following text editor feature is designed to simplify this task Bring a text editor window to the front and select highlight some analysis results Tidy Selection Text gt Tidy Up Selection Turn on the Delete blank lines check box to delete all empty lines in the selected text Lines that contain only space and tab characters are also deleted This closes up the selection 3 Delete blank lines spaces or tabs only O Delete text lines non numeric text only Turn on the Delete text lines check box to delete all line
51. the Finder then select AxoGraph 4 4 File gt Get Info gt Memory show Memoy 7 AxoGraph uses its internal 9 9 MByte memory partition for graph style and layout Memory Requirements information and external system memory for graph data Kind application program Suggested Size 9953 K Minimum Size 2465 K K When a graph file is opened all data is Preferred Size loaded into system memory and style 5 ES 7 information is loaded into AxoGraph s e erred de ru memory partition The defaut memory memory is turned on in the Memory control panel partition is 9 9 MBytes reduces to 8 MBytes if virtual memory is turned on This is sufficient to open and manipulated files with a total of 3 500 traces To handle additional traces increase the size of AxoGraph s memory partition by 2 MByte per 1 000 traces increase Preferred size in the above dialog AxoGraph must be re launched for the new memory partition size to take effect 3 2 The Log Window When AxoGraph is started up a text editor window is opened automatically This log window will receive analysis results from graph H ill 0 AxoGraph 1 This is the log window Use it to make notes perform calculations or to write data analysis programs windows It can also be used to enter notes edit data perform calculations and write or execute Calculations mudo Type in an arithmetic expression then press ente
52. the current directory and is a digitized data file IsATextWindow window Returns True when the specified window is a text file IsAGraphWindow window Returns True when the specified window is a graph file IsADigitizedWindow window Returns True when the specified window is a digitized file 88 NewText fileName NewGraph fileName Create a new file Returns the window number If no fileName parameter is supplied a default name is used OpenText fileName OpenGraph fileName OpenDigitized fileName Open the file with the specified name and type It must be in the current folder If no fileName parameter is given a standard file dialog is presented Open returns the window number of the newly opened file If the file is already open its window number is returned If the file is not found return 1 If the file is not of the specified kind return 2 IndexOpenText index afterTime IndexOpenGraph index afterTime IndexOpenDigitized index afterTime Open a file by index Position in the current folder s file list Only open if it is was modified later than afterTime The optional afterTime parameter is given in seconds since Jan 1st 1904 Mac standard A reference time can be obtained using the GetTime function IndexOpen returns the window number of the newly opened file If the file is not found return 1 If the file is not of the specified kind return 2 If the file wa
53. update the Program menu Each of the hierarchical items under the Program menu Copy and Paste through to Statistics correspond to sub folders of the Plug In Programs folder Most of these folders should be retained as an integral part of AxoGraph but startup time and menu clutter can be reduced by removing any unused folders The Electrophys Tools and Statistics folders contain more specialized programs that may not be needed To remove the Statistics sub menu drag the Statistics folder out of the Plug In Programs folder then select Program gt Reload Plug Ins AxoGraph ships with many specialized analysis programs that are not pre loaded These include an event detection package several electrophysiology analysis programs a chemical kinetic modeling package and many other utility programs To activate the event detection programs open the Event Detection Package folder and drag the Event Detection folder into the Plug In Programs folder then select Program gt Reload Plug Ins 14 3 Commands Under the Program Menu Program gt Load or Evaluate This command takes the selected highlighted text from the front text window and loads any programs or functions from the text into memory for future execution It then takes any remaining text from the selection and attempts to execute it immediately If the remaining text contains one or more arithmetic expressions they are evaluated and the numerical result appended to the text window If no t
54. using the Shift Cmd N key combination When dialog bypass is active the Cursor Measurement dialog can still be accessed by selecting Analyse gt Cursor Measure 33 9 8 Peak Amplitude Measurement Analyse gt Peak Measure The largest amplitude peak in each trace is detected and the amplitude and shape X Measure amplitude at peak O Average the amplitude parameters are measured The dialog controls which of these parameters are reported Switch on the Measure um punis DERMEE amplitude at peak check box to display the amplitude of io paintis after peak each peak If the signal contains high frequency noise the peak measurement may overestimate the amplitude of the underlying signal A more reliable estimate may be obtained by averaging the amplitude around the peak To measure average peak amplitude switch on the Average the amplitude check box and enter the number of data points before and after the peak to include in the average Click OK The peak is automatically detected and marked on the graph The peak amplitude is displayed on the graph and written to the log Peaks are only detected in the visible x axis range If an artifact is present that is larger than the signal peak adjust the x axis range to exclude the artifact 9 9 Peak Shape Measurement Also Measure L Location of peak g Risefram a Je ip ja Je O Width pe J of peak O Onset a s Je of peak The remaining shape measurement options assume
55. 10 x Log to the base 10 trunc x Next lowest integer round x Nearest integer pi Returns the value of pi 3 1415926 Sin x Sine Cos x Cosine Tan x Tangent Sinh x Hyperbolic Sine Cosh x Hyperbolic Cosine Tanh x Hyperbolic Tangent ArcSin x Inverse Sine ArcCos x Inverse Cosine ArcTan x Inverse Tangent 44 Analyse gt Fit General Enter the equation to fit in the f x text edit box The equation shown here a exp x c 2 b Y describes a Gaussian curve General Curve Fit The Precision of the fit Free parameters are a b c 2 j Expression is a function of x should generally be set Mod o to less than 596 The f x a eup u c 2 b smaller the value the l more accurate the fit but Minimize the longer it will take A value of 096 will give the best possible fit 4 Guess starting values Precision 5 Js Sum of Sq Errors Q Chi Squared The simplex algorithm will Minimize either the Sum of Squared Errors or Chi Squared Sum of squared errors minimization should be used when the noise on the data is independent of the amplitude of the data Chi squared minimization should be used when the noise on the data is approximately proportional to the amplitude of the data The fitting algorithm works best when reasonably good starting guesses are supplied for the parameter values Turn on the Guess starting values check box and enter starting guesses i
56. 39 10 6 Load Vertical Lists into Arrays seeeeseeseeeeeeeeeene enne tenne eene treten nennen trennen 39 10 7 Text Editor Display Options one nIdeeesu CIPRO Pe RET UU 39 AL Data Analysis Curve Fit s cscsscssevssnsssscsoeseoscedecsosssusenasussnasusdondsoesessundensososnsoensassongnsdestesossnesussudsoaswasessease 41 DLL Fat EC C 41 TEZ FEitamn Exponential mn s setae siccecsdesencesteceshes iaa a O E EEEE E 42 11 3 Fit a General Equation 5 eee eter tre tt OUR EE EEEE rete reete ree 43 TIA Fita User Defmed Function 2 none temo Dn RO ette e nter et o RES 45 12 Data Analysis Multi Episode c eeeeee eee e entente eerte eene ene en senses essa estos senses estne en es estes es es ens tnseo 46 12 1 Remove Corrupt Episodes from Analysis and Display seen 46 12 2 Average Mul Bpisode Data ciet teer ER ERE ERE QUEEN E TE ESPERE et ERE eR eor R Eras 47 13 Analysis Case Studies icsscscscscscicasiccsscccssectccsedecesuscouscscscsascunectessccdecsedacessccsscuanssucsccneesseuscesseuaseassenseesseusceuscei acss 48 13 1 How to Use the Case Studies sese netnenrtne nennen eene 48 13 2 Construct a Current Voltage Curve from Voltage Clamp Step Data sess 48 13 3 Calculate an Amplitude Histogram and Open Time Histogram from Single Channel Data 49 14 The Program Mer eee etre erre teer euis
57. Analysis Marks Title amp Notes General Fit 4 Display second toolbar The Hitting any key interrupts graph drawing change takes effect after restarting AxoGraph check box is self explanatory This option is only useful when dealing with very large data Cx files that take a long time to draw A draw operation that is interrupted can be restarted by typing the clear key or selecting Edit gt Clear AxoGraph automatically handles Standard International SI units if they are present in the axis title For example if the initial units are pA and the axis range is 1500 pA to 2000 pA then the displayed axis range will automatically switch to 1 5 nA to 2 0 nA The axis range threshold above which the automatic conversion takes place can be set in the Convert units when number 2 text edit box Turn off the Automatic SI unit conversion check box to disable the unit conversion feature In addition AxoGraph automatically converts a time axis from seconds to minutes or hours if a very long axis range is specified Turn off the Automatic time conversion check box to disable this feature The two check boxes in the Log Output box determine what information will be written to the log window during data analysis When the Suppress name of analyzed file and Suppress type of analysis check boxes are on analysis results will be less cluttered with information This may help to construct a compact table of results See also sectio
58. AxoGraph X User Manual ae Note For Acrobat Reader Users For the best figure quality when reading this document onscreen the zoom setting should be 147 If the zoom setting has changed type 147 96 into the zoom field in the toolbar at bottom left e Search for a key word by clicking on the binoculars icon dd in the toolbar at top right Navigate this document using the bookmarks at left The bookmarks can be toggled in and out by clicking the toolbar icon kp at bottom left Alternatively navigate using the table of contents on following pages Clicking on a chapter name will jump to that chapter in the main text Copyright Copyright 1991 2005 by Dr John Clements All rights reserved Axon Instruments Inc 3280 Whipple Road Foster City CA 94587 U S A Disclaimer All product names mentioned in the AxoGraph X User Manual are registered trademarks of their respective manufacturers Mention of specific products should not be construed as an endorsement by Axon Instruments Chapters Introduction and Overview Terminology Operating Principles Open Import Save and Export Files Browse Graph Data Display Options Traces and Groups Print or Export a Graph Data Analysis Graph Data Analysis Text Data Analysis Curve Fit Data Analysis Multi Episode Analysis Case Studies The Program Menu Plug In Analysis Programs Writing Programs Manipulating Graph Data and Display Parameters Math String and Arr
59. KaleidaGraph CricketGraph Text Data AxoGraph Digitized AxoData pClamp ABF MacLab Chart O Default Dis File Format Options Other Digitized Data Setup 2 Data CED Igor Wave Igor Series Igor Expt 4 5 Default Display Settings in the Open File Dialog File gt Open then change the File Format popup setting to AxoGraph Use the standard file dialog to find the Example Data folder then select and open an AxoGraph data file 2 Pulses for example If another file is opened then the File Format will default to its most recent setting AxoGraph in this case The current display format settings symbols and colors trace grouping axis ranges etc are saved when a graph is closed and restored when it is re opened Default Display Settings To override the stored settings turn on the Default Display Settings check box in the File Open dialog When a graph is opened the default display settings will be used instead of the display settings saved with the file The defaults are the last settings that were manually selected in an open graph window When a file is opened the default display settings will be used instead of the display settings saved with the file The defaults are the last settings that were manually selected in an open graph window 4 6 Import a KaleidaGraph or CricketGraph Data File O Default Display Settings File Format KaleidaGraph h File Open t
60. Number of A D Channels 1 Binary This determines the byte order of the digitized data PCs based um it Chan MRN on Motorola chips DEC Gain pA per 1 Volt at A D C VAX Atari generally use lt Previous P CILE Offset pA for 0 Volts at t D 0 the same byte order as the Macintosh while R D Converter machines based on Intel CPU chips IBM and Holtage hange 3 19 Pone clones e reverse Precision 12bit s 2047 Data ficquisition Parameters R D Channel 0 Next gt Set the Voltage Range to 10 Volts and the Precision to 16 bits The voltage range is the minimum to maximum voltage at the input of the A D converter and the precision is the maximum numerical range of the digitized values It is not strictly the precision Some 12 bit A D converters left shift the converted values for compatibility reasons the 4 low order bits are set to zero In this situation Precision should be set to 16 bit 12 Set the remaining parameters as shown above The acquisition parameters only need to be set once The next time the file is opened they will be applied automatically Also they become the default parameters aiding import of multiple files with similar acquisition parameters The Acquisition Parameters dialog can be accessed after a file is opened by selecting Edit gt Acquisition Params 4 12 Save a File There are three items under the File menu that can be used to save the front graph or text window to a
61. S Average of array elements Alternative average Standard deviation s Alternative standard deviation Standard error Alternative standard error Variance Minimum array element Maximum array element 81 count arr from to Number of elements processed copyArray arr from to Return a copy of all or part of the array Note The from and to parameters are optional They specify a range of array elements to be processed If they are omitted the entire array is processed Array indexing in C starts from zero but in all other languages it starts from one 18 6 Advanced Array Functions WriteArray arr from to Write the contents of the array to the front window Stats arr from to Calculate the following summary statistics for the specified array and output the results to the front window Mean Variance S D S E M Minimum Maximum and Count Note the from and to parameters are optional They specify a range of array elements to be processed If they are omitted the entire array is processed FitLine xArr yArr slope yIntercept correlation Performs a linear regression of xArr and yArr and returns the result in the parameters slope yIntercept and correlation FitExp dx yArr amplitude timeConstant Fits a single exponential to the data in yArr and returns the result in the parameters amplitude and timeConstant The parameter dx
62. SI units instead of currently displayed units for example V instead of mV The following procedures are identical to the above except they return their parameters in the currently displayed units DisplayedXTitle w xStr copy the x axis title DisplayedYTitle w t yStr copy the trace s y axis title DisplayedGroupTitle w g yStr copy the group s y axis title DisplayedXUnits w xStr copy the x axis units DisplayedYUnits w t yStr copy the trace s y axis units DisplayedGroupUnits w g yStr copy the group s y axis units DisplayedXRange w xMin xMax copy the x axis range DisplayedYRange w t yMin yMax copy the trace s y axis range DisplayedGroupRange w g yMin yMax copy the group s range DisplayedXScale w scale DisplayedYScale w t scale DisplayedGroupScale w g scale Returns the x or y axis display scale factor This is the scaling between absolute units e g A and displayed units e g uA gt scale 1 000 000 17 7 Manipulate Graph Windows NWindows WindowTitle w Save w Close w GetComment w aStr SetComment w aStr GetNotes w aStr SetNotes w aStr AddNotes w aStr ShowNotes w display AddTag w tagPoint ShowTags w display NIntervalBars w Returns the number of open windows Returns a window s title Save any changes to a window Close a window Get a window s comment Set a window s comment Get a window s notes Set a window s note
63. The population spike is the a transient in an extracellular field recording due to the synchronous firing of a population of neurones The spike is typically negative going when the recording electrode is in a region containing predominantly cell bodies The population spike is superimposed on a slower field epsp and this is typically positive going Thus the population spike appears as a brief negative peak in the middle of a broad positive hump This creates two small positive peaks and one large negative peak The program Population Spike Analysis automatically identifies the two small positive peaks and the negative population spike peak A line is extrapolated between the two positive peaks and the population spike amplitude is estimated relative to this line This program also estimates the amplitude of the underlying EPSC by calculating the maximum slope on the rise of the EPSP field Quantal Analysis The program Simulation generates a synaptic amplitude histogram using a binomial model of transmitter release The model incorporates quantal variability and stimulus failures The program also generates a simulated list of amplitude measurements and the theoretical distribution from which the amplitudes were sampled The program Analysis fits a theoretical curve to an amplitude histogram It then tests the adequacy of the fit using the Chi Squared statistic The theoretical curve uses a binomial model to describe trans
64. YData YData Se H Rd just Y Axis then select a graph file 2 Pulses in the Example Data folder for example Analyse 2 Scale and Offset Y This opens the Scale and Offset dialog Turn on the multiplication radio button and enter a scale factor of 2 and an offset of 0 Make sure the Adjust Axis check box is on so that the y axis range will change to accommodate the scaled data Watch the y axis of the front group and click OK The scale and offset factors are applied to all traces in the front group Analyse gt Scale and Offset X A similar dialog is presented but the scale and offset factors apply to the x axis data only 9 2 Change X Axis Zero Location Selector Set new H zero Analyse Change X Zero A location marker vertical line is drawn at the x axis Zero point and a location selection dialog appears in a movable window A new x zero location can be entered by typing a value or the location marker can be moved using the mouse Point to a new x zero location in the graph window and click the mouse button Click OK and the x axis data is offset so that zero is now at the selected location The x axis range is adjusted automatically 30 9 3 Normalize Peak Amplitude Analyse gt Normalize Each trace in the front group will be scaled so that its peak amplitude is set to 1 This dialog controls whether positive or negative peak amplitudes are normalized Normalize Peak Amplitude Peak Detect
65. a File There are four items under the File menu that can be used to open files Open can be used to open graph digitized text or foreign files Open Digitized will only open digitized data files Open Text will open text files into a text editor window Import will open and import foreign data files Igor Pro etc The File Open dialog remembers the format of the last file opened via a given item This helps when files of several different format graph digitized text and foreign need to be opened consecutively in a single session For information about the plug in programs that import foreign data files read About Foreign File Import in the Foreign File Import Export folder which is in the Extras folder It is possible to import any foreign data file format via an import program 4 2 Create a New Text Editor Window File New Text creates a new text editor window that can be used to enter numeric data perform calculations and write or execute programs 4 3 Open a Text File for Editing File gt Open Text then find the Example Data folder and open the text file Current Voltage Tab Text The file opens into a text editor window and contains two tab delimited columns of numeric data 4 4 Open an AxoGraph Data File 2 Pulses Oy Average All k Current Voltage AxoGraph Dose Response Oy Igor Experiment Operational Model Data fe Variance Mean Plot All Files Text Edit v AxoGraph
66. a dash between the lowest and highest numbers in the range For example 1 3 5 8 would average episodes 1 3 5 6 7 and 8 Calculate the Average Episodes Q RII amp Select Ensemble MM Average L1 S D M S E M L Variance L Residual l ariance O Trial to Trial Variance Turn on the check boxes to calculate Average ensemble average S D ensemble standard deviation S E M ensemble standard error of the mean Variance ensemble variance Residual Variance ensemble variance after subtracting the optimally scaled average from each episode Trial to Trial Variance ensemble variance of Ej Ej 1 Ej 1 2 where Ej is the jth episode The resulting traces are displayed in a new graph window The ensemble S D S E M and variance all provide information about episode to episode variability If this variability is due to background recording noise the ensemble variance should be approximately flat If some of the variability is due to episode to episode changes in the signal this will show up as a positive deviation in the ensemble variance waveform If the signal has a constant waveform but varies in amplitude the ensemble variance time course will be approximately the signal squared If the signal has a constant amplitude but varies slightly in waveform the ensemble variance time course will be approximately the derivative of the signal Ensemble S E M is useful for constructing an average with error ba
67. am gt Current Voltage Analysis gt I V Setup and Run Turn on the Voltage Data was Recorded check box and turn off the Leak Data was Recorded check box Specify the current and voltage group numbers 1 and 2 respectively for the VoltageClamp DAT example file Select the range over which to measure the current and the voltage The measurements are made by averaging the data points over the selected range for each episode The resulting current measurements are plotted against the voltage measurements in a new graph window The axis is automatically set to zero crossing which is the standard for I V curves The Setup process only needs to be done once To perform I V analysis on another file select Program gt Current Voltage Analysis gt I V Run Voltage data is not required It can be filled in by the I V analysis programs File Open Digitized then select a multi episode digitized data file containing no voltage traces Current TTX DAT in the Example Data folder for example 66 Program gt Current Voltage Analysis gt I V Setup and Run Turn off the Voltage Data was Recorded check box Specify the current group number 1 then specify the voltage for the first voltage clamp step 60 mV and the step increment 5 mV Select the range over which to measure the current The current measurements are made by averaging the data points in the selected range for each episode The resulting current measurements are plotted against the calcul
68. an artifact that occurs in a sloping region of a trace Program gt More Utilities gt Zero Artifact A dialog appears which is used to select a region over which to zero the trace Select a range then click OK All data points in the selected region are set to zero This simple behaviour may be preferred to Blank Artifact under some conditions Program gt More Utilities gt Normalize Over Range A dialog appears which is used to select a range over which to normalize Select a range then click OK For each trace in the front group the average is calculated over the selected range and the amplitude of the trace is divided by the average This is useful for normalizing noisy traces Program gt More Utilities gt Align at Onset A dialog appears asking for an onset threshold relative to baseline standard deviation Select a value typically 10 to 20 then click OK A second dialog appears which is used to select a region of baseline that is common to all traces in the front group Select a range then click OK For each trace in the front group the onset of the response is identified first data point with absolute value greater than the threshold and the trace is adjusted so that the onset occurs immediately after the last point of the common baseline region This aligns all the traces so that their onsets occur at the same time Program gt More Utilities gt Average Front Group An average is constructed of all traces in the front group
69. are displayed in a final dialog and are also sent to the log window Program gt Statistics gt Compare Two Data Sets Test the hypothesis that two data sets are drawn from the same distribution The test uses the Kolmogorov Smirnov statistic The data sets can be arranged as two sequences of unbinned data points two binned histograms or two normalized cumulative histograms A dialog appears asking for the window and trace number of each data set Cancel and select Trace gt Group to help determine the correct trace numbers Enter the window and trace numbers and click OK A second dialog appears asking for the data format Both data sets should be in the same format If the traces contain a sequence of unbinned data values then chose the Unbinned Data radio button If the data values have been binned into histograms then chose the Binned Data radio button If the histograms have been scaled to give a probability density estimates area under the curve 1 then chose the Probability Density Estimate radio button If the histograms have been integrated and normalized then chose the Normalized Cumulative Histograms radio button The results of the Kolmogorov Smirnov analysis are displayed in a final dialog and are sent to the log window 14 13 Trace Appearance The commands in the Trace Appearance sub menu alter the display style color symbol line thickness of several traces simultaneously Program gt Trace Manipulations gt Set Colo
70. ated voltages in a new graph window Leak subtraction can be performed by the I V analysis programs File gt Open Digitized then select a multi episode digitized data file containing leak current data CurrentTTX DAT in this example Open another digitized file containing the currents of interest Current DAT in the Example Data folder for example Make sure the data of interest is in the front window and the leak data in another window Program gt Current Voltage Analysis gt I V Setup and Run Turn on the Leak Data was Recorded check box Specify the current group number 1 then specify the voltage for the first voltage clamp step 60 mV and the step increment 5 mV Specify the numbers of the windows containing the test Current DAT and leak Current TTX DA T current traces Select the range over which to measure the current For each episode the leak current trace is subtracted from the test current trace and The resulting current measurements are plotted against the calculated voltages in a new graph window Population Spike Analysis File gt Open then select a multi episode digitized data file containing population spike data Pop Spike AxoData in the Example Data folder for example Program gt Population Spike Set Regression points for max slope to 7 and click OK Select the region for the population spike analysis This region must exclude the stimulus artifact and include two positive going peaks on either side of a
71. ay Functions File Management Functions and Commands Technical Information p d mow PA AA MN WN Do p p p j pd ell el eee SMU ANINAN BWW Contents 1 Introduction and Overview scccssccsssecscssssssscsssscsssssessesscsssscsssccesesecseseessssessseseseseesssessssessessseessssessnseseeses 1 1 Time Saving Information eese enne ene treten EEEE EEEE 1 2 About This Manual 6 gonesotene ue DUI ROBO ona eee me OE gus 1 3 Supported File Formats sess enne tntnennenen nenne tenni treten entre trente nennen nnne 1 4 Axon s Data Acquisition Software for MS Windows and MS DOS sse 1 5 System Requirements ose tte teer RH Re re th trt eee e Hi Eee EI Het thee rete 1 6 B g Reports nhe op PR OR RO RE PO EE ts Ds LELMIN OOTY 2 Er T M 2 1 Digitized Graph and Text Files sess enne tnnt nenne nennen 22 Continuous and Episodic Data neto n e ORO EO TERCER EE REP UR beasties 2 3 Traces unde I M P m S Operating Principles E n 3 1 AxoGraph Memory Management esses nennen trennen tenente trennen nenne nennen 3 2 The Log inn E 333 Ihe Window Men fe eode bite TER REUNIR 34 The Program Men eee eH IR HE RE DUREE ESTER E ene ee e RES 3 5 AxoGraph Preferences ee oO
72. ce is added to the front group Fit General uses a Simplex optimization procedure to minimize the sum of squared errors between the general equation and the data trace See Chapter 19 Technical Information for details The general equation is compiled into memory for maximum speed A general curve fit can be used perform a linear or exponential curve fit with one or more parameters held constant during the fit For example a exp x 2 is a single exponential with a fixed time constant of 2 This is not possible with the dedicated linear and exponential fits described above section 11 2 and 11 3 45 11 4 Fit a User Defined Function Any user defined function can be fitted to a trace or to several traces simultaneously using the Minimize procedure Some simple programming is required but this approach is completely general Three standard situations where Fit General may not be adequate are 1 A goodness of fit criterion other than sum of square errors or Chi sq is required 2 Fit a single equation to several traces simultaneously 3 Fit a function that cannot be expressed as a simple equation For example f x a x whenx lt 0O f x b x whenx gt 0 An example program demonstrating the Minimize procedure is included with AxoGraph It is in a file called Simultaneous Exp Fit in the More Analysis Programs folder The program uses a weighted sum of squared errors WSSE to simultaneously fit two exponentials with add
73. cond group contains three commands that control the programming environment and display its current state The first two groups of items are described in section 14 3 The next three groups contain hierarchical menu items These lead to sub menus that contain all the plug in analysis programs that are currently loaded in memory Selecting an item from these sub menus runs the associated analysis program The last group contains two items that link to online documentation Sections 14 4 to 14 15 describe all the default plug in analysis programs that ship with AxoGraph These programs appear under the Program menu after AxoGraph is installed but can be customized by the user Section 14 2 describes how to customize the Program menu The last group contains two times that link to online documentation When the data acquisition package is loaded an additional group of hierarchical menu items is added to the Program menu See the online Data Acquisition Manual for information on the acquisition package Section 14 16 describes two methods for loading new programs into the Program menu manual load or via the Plug In Programs folder 51 14 2 Customize the Program Menu The Program menu can be customized by dragging files or folders into or out of the Plug In Programs folder Changes will be reflected in the Program menu next time AxoGraph is launched If AxoGraph is already running then select Program Reload Plug Ins to dynamically
74. ct only peaks 0 34 Analyse gt Peak Detection The dialog specifies the basic peak detection options Peaks are only detected within the visible x axis range Select the Auto radio button to detect peaks in the direction of the largest absolute amplitude Select the Maximum radio button to detect the largest absolute peak amplitude in the selected direction Select the First radio button to detect the first peak that satisfies the peak detection criteria see below Select the All radio button to detect every peak that satisfies the peak detection criteria see below Turn on the Relative to first point in trace check box to subtract a baseline from the data before peaks are detected This option may be useful when the signal is offset from zero It is only provided as a convenience feature and it will generally make more sense to manually subtract a baseline from the signal Analyse gt Baseline before using the peak measurement features When the Relative check box is on the baseline level is averaged over a small region at the left end of the visible x axis range then subtracted from the signal The baseline level is added back to the signal before measuring the peak amplitude or other peak parameters Peak Threshold Detect peaks greater than of max peak Onn Valley Threshold Separating valleys are less than of adjacent peaks Q nR of the maximum peak amplitude When the F
75. cy of the mathematics fit However the averaging tends to reduce the noise component if one exists The noise is usually distributed over the coefficients not necessarily equally Thus summing coefficients and using the averaged values has a significant ameliorating affect on noise It is probably true that this is not the best way to sum over the coefficients because the transform does not produce a flat spectrum if white noise is presented to it There are probably more sophisticated smoothing windows that could be run across the coefficients to reduce noise even more
76. d and AxoGraph ssdrolumi ities X Column Number attempts to guess the data layout To manually specify the layout turn off the Automatic check box Turn on the Read column titles check box if column titles are present Specify how many lines to skip at the start of the file usually 0 and the column delimiter Tab Comma or Space Enter the number of the x axis data column If there is no x data column click on the Y only radio button then specify the sample interval data points are assumed to be uniformly spaced Click OK pe Cohens Detimiter i Tab Q Space Comma A new graph window is created and the selected text data is plotted This technique may be applied to any text data in any text editor window For example data analysis results in the log window can be conveniently plotted in this way 37 Data to be graphed can also be organized as a series of lists one below the other For example X s 0 1584 0 2988 0 4437 Y nA 2152 570 4 124 3 To plot the list of three Y values in the second list against the list of three x values in the first list select both lists then Text Graph Selection 10 2 Simple Statistics in the Text Editor Bring a text editor window to the front and select a list of data values Do not select the column titles Select only the numerical values to be analyzed Text gt Selection Stats Summary statistics are calculated for the data values in each column of selected
77. d file will automatically be opened after it is created x 14 5 Browse Graph Data 5 1 Open a Graph Window 5 2 Zoom and Unzoom 5 3 Scroll 5 4 Review Multi Episode Data 5 1 Open a Graph Window All sections in this chapter assume that a graph window is open File gt Open then select a graph file 2 Pulses in the Example Data folder for example 5 2 Zoom and Unzoom Move the mouse cursor into the middle of a graph It changes to a magnifying glass Hold the mouse button down and drag a zoom box around a feature of interest The selected feature is enlarged and the x and y axes change accordingly With the mouse cursor still in the middle of a graph double click the mouse button to zoom back out Each double click zooms further out until the default axis range is reached Move the mouse cursor over one of the axes It changes to a cross hair Hold the mouse button down and drag a zoom bar besides or below a feature of interest Only one axis is zoomed Display Default Axis Ranges sets the x and y axis ranges back to their default values 5 3 Scroll Zoom in on a feature of interest in a graph window then use the keyboard arrow keys to scroll the graph Alternatively use the mouse to activate the scroll buttons in the bottom left hand corner of the graph window These provide smoother faster scrolling than the keyboard 15 5 4 Review Multi Episode Data File gt Open Digitized the
78. dialog can still be accessed by selecting Analyse Mouse Measure 32 9 7 Cursors Measurements Analyse Cursor Measure The dialog specifies the type of cursor measurement to make Cursor Measurement Q Measure amplitude at a single cursor Measure between two cursors Select the Measure amplitude at a single cursor radio button to measure the amplitude of each trace at a single point only W Average amplitude 0 Standard Deviation L Area O Standard Error of Mean O Number of points L Variance O Minimum L Maximum O 28N bypasses dialog o Select the Measure between two cursors radio button to perform measurements on each trace over a selected range of the x axis All data points located between the two cursors are analyzed Turn on one or more of the check boxes Average amplitude Area etc to indicate which measurements to perform Location Selector If the Measure amplitude at a single cursor Measure Amplitude All Groups radio button is selected a location marker vertical line is drawn at the default amplitude At 7 measurement point and the Location Selector dialog is presented The measurement location can be entered by typing it in or it can be selected using the mouse Point to the desired location in the graph window and click the mouse button once By default all traces in the front group are measured To measure all visible traces turn on the All Groups check box Click OK a
79. disk file Save saves to an existing file Save as saves all graph data a new file then switches to that file Save a Copy saves only the displayed graph data to a new file For graph windows the Save as and Save a Copy items let the user specify the format of the new data file AxoGraph KaleidaGraph etc The Save and Save as items do not work for digitized data files This protects these valuable files from being overwritten Use Save a Copy to save changes to digitized data files 4 13 Export a Graph Window to a KaleidaGraph File Open the AxoGraph file 2 Pulses File gt Save as then change the File 2 Pulses d 63 Format setting to KaleidaGraph as shown Qi Average All SEITE UTI Also change the file name so that the AxoTapeTags DAT NM eee original file is not overwritten Current Yoltage AxoGraph Be Current Voltage Comma Text 3 Save this document as AxoGraph v KaleidaGraph CricketGraph Text Data MacDraw PICT File Format Now click on the Options button If the Created by AxoGraph check v Include Column Titles box is turned on the new file s creator flag will be set to AxoGraph Created by AxoGraph otherwise it will be set to KaleidaGraph This determines which program is run when the file s icon is double clicked EG 13 4 14 Export a Graph Window to a Text Data File Graphs can be exported to tab delimited text files Thi
80. displayed in the dialog Subsequent parameters are optional but if present they must repeat the sequence text string then a boolean variable then boolean expression After executing this procedure the boolean variable a b corresponding to the item selected by the user will be set to true and all others will be set to false Report Report Message This procedure opens a temporary report window modeless dialog that displays a message in bold blue text It remains open until it is closed by calling the procedure CloseReport It provides a mechanism for informing the user that a procedure will take some time to complete CloseReport Closes the report window 18 10 Array Procedures WriteArr arr from to Write the contents of the array to the front window Stats arr from to Calculate the following summary statistics for the specified array and output the results to the front window Mean Variance S D S E M Minimum Maximum and Count FitLine xArr yArr slope yIntercept correlation Performs a linear regression of xArr and yArr and returns the result in the parameters slope yIntercept and correlation FFT arr Performs a fast Fourier transform on the contents of arr and returns the result in the same array e ComplexFFT realArr imaginary Arr Performs a complex fast Fourier transform on the contents of realArr and imaginaryA
81. e decay constants for each exponential component of the fit The decay constants and amplitudes of each exponential component the added constant and the goodness of fit usually SSE are sent to the log window 43 The Fitted Function dialog appears that specifies how to handle the fitted exponential curve By default the curve is not saved To incorporate the fitted curve into the graph turn on the Append to current file radio button and click OK The fitted curve is appended to the graph window and the new trace is added to the front group The graph and fitted curve can now be printed or exported When analysing a series of files the repeated presentation of the Exponential Fit dialog is unnecessary To bypass this dialog turn on the Shift Cmd E bypasses dialog check box and initiate exponential fits using the Shift Cmd E key combination The Exponential Fit dialog can still be accessed by selecting Analyse Fit Exponential 11 3 Fit a General Equation A general equation can be fitted to the front trace or group of traces The equation should be a function of x and the free parameters should be selected from a b c P i e a maximum of 10 free parameters is supported The equation can include any of the following abs x Absolute value sqrt x Square root sqr x Square exp x Exponential e raised to the power x In x Natural Log to the base e exp10 x 10 raised to the power x log
82. ed Episodes All currently masked episodes are unmasked and all currently unmasked episodes are masked As an example this program can selectively display and analyse all large events when applied following the Mask Large Events program Program gt Select Events gt Unmask All Episodes All episodes in the front graph window are unmasked 14 11 Electrophys Tools The commands in the Electrophys Tools sub menu are designed to help measure and analyse synaptic currents recorded in whole cell patch clamp recording mode Both high frequency noise contamination and synaptic latency jitter contribute to synaptic amplitude measurement errors These errors can be reduced by calculating the average amplitude over a period that includes part of the rising and falling phases of the synaptic current The average amplitudes will always be less than the peak amplitude and generally need to be scaled up to match the peak synaptic current One of the following programs performs this two stage amplitude measurement When synaptic amplitudes are large gt 1 nA they can be distorted by voltage clamp error due to the patch electrode series resistance Rs If the series resistance is known then it is possible to compensate the recorded currents for this distortion Two of the following programs measure Rs and generate a series resistance compensated synaptic current Program gt Electrophys Tools gt Measure Rs This program assumes that the front window con
83. ed constants to two data traces in the front graph window The WSSE is calculated as WSSE Sum of Data Fit 2 abs Fit The second fitted exponential is a scaled version of the first The first exponential is fitted to the first trace and the scaled exponential is fitted to the second trace The WSSE is calculated by adding together the WSSE s for each trace The combined WSSE is a function of four free parameters the exponential amplitude and time constant the added constant and the scaling factor One approach to this optimization problem is to fit the first three parameters to the first trace then hold these parameters constant and find the optimal scaling factor to fit the second trace A better approach is to fit both traces simultaneously and to minimize the combined WSSE All four free parameters are optimized simultaneously in this approach To demonstrate the Simultaneous Exp Fit program it needs to be loaded Drag the program from the More Analysis Programs folder into the Plug In Programs folder then select Program gt Reload Plug Ins Bring a graph window with at least two traces to the front 2 Pulses for example Program gt Simultaneous Exp Fit A dialog appears requesting the numbers of the data traces to be fitted and the numbers of traces to receive the fitted exponentials Click OK The next dialog requests starting guesses for the exponential parameters Click OK A dialog appears requesting the region over which the
84. ed episodes ReviewEpisodes w el e2 Review the specified episodes EraseEpisodes w Erase all episodes EpisodeIsDisplayed w e True if the episode is currently displayed EpisodeIsMasked w e True if the episode is masked SetEpisodeDisplayed w e displayed Displays the episode if displayed True SetEpisodeMasked w e masked Masks the episode if masked True Get YLocation w t loc size Set YLocation w t loc size Get or set a group s vertical location and size GetSymbol w t type size SetSymbol w t type size Get or set a trace s symbol type and size type is a number from 1 to 12 0 no symbols size sets the symbol diameter in pixels GetSymSpacing w t space SetSymSpacing w t space Get or set the spacing between adjacent symbols If space is positive the separation is defined as number of data points If space is negative the separation is in number of pixels GetLine w t width dash SetLine w t width dash Get or set the line width and style of dashes width is the line width in pixels 0 no line For a fine line on a laser printer set width 0 25 dash is the style of dashed line 1 no dash GetHistogram w t type separation SetHistogram w t type separation Get or set the histogram type and bin separation type 2 0 no histogram is drawn type indicates a histogram with filled bins type 2 indicates a histogra
85. ed using commands and functions described in Chapters 16 17 and 18 10 7 Text Editor Display Options The appearance of text windows can be altered via the following items under the Text menu Line Wrap Text gt Line Wrap toggles the Line Wrap setting When Line Wrap is active v Auto Indent text lines longer than the current text editor width will automatically wrap to Show Invisibles a new line standard behavior for a word processor Show Page Breaks Tab Width Text gt Auto Indent toggles the Auto Indent setting When Auto Indent is Test Width active and a line starts with one or more spaces and tabs then typing the Use As Default Style return key automatically inserts the same number of spaces and tabs at the start of the new line This is particularly useful when writing AxoGraph programs Text gt Show Invisibles toggles the Show Invisibles setting When Show Invisibles is active tab and return characters are displayed using special symbols Text gt Show Page Breaks toggles the Show Page Breaks setting When Show Page Breaks is active page breaks between pages are shown as dashed lines Calculating and displaying the page breaks slows the performance of the text editor 40 Text gt Tab Width Specify the separation between the tab markers Tab spaces relative to a space character A setting of 20 to 30 spaces is usually adequate to separate data columns Tab markers are equally spaced ok
86. eeeeseesceceecsceecseesecaeeecseasseceessseessceaseasesseeaees 62 15 Plug In Analysis Program cccscsscsscsssscssssscesssssessesscessessessessessesssssessessessessccssessessesessessessessessessesseree 63 15 1 The Analysis Programs 22 csccselecscicsstsssnnctescadavtusessnscosdsscbes isseseteteeensevacactrechesssectesadsausetasubestesnvaeseeeees 63 15 2 Case Studies Using Analysis Programs eese nemen ene enne 65 16 Writing Programs eeiete secedere aen nena eere none eta aea o Sce enun ases epaa spore Dua eas ropa a pea UE e Det nada epa o oaeeo Ee esok Toes 67 16 1 Introduction to Programming AxoGraph sse nennen een nene ener 67 16 2 A Programming Example in Basic essent enne enne emet ene trennt 68 16 3 A Programming Example in Fortran esee nennen eene trennen eene 68 16 4 A Programming Example in Pascal eese see etetnttete nete tte stent tete tete nonet nennen 69 16 5 A Programming Example in C sssessssssseeeeeeeeeeteenne nennen nenne tremere trennen 69 17 Manipulating Graph Data and Display Parameters 4 ee eeee eese ee eee eene e eene en senten es enn etos ens tnsetntnae 70 LL Accessa Data TTa 6 5n tt de tano pee EO OD RET et metodi 70 17 2 Graphs with Multiple X Axis Data Columns eeeeseseeeeeeeeeeenee tenete nennen ener 71 17 3 Access Section Of a Data Trace eei ocoo teer e p ede
87. efers to the position of a data column in that list NXColumns w is the number of X data columns XColumn w xc is the XCth data column in the X data column list GetTraceXColumn w t xc returns the list position xc of the X column assigned to the specified trace SetTraceXColumn w t xc assign the specified X data column xc to the specified trace 72 The following commands apply to the list of all data columns The parameter c refers to the position of a data column in that list NColumns w is the total number of data columns Column w c is the Cth data column GetColumnTitle w c cStr returns the column s title string GetXColumn w t c returns the number of the data column assigned to the X axis of the specified trace SetXColumn w t c assigns the Cth data column to the X axis of the specified trace GetYColumn w t c returns the number of the data column assigned to the Y axis of the specified trace SetYColumn w t c assigns the Cth data column to the Y axis of the specified trace GetErrColumn w t c returns the number of the data column assigned to the error bars of the specified trace SetErrColumn w t c assigns the Cth data column to the error bars of the specified trace 17 3 Access a Section of a Data Trace Xrange w t minRange maxRange Yrange w t minRange maxRange Erange w g e minRange maxRange RErange w g r e minRange maxRange where minRange and maxRange define a range of x
88. emical Kinetic Model folder contains a package of programs for modeling the evolution in time of a chemical kinetic model Markov model This package can be used to model the properties of voltage and ligand gated channels with complex reaction schemes It can also be used to estimate reaction rate constants by fitting the predicted current to a recorded current transient The Electrophysiology Models folder contains the following three modeling packages MK 801 Model Vesicle Release Model Operational Model Fit A brief overview of these packages is supplied below The Foreign File Import Export folder contains programs for importing and exporting data files in various formats It also contains documentation on how to write programs of this type The More Analysis Programs folder and the Utility Programs folder contain programs for analyzing and manipulating data that are not likely to be widely useful They are provided here as examples and potential starting points for people wishing to write custom analysis programs in AxoGraph 64 Current Voltage Analysis The analysis programs in this package are intended for data recorded with a voltage clamp step protocol A typical protocol starts each episode at a constant command potential holding potential then steps to a test potential that changes from episode to episode An I V curve is constructed by plotting the clamp current vs the voltage during the step Population Spike Analysis
89. ent on the Y axis against column 1 Membrane Potential on the X axis Each data point will have an error bar attached with length specified in column 3 Error The Err Bar column numbers are optional and can be left blank or set to zero 4 Move selection down after clicking ona column name lt Display gt X vs Y Brings up the same dialog but with the error bar settings hidden This dialog is simpler to work with when error bars are not required O Delete unused columns When error bar columns are specified the error bar display format can be controlled as follows Turning on the check box to displays a bar above each data point turning on the check box displays a bar above each data point and the length in pixels of the cross bar at the end of each error bar can be entered in the Width field The specified error bar format will be applied to all traces To set a different error bar format for each trace see the following section 6 11 Column Numbers in the right hand panel can be entered automatically by clicking on a Column Name in the left hand panel The Move Down check box is on the text entry selection will move down after clicking on a Column Name otherwise it will move to the right Click on the Next button to move down or to the right without altering the currently selected item Click on the Delete button to mark the currently selected trace for deletion A marked trace has X and Y col
90. ent with an amplitude greater than a specified threshold are masked This permits the selective display and analysis of small events Large events can be displayed and analysed by subsequently selecting Swap in Masked Episodes A dialog is used to select the amplitude threshold A second dialog is used to limit the range over which to search for events Program gt Select Events gt Mask Misshaped Events All episodes in the front group are examined Episodes that contain an event with shape parameters outside a selected range are masked This permits the elimination of misshaped events glitches noisy traces etc A dialog is used to select the shape parameters to be tested The shape parameters that can be used are peak amplitude 20 80 rise time half width event onset 2046 of peak baseline standard deviation and baseline offset A histogram is constructed displaying the distribution of each parameter and cursors are used to limit the acceptable parameter range Program gt Select Events gt Mask Even Episodes All even numbered episodes are masked Program gt Select Events gt Pick One Episodes This program picks a single episode out of a the displayed episodes A dialog appears requesting a mouse click point The episode that passes closest to that point is displayed and the episode number is shown This is a convenient method for identifying an individual episode to be analysed or masked Program gt Select Events gt Swap in Mask
91. esidual variance V i is the ensemble variance of the N subtracted episodes SY i Sum over j Y ij SYY i Sum over j Y ij Y ij Vu SYY n SYi SY3 N N Trial to Trial Variance The trial to trial variance of selected episodes in a digitized data file is calculated as follows The first and last episodes in the selected group are not processed For each of the remaining episodes the average of the previous and the following episodes is subtracted Yij Yij YiG 1 Yig l 2 forjz2to N 1 The trial to trial variance V i is the ensemble variance of the N 2 subtracted episodes SY i Sumoverj Y j forj 2 to N 1 SYY i Sum overj Y ij Y j forj 2 to N 1 V iz SYY SY i SY 1 N 2 N 2 Linear Fit Linear regression is a standard procedure described in many numerical recipe books The slope A and y axis intercept B of the fitted line are calculated as follows Let Xi Y1 be the ith of N data points 97 SX Sum over i Xi SY Sum over i Yi SXY Sum over i Xi Yi SXX Sum over i Xi Xi A SXY SX SY N SXX SX SX N B SY A SX N The correlation coefficient CC of the fitted line is calculated as follows Let Fi be the ith point of the fitted line Fi A Xi B SY Sum over i Yi SF Sum over i Fi SYY Sum over i Yi Yi SFY Sum over i Fi Yi SFF Sum over i Fi Fi Co Square Root SYY SY SY N SFF SF SF N CC
92. ext is selected the single line of text that contains the flashing cursor is processed as above Hitting the enter key is the standard method of executing this command The Load or Evaluate menu item is only included as an aid to new users This command can be used as a calculator For example type 2 2 into the log window then hit the enter key The result 4 will appear immediately below It is important that new programs functions and global variables are given unique names The three items under the Program List menu can be used to check whether a function name or global variable name has been defined Program gt List gt Global Variables The names and values of all global variables currently in memory are listed to the front text edit window Program gt List gt Programs and Functions The names of all currently loaded programs procedures and functions are listed to the front text edit window Program gt List gt Built In Functions The names of all built in procedures and functions are listed to the front text edit window 52 Program gt Programming Prefs This dialog sets the default behaviour of the programming environment Most importantly it selects the default programming Language It also specifies whether the arguments to trigonometric functions are in Degrees or Radians and the number of Significant figures to be used when numerical results are output to the text window Programming Options Lang
93. ffset Returns the current window and character offset where the next Read or ReadLn will start from The offset parameter is optional ReadLn aStr Read a line from a text file into the string aStr The next Read or ReadLn will begin at the start or the next line Read aVar bVar Read numeric values from a text file into the variables aVar bVar The next Read or ReadLn will begin at the start or the next line Numeric values should be comma tab or space delimited Non numeric text in the input line will be skipped automatically If there are too few values to satisfy the parameter list an error will result WriteTo window offset The next program output e g from a WriteLn will be directed to the specified window and character offset The output buffer of the current window is flushed If the offset parameter is omitted the end of the current selection is used GetWriteTo window offset Returns the current window and character offset where the next output will be directed too The offset parameter is optional EndOfFile window Returns True when the current selection offset is at the end of the text in the specified window TextLength window Returns the total number of characters bytes in the text in the specified window 91 Write A string a b c WriteLn A string a b c Print A string a b c Printf A string a b c These
94. figures for output SetSigFigs sf Set the default number of significant figures for output GetKeyDown Returns the ASCII code of any key that is currently down or zero if no keys are down Useful for interrupting a loop FlowControl Background SpinCursor CheckKeys TempMem Set parameters that control the execution of large programs within AxoGraph The four boolean parameters are Background AxoGraph can switch to background while a program is running SpinCursor Display a spinning cursor while program is running Suppress this for time critical programs CheckKeys Check for Cmd period escape and other key strokes TempMem Use temporary system memory when creating arrays etc Unload varName programName procedureName functionName Unload all the specified global variables programs procedures and functions This frees up memory space allocated to the arrays and procedures It also removes the specified program names from the Program menu This procedure may be useful for freeing up memory associated with large global data structures but in practice it is rarely used 18 8 Create Array and String Variables NewArray variableName arrSize This procedure creates a new array with arrSize real elements and sets all elements to zero It provides an alternative to formally declaring an array variable at the start of a program e Copy Array theArray from to Returns a copy of a subrange of theArray The para
95. files but some minor distinctions are retained Raw digitized data is valuable and should not be altered by an analysis program For this reason digitized files are copied when they are opened and all processing is applied to the copied data To save the processed data it must be exported to a graph file 2 2 Continuous and Episodic Data Digitized data files are of two types Continuous one long data trace recorded without interruption Episodic several short data traces synchronized to a stimulus Continuous acquisition is used to record spontaneous or unsynchronized events Episodic acquisition is used to record evoked or synchronized events Some display and analysis features Review Average only work with episodic data AxoScope AxoTape and Fetchex record continuous data Clampex 6 and AxoData record episodic data The AxoGraph data acquisition package and Clampex 7 and 8 record both continuous and episodic data A graph file can be continuous or episodic If two or more traces are combined in a group see following section then the file is treated as episodic 2 3 Traces and Groups For a graph file a Trace is a single array or column of data For a digitized file a Trace is a single episode recorded on one A D channel Current nA X c c o he k 2 o 2 Pulses See 2 Time s Traces can be organized into numbered Groups When more than one
96. flect is doubled and Xreflect is replaced by Xexpand 2 Xreflect Xo Contraction is used when reflection fails The maximum vertex is replaced be the point half way between itself and the centroid so Xmax is replaced by Xcontract 0 5 Xmax 0 5 Xo The search procedure can be viewed as the reflecting expanding and contracting motion of the simplex toward the minimum This motion is accomplished in the following way 1 An initial simplex is formed the vertices are guessed and the function is evaluated at each of the vertices 99 2 The points Xmax X2nd Xmin are determined and the centroid Xo is calculated The reflection operation is performed and the function is evaluated at Xreflect Depending on the value of f Xreflect one of the steps 3 4 or 5 is performed 3 If f Xreflect lt f Xmin the reflection was successful so the simplex is expanded in the new direction and the function evaluated at Xexpand If f Xexpand lt f Xmin the expansion was successful and Xmax is replaced by Xexpand otherwise Xmax is replaced by Xreflect In either case the search procedure returns to step 2 4 If f X2nd 2 f Xreflect 2 f Xmin the reflection was a partial success so Xmax is replaced by Xreflect and the procedure returns to step 2 5 If f Xmax gt f Xreflect gt f X2nd the reflection was a partial failure so Xmax is replaced by Xreflect and contraction is tried and the function evaluated at Xcontract But if f X
97. g highlighting it in the text editor window then hitting the enter key program Example Program 0 printf Calculate factorial numbers from 1 to 10 f 1 for i 1 i lt 10 i f i printf Factorial ji f If the program loads successfully Example Program should now be the last item under the Program menu To run the program select Program gt Example Program 70 17 Manipulating Graph Data and Display Parameters 17 1 Access a Data Trace 17 2 Graphs with Multiple X Axis Data Columns 17 3 Access a Section of a Data Trace 17 4 Graph File Management 17 5 Get a Location Range or Point via a Dialog 17 6 Set the Axis Title and Range 17 7 Manipulate a Graph Window 17 8 Manipulate Traces and Groups 17 9 Temporary Analysis Feedback 17 1 Access a Data Trace AxoGraph incorporates several languages Basic Pascal Fortran and C In addition to the standard features of these languages AxoGraph implements many language independent commands and functions which can be used to access and manipulate data Data is organized in columns as in a spread sheet A column is a list of numbers and is equivalent to a one dimensional array A data column can be passed to data array in one of the programming languages One or more columns typically just one contains x axis data values The remaining columns contain y axis data values Each trace on the screen represents a y axis data column plotted agains
98. g sub windows The best way to visualize the two types of Tile is to try them Trace gt Offset All active groups are rearranged with a small offset between each group This stacked arrangement is most useful when the y axis titles are displayed horizontally see section 6 7 because the titles will then form a list in the graph window 7 4 Next Group Trace gt Next The front group is sent to the back and the next group is brought to the front This option is only active when there is more than one group in the graph window 7 5 Show All Traces Trace gt Show All Hidden groups are moved back into the graph window then all groups are rearranged with a small offset between each group 7 6 Hide Traces Trace gt Hide This dialog permits one or more trace to be hidden While a trace is hidden it will also be excluded from any analysis For example turn on the check box corresponding to trace 1 then click OK Trace 1 is moved to the hide region This dialog can also be used to move traces out of the hide region by turning their check boxes off Traces to Hide O 1 Normalized Binding O 2 Normalized Peak O 3 Normalized Equilibrium cancer ox When several traces are combined in a group individual traces can also be hidden using the Display gt Mask Selected feature see section 12 2 7 7 Delete Traces Trace gt Delete This dialog permits one or more traces to be permanently deleted from the graph It i
99. gram Online User Manual A documentation window appears with information about the two online user manuals These include the manual that you are currently reading 14 17 Add Custom Programs to the Menu Manual Load Programs can be written in Pascal Fortran Basic or C then loaded into memory and run at a later time The document containing the source code does not need to remain open once the program or function has been loaded Programs functions and procedures are loaded manually by selecting the source text using Edit gt Select All then pressing the enter key When a program is loaded its name is appended to the bottom of the Program menu See the next chapter for more information about how to write a program in AxoGraph A complete list of currently loaded programs functions and procedures can be obtained by selecting Program gt List Programs Automatic Load When AxoGraph starts up it looks for a folder called Plug In Programs located in the same folder as the AxoGraph application and opens any program or compiled module files that it finds in this folder It also searches sub folders in the Plug In Programs folder for additional files The contents of each file is loaded into memory and the file is closed The loaded programs have their names added to the Program menu Files are loaded in alphabetical order and programs are added to the menu in the order they are loaded 63 15 Plug In Analysis Programs 15 1 The Analysis
100. group is present in a graph window each is displayed in an outlined sub window with a numbered tag in the top left corner A group can be moved by dragging its tag It can be resized by dragging the handle small black square in the bottom right hand corner A group can be hidden by dragging its tag into the gray Hide region at the bottom left of the graph window Analysis functions are not applied to hidden groups Groups have a front to back order which affects both display and analysis The front group is outlined in black and the tag of the front group is dark red group 1 above All other tags are gray Some analysis functions only apply to the front group although most functions permit optional processing of all groups To bring a group to the front click once on any part of its sub window To send the front group to the back click on its dark red tag For a graph file traces can be grouped in any combination see chapter 7 For an episodic digitized file all traces episodes corresponding to a given A D channel are automatically combined into a group and this grouping cannot be altered 3 Operating Principles 3 1 AxoGraph Memory Management 3 2 The Log Window 3 3 The Window Menu 3 4 The Program Menu 3 5 AxoGraph Preferences 3 1 AxoGraph Memory Management D SSS AxoGraph 4 4 Info SB Toreview or edit AxoGraph s memory partition highlight the AxoGraph zx AxoGraph 4 4 application icon in
101. groups Group 1 shows the whole cell voltage clamp current and group 2 shows the corresponding voltage clamp step commands Display gt Review Episodes Turn on the All radio button and click the Scan button Wait until all 10 episodes are displayed Optionally zoom the y axis of group 1 to see the clamp current traces more clearly Analyse gt Cursor Measure Turn on the Measure between two cursors radio button switch on only the Average amplitude check box and click OK A Range Selector dialog is presented Enter a range from 48 to 50 ms switch on the All Groups check box then click OK In the Cursor Measurements Complete dialog click OK again Switch to the log window click on the AxoGraph 1 title bar or select Window gt Show Log Select the two lists of amplitude measurements including the titles Text gt Selection to Table The two measurement lists are converted to a tab delimited table with current and voltage columns Text gt Graph Selection The Text Import dialog appears Turn on the Automatic check box turn on the X vs Y radio button and set the X Column Number to 2 Click OK and a new graph window appears Display gt Type of Axes gt Zero Crossing The axes now cross at 0 0 the standard format for a current voltage plot Display gt Change Axis Range Set the x axis range to extend from 60 to 50 mV and set the minor x tick interval to 10 mV and the major x tick interval to 30 mV Click OK
102. guageName where LanguageName is Pascal Basic Fortran or C Many useful programs are supplied in the Plug In Programs folder the Electrophysiology Analysis folder the More Analysis Programs folder and the Extras folder These programs can be customized or adapted to perform new tasks AxoGraph s startup time and memory overhead can be reduced by removing unused programs and folders from the Plug In Programs folder Programs can manipulate data and change display parameters in any open graph window Programs can interact with the user via standard dialogs Frequently used programs can be assigned names which are then appended to the Program menu Programs can be automatically executed or loaded onto the menu each time AxoGraph is run A minimal knowledge of one of the above programming languages and of AxoGraph s built in functions is required to program AxoGraph effectively The time invested leaning to program AxoGraph greatly extends analysis flexibility and can lead to automation of routine data analysis The programming environment can be used as a sophisticated scientific calculator Language syntax has been relaxed to make numerical calculations easier Calculations are performed by typing them into a text 67 window then pressing enter A record of the calculation is maintained that can be cross checked or printed Standard scientific functions sin cos exp and many statistical functions are supported Several array functions
103. hat contain program source code into or out of the Plug In Programs folder then re run AxoGraph See Chapter 14 for a complete description of the Program menu Select Program gt Programming Help to access online documentation describing AxoGraph s multi language programming environment 3 5 AxoGraph Preferences AxoGraph Preferences Edit gt Preferences A dialog appears which controls global aspects of AxoGraph s operation including Redrawing graph windows Automatic unit conversion in axis titles Numerical precision of results sent to log window Default colors in graph windows Whether to display a second toolbar Graph Window W Fast graph refresh uses more memory O Hitting any key interrupts graph drawing Axis Titles 4 Automatic SI unit conversion Convert units when number 2 4d Automatic time conversion min hour Log Output O Suppress name of analyzed file O Suppress type of analysis Significant figures for results 5 Color BackGround Axis Labels Linear Fit When the Fast graph refresh check box is switched on AxoGraph takes a snapshot after it has finished drawing a graph and uses the snapshot to refresh the graph when parts of it are covered then uncovered again Using the snapshot is much faster than redrawing the graph but takes up additional memory To gain extra free memory at the cost of slower performance turn this check box off before opening any graph windows
104. he new traces or Trace gt Combine to combine old and new traces into a single group 4 10 Open an AxoData or pClamp Digitized Data File File gt Open Digitized then change the File Format setting to AxoData or pClamp Open a digitized data file Current DAT for example The graph window is titled Current DAT Copy because AxoGraph is working with a copy of the original data Any changes made to the data cannot be saved back to the original file This protects the raw digitized data Use Save a Copy to save changes to digitized data files 4 11 Open a Foreign Digitized File File Open Digitized then change the File Format setting to Digitized Data and turn on the Default Display Settings check box in the File Open dialog Select and open the file ForeignDigitized DAT This single run foreign data file was digitized on an IBM PC and written to a disk file with no header information When a digitized data file which lacks a pClamp or AxoData header is opened AxoGraph presents the user with a dialog where the acquisition parameters sampling rate gains etc can be specified Set Skip bytes at start of file to 0 since this file has Sampling no header If a foreign file Continuous Skip bytes at start of file had a 512 byte header Episodic then this parameter would Points per Episode 2048 204 8 ms i G 2046 be set to 512 Data Format Sample Interval j 100 us IBM Binary Set Data Format to IBM Mac Binary
105. he other rates docking and loading are invariant This model describes release from a single terminal If several terminals are present and if all terminals are identical contain the same number of reservoir vesicles and release complexes then the parameters for all terminals can be lumped into a single meta terminal In this case the parameter values describe the meta terminal Operational Model Fit Two dose response curves are simultaneously fitted using the operational model of agonism The parameter tau may be different for the two dose response curves The operational model is defined by the equation Em tau n A 4n Ka A 4n tau n A n where A Agonist concentration Em Maximum amplitude of response tau Transducer ratio Ka Affinity n Hill coefficient index of cooperativity 15 2 Case Studies Using Analysis Programs Case studies are presented for two of the analysis programs Current Voltage Analysis and Population Spike Analysis The case studies refer to files supplied in the Example Data folder Current Voltage Analysis The commands in the Current Voltage Analysis sub menu generate current voltage I V curves from multi episode data These programs can be applied to data recorded with a variety of different protocols File gt Open Digitized then select a multi episode digitized data file containing current and voltage traces VoltageClamp DAT in the Example Data folder for example Progr
106. hen change the File Format setting to KaleidaGraph or CricketGraph Find the Example Data folder then select and open a data file Current Voltage KaleidaGraph for example 10 4 7 X Axis Data in Imported Files AxoGraph data files are always organized with the x axis data in the first column Imported data files may not share this organization Some have no x axis data and others may place it in a different column File gt Open then change the File Format setting to KaleidaGraph or CricketGraph File Format KaleidaGraph h t Click on the Options button in the File Open dialog O Default Display Settings The text import dialog appears Enter the number of the x axis data Data Organization column in the imported file If there is no x data click on the Y only amp Xvs Y radio button then specify the sample interval data points are assumed Q Y only to be uniformly spaced X Column Number n 4 8 Open a Text Data File AxoGraph can import text data files containing lists of numbers The numerical data columns can be separated by tabs or commas There can be several lines of description at the start of the file and the data columns may or may not have text titles AxoGraph will try to guess the layout of the text data If this does not work the layout can be specified in detail as follows File gt Open then change the File Text Format Data Organization Format setting to Text Data see section Bi Aut
107. ime s E H ES H A H FR z gt l El n E 6 14 Raster Display 0 SS singleChannel DAT Copy lelo lelo ME Lt Pars feel bol 22 E Open the file SingleChannel DAT in the Example Data folder This graph is displayed in raster mode The continuous data trace is plotted in 4 separate segments one below the other The advantage of this display mode is that it sacrifices redundant y axis resolution for improved x axis temporal resolution Raster Display Y axis units Offset between sweeps nA Width of each sweep S 3 Ruto ad just Y range EJ Force scale bar display Specify raster offset using Q Percentage of display height Display gt Type of Axes gt Raster Display Each trace is divided into several sections sweeps and the sweeps are displayed vertically offset from one another The offset is specified in the Offset between sweeps text edit box and the width of each sweep and hence the number of sweeps is specified in the Width of each sweep text edit box When the Auto adjust Y range check box is turned on the Y display range automatically increases to accommodate the raster sweeps When the Force scale bar display check box is turned on the display parameters are automatically adjusted to hide the axes and show scale bars Note Raster display disables some analysis features ok Episodic data is displayed with episodes offset
108. indow indicating the progress of the analysis The following functions permit drawing of lines symbols and text into a window but these features are temporary and should be erased when analysis is complete Drawlnitialize Sets up for drawing to the front window Must be called first before other Draw commands DrawMove x y Move current position to x y DrawLine x y Draw a line from current position to x y x and y are in absolute axis units not displayed units DrawPixelMove x y Increment the current position by x y screen pixels DrawsString aStr Display a character string at the current position aStr is a Pascal style string up to 255 characters DrawSymbol sym size Draw a symbol at the current position DrawErase Erase all temporary feedback DrawClear Same as DrawErase DrawSetLine thick Line thickness for subsequent calls to DrawLine DrawSetDash dash Dash setting for subsequent calls to DrawLine DrawSetFont font Set text font for subsequent calls to DrawString DrawSetSize size Set text size for subsequent calls to DrawString DrawSetStyle style Set text style for subsequent calls to DrawString DrawSetColor red green blue Change the color used by subsequent Draw commands aStr is a Pascal style string up to 255 characters red green and blue are set to values from 0 to 1 and specify the relative intensity of each color If all are set to O the trace is black 78 18 Math String and Array Functions
109. ion Auto Positive Negative 4 Adjust Axes Select the Positive radio button to detect only positive peaks Select the Negative radio button to detect only negative peaks Select the Auto radio button to detect the largest absolute peak Peaks are only detected in the visible x axis range If an artifact is present that is larger than the signal peak adjust the x axis range to exclude the artifact By default the y axis range is automatically adjusted to display the normalized traces To disable this adjustment turn off the Adjust Axes check box 9 4 Subtract Baseline Range Selector Analyse gt Baseline Two Calculate Baseline Over the Range O AII Groups range delimiters vertical lines are drawn and a range selection From To dialog appears Dashed horizontal lines indicate the baseline offsets average amplitude of each trace in the selected range Click OK and the baseline offset is subtracted from each trace The y axis range is adjusted automatically The baseline range can be entered by typing minimum and maximum values or the range delimiters can be positioned using the mouse Point to one end of the range in the graph window and click the mouse button once Alternatively hold the mouse button down and drag the delimiter to the desired position To switch to the other range delimiter hit the tab key or double click the mouse button To restrict baseline subtraction to traces in the fro
110. ions and when the tally reaches 8 the simplex search converges 20 5 Chebyshev Algorithm AxoGraph incorporates a very fast exponential fitting algorithm based on Chebyshev polynomials This algorithm was written by George Malachowski of Melbourne University The general fitting routine uses the discrete Chebyshev transform and the Chebyshev recursion algorithm to fit discrete data with a set of harmonics or exponentials The method attempts to fit the functions f j a0 al exp t taul a2 exp t tau2 The fit transforms the input time series data into Chebyshev polynomial coefficients via the summation 100 d k Sum over j f G TkG Rk where Rk is a normalization factor given by Rk Sum over j TkG TkG where the sum is performed from 0 to N N 1 is the number of data points and Tk j is the kth order discrete Chebyshev polynomial The method exploits the fact that the Chebyshev transform linearizes the problem of fitting exponentials If the input data is an exact exponential then the resulting Chebyshev coefficients have the following interrelationship d j k N j 2 d j 1 2 j 3 d j N j 1 d j 1 2 3 1 k 1 exp 1 tau 1 exp 1 tau This expression holds for all the d j Thus k can be directly calculated by regressing over all j The Two Exponential Case The case of multiple exponentials is a more complex Let f t al exp t taul b exp t tau2 Transform the data via the
111. irst or All radio buttons are selected the dialog expands to offer additional peak detection options The additional detection criteria help select only the peaks of interest when they are embedded in a noisy signal To be a significant event a peak must rise out of the background noise and it must be separated from an adjacent peak by an intervening valley that is deeper than the peak to peak noise Use Peak Threshold to set the minimum amplitude that a peak must reach before it can be detected The peak thresholds can be specified in y axis units or as a percentage Use Valley Threshold to set the amplitude that the signal must fall below in order to separate two peaks When the of adjacent peaks radio button is on a valley will be detected if it dips below the selected percentage of the smallest of the two adjacent peaks 9 11 Convert to Histogram Convert to Histogram Histogram Style Simple Histogram Probability Density Function Estimate Bins Width CENE nR Cancel amp Zero Centered Zero Aligned 35 Analyse gt Convert to Histogram The data points in each trace are grouped into bins based on their amplitudes The number of data points in each bin is then plotted against amplitude The width of the bins can be specified using the Bin Width text edit box Select the Zero Centered radio button to construct bins with boundaries that fall on half bin widths For example if the bin
112. is intercept and correlation coefficient and p value for the fitted line are sent to the log window The p value is the probability that the data points in the selected range are uncorrelated If p 0 05 the points are significantly correlated 42 11 2 Fit an Exponential An exponential function can be fitted to the front trace or group of traces The simplest equation that can be fitted is a single exponential a exp x t Additional exponential components and an added constant can optionally be included The equation for a triple exponential with added constant is al exp x tl a2 exp Cx t2 a3 exp x t3 c Analyse Fit Exponential The Exponential Fit dialog specifies the number of exponentials to fit and the method used to fit them Turn on the One Two Three or More radio button to select a fit with one two three or more exponential components If the More button is used type in the number of components Use the smallest number of components that gives an adequate fit to the data Turn on the Added constant check box to include a constant in the fitted equation when the data does not decay to Zero Exponential Fit Fitting Algorithm Chebyshev very fast Number of exps One More O Tun Q Three EJ Added constant Q Simples accurate amp robust Precisian 8 881 pe 001neS9 8f E E O 38E bypasses dialog i Q Sum of Squared Errors i Q Chi Squared i Like
113. is read into or written from dataArray after optional processing The processing is specified by kDataType which can take the following values Integer int Longint long Real float IBM Integer IBM Longint IBM Real Pascal String C String Double Extended OMAN DNFWNK 0 The Integer types are 2 bytes per data value The Longint and Real types are 4 bytes per data value Double is 8 bytes per floating point data value Extended is 10 bytes per floating point data value IBM types have their byte order reversed as they are transferred Pascal Strings begin with a length byte C Strings terminate with a null byte 93 20 Technical Information 20 1 AxoGraph File Format 20 2 Floating Point Data 20 3 Formulas and Algorithms 20 4 Simplex Optimization Algorithm 20 5 Chebyshev Algorithm 20 1 AxoGraph File Format There are two different AxoGraph file formats one for graph data and one for digitized data Versions of AxoGraph prior to 4 1 only support the graph file format The digitized file format was introduced in version 4 1 to store acquired data more efficiently The formats of both files are very simple Graph style information line thickness symbols color trace grouping etc is stored in the resource fork of the file Graph and digitized files do not require a style resource and files that lack a resource fork will be opened with the default style settings The file format for graph
114. k Detection Select the All radio button in the Detect cluster turn off the Baseline at left check box select the Positive radio button in the Direction cluster set Detect peaks greater than to 5096 and Separating valleys are less than to 50 These settings implement a 50 crossing algorithm for detecting a channel opening Click the Peaks button The Peak Measurement Options dialog appears Turn off all the check boxes except for Width and set Width at to 50 of peak then click OK Switch to the log window click on the AxoGraph 1 title bar or select Window gt Show Log Select highlight the list of open time measurements including the column title Text gt Graph Selection The Text Import dialog appears Turn on the Automatic check box and the Y Only radio button set the X Sample Interval to 1 then click OK The resulting graph shows the open times as a function of opening number This graph is useful for checking stationarity of channel properties with time It can be converted to an open time histogram as follows Analyse gt Convert to Histogram Select the Simple Histogram radio button set the Bin Width to 0 04 sec and select the Zero Aligned radio button then click OK A range selector dialog appears Select all the data and click OK Set the x axis range from 0 to 700 ms for a clearer display Analyse gt Fit Exponential Turn on the Simplex radio button the Chi Squared radio button and set Precision to 0 In the
115. k1 k2 and x2 k1 k2 d j x1 db j x2 dbb j 0 for all j In general where f t is not an exact sum of exponentials the equation d j x1 db j x2 dbb j is regressed against x1 and x2 to yield the linear equations in x1 and x2 Sum d j db j x1 Sum db j db j x2 Sum dbb j db j Sum d j dbb j x1 Sum dbb j db j x2 Sum dbb j dbb j These equations can be solved and the solution of the polynomial 102 k 2 x1 k x2 0 Gives solutions k1 and k2 which are related to the time constants taul and tau2 via kl 1 k1 1 expC 1 tau1 1 exp 1 taul k2 1 k2 1 expC 1 tau2 1 exp 1 tau2 Calculate the Exponential Amplitudes The amplitudes are obtained by regressing the original coefficients d j against coefficients that are the Chebyshev transform of the exponentials exp t taul and exp t tau2 These coefficients are obtained from a method called inward recursion which is detailed in Abramovitz and Stegun Basically the method relies on the fact that the transform of an exponential satisfies the expression d j 2 kK 1 k N j 2 d j 1 2 j 3 d j N j 1 dfj 1 2 j 1 where k 1 k 1 exp C I tau 1 exp 1 tau The equation can be reorganised by taking the d j on the right over to the left side and dividing through by 1 k d j K 1 k N4j 2 dGj 1 2 j 3 N j4 D d j 1 2 j 1 Then by further reorganising d j 1 1 k 2 j 1 d j N j
116. le linear regression The linear equations are formed Sum d j d taul j al Sum d taul j d taul j a2 Sum d taul j d tau2 j Sum d j d tau2 j al Sum d taul j d tau2 j a2 Sum d tau2 j d tau2 j and the a1 and a2 amplitudes are calculated Calculate the Added Constant The input data can be expected to have an offset f t offset al exp t taul a2 exp t tau2 Here another property of the Chebyshev transform can be exploited The offset term only appears in d 0 Nowhere else Since tau and tau2 now known as well as the amplitude then d 0 offset al c taul 0 a2 c tau2 0 Thus the offset is obtained by rearranging this equation offset d 0 a1 c tau1 0 a2 c tau2 0 Smoothing Window In order to reduce noise and non linear noise effects on the statistics of the fit a smoothing window is carried across the raw coefficients d j and the derived coefficients db j dbb j etc This is a window that averages the coefficients The usefulness of this window is that because the expression for the fit parameters is linear e g in the case of a double fit d j x1 db j x2 dbb j then the expression is also true of a sum of these over j d j d j 1 x1 db j db j 1 x2 dbb j dbb j 1 The summation is carried over a window of usually ten coefficients The actual window size is calculated using window Min Number of Data Points 10 The averaging does not alter the accura
117. lihood Estimator The exponential equation can be fitted to the data using two approaches The first is based on Chebyshev polynomials and is extremely fast The second is based on a simplex optimization procedure and is slower but more robust Turn on the Chebyshev or Simplex radio button to select the fit method See Chapter 19 Technical Information for details about these two methods The Simplex fit can be fine tuned by setting the precision of the fit and by selecting the best goodness of fit measure for the data Precision should generally be set to less than 5 The smaller the value the more accurate the fit but the longer it will take A value of 0 will give the best possible fit There are three goodness of fit measures to choose from If there is no correlation between noise and signal amplitude turn on the Sum of Squared Errors radio button If the noise variance is approximately proportional to the signal amplitude select the Chi Squared or Likelihood Estimator radio buttons Likelihood Estimator is slightly superior in theory but Chi Squared is usually more familiar and results in a statistically meaningful parameter Click OK and the Range Selector dialog appears Use it to select the range of data values to be fitted See section 11 2 for details on how to select the range Click OK and an exponential curve is fitted to each trace in the front group The fitted exponential s are drawn to the screen together with th
118. m with open bins type 3 indicates a cityscape histogram separation is the separation between adjacent bins as a percentage of bin width 0 100 GetColor w t red green blue SetColor w t red green blue Get or set the trace color red green and blue are set to values from 0 to 1 and specify the relative intensity of each color If all are set to 0 the trace is black GetGraphStyle w axisType axes yTitle y Title90 scaleBars SetGraphStyle w axisType axes y Title y Title90 scaleBars Get or set parameters defining the graph s style axisType 0 indicates standard axes axisType 1 indicates zero crossing axes axisType 2 indicates log linear axes axisType 3 indicates linear log axes axisType 4 indicates log log axes axisType 5 indicates raster display axes is true indicates axes are displayed yTitle is true indicates y axis titles are displayed y Title90 is true indicates y axis titles rotated 90 scaleBars is true indicates scale bars are displayed GetGroupRaster w g offset width SetGroupRaster w g offset width offset is the offset between raster sweeps width is the width of each raster sweep If width 0 the full width of a trace is used This is the typical setting for episodic data 17 9 Temporary Analysis Feedback When creating a custom analysis program it is sometimes useful to display some feedback in a graph w
119. meters from and to specify the subrange to copy If these parameters are omitted the entire array is copied SetArraySize variableName arrSize This procedure changes the size of an existing array to arrSize elements and does not alter the values of existing elements 84 NewString variableName This procedure creates a new 255 character string It provides an alternative to formally declaring a string variable at the start of a program String variables can also be created on the fly aString abc 18 9 User Dialogs Alert A string a b c This procedure converts its parameter list to a line of text as for Writeln Printf etc and sends the text to an alert dialog String numeric and boolean parameters can be included in the list Any number of parameters may be present and they may appear in any order e PoseDialog Introduction Describe A a Describe B b This procedure requests parameter values from the user via a standard dialog The parameter list must have the following format The 1st parameter is a string containing general information The 2nd parameter is a string describing the first requested variable The 3rd parameter is a numeric boolean or string variable which will receive the requested value Subsequent parameters are optional but if present they must alternate text string then numeric boolean or string variable After executing this
120. mitter release statistics and incorporates quantal variability and stimulus failures MK 801 Model This program models the progressive block of an NMDA receptor mediated synaptic current in the presence of the irreversible open channel blocker MK 801 Itis assumed the synaptic transmission is mediated by two populations of synaptic terminals with high and low probability of transmitter release Each time a terminal releases transmitter in the presence of MK 801 a fraction of the postsynaptic NMDA receptors are irreversibly blocked The peak amplitude of the synaptic current is plotted as a function of stimulus number It typically has a double exponential form in the presence of MK 801 Vesicle Release Model This program models the kinetics of vesicle release It assumes that each terminal contains a reservoir of vesicles and a cluster of proteins and cytoskeletal elements attached to the terminal membrane that together constitute the release complex Each release complex has several vesicle docking sites These serve to bind and hold vesicles in a position close to a single release machine One vesicle at a time can be loaded from a docking site into the release machine The loading rate is proportional to the number of vesicles docked to the complex Loaded vesicles are released at a very slow rate under control conditions However the release 65 rate is a time dependent variable and can be increased during a stimulus period T
121. n 10 4 Tidy Selected Text The Significant figures for results field determines how many significant figures will be used when sending numerical results to the log window A setting of 5 is recommended The buttons in the Color box set the default colors for displaying graphs and fitted curves Default Trace and Background set the graph trace color and background color Default Trace and Background set the graph trace color and background color Axis Lines Axis Labels and Title amp Notes set the colors of the corresponding elements of a graph Analysis Marks sets the color of measurement feedback marks baseline and measurement cursors etc By default two toolbars are displayed at the bottom of the screen The second toolbar can be removed to reclaim some screen space by turning off the Display second toolbar check box 4 Open Import Save and Export Files 4 1 Open a File 4 2 Create a New Text Editor Window 4 3 Open a Text File for Editing 4 4 Open an AxoGraph Data File 4 5 Default Display Settings in the Open File Dialog 4 6 Import a KaleidaGraph or CricketGraph Data File 4 7 X Axis Data in Imported Files 4 8 Open a Text Data File 4 9 Append a Graph File to an Open Graph Window 4 10 Open an AxoData or pClamp Digitized Data File 4 11 Open a Foreign Digitized File 4 12 Save a File 4 13 Export a Graph Window to a KaleidaGraph File 4 14 Export a Graph Window to a Text Data File 4 15 Export Only Part of a Graph Window 4 1 Open
122. n select and open the file VoltageClamp DAT in the Example Data folder This is a Clampex file containing 10 episodes sampled on two A D channels Display Review Episodes By default all episodes are reviewed To review only selected episodes enter the episode numbers separated by commas or spaces A range of episodes may be reviewed using the first and last episode number separated by a dash Review Episodes QAM Select O Erase between episodes Click the Short Cuts button and a dialog appears listing keyboard techniques for browsing multi episode files Keyboard short cuts for reviewing multi episode files return scans through multiple episodes starting from the current episode Hit any other key to halt the scan space bar displays the next episode shift space bar displays the previous episode tab erases the graph then displays the next episode shift tab erases then displays the previous episode delete erases then displays the current episode lt or jumps to the first episode gt or jumps to the last episode 6 Display Options 6 1 Axis Range and Title 16 6 2 Comment Title and Notes Options 6 3 Symbol and Line Display Options 6 4 Symbol Legend 6 5 Automatic Axis Units 6 6 Font Size and Style 6 7 Axis and Scale Bar Display Options 6 8 Axis Tick Length and Line Thickness Options 6 9 Interval Bars 6 10 Set the X and Y Axis and Error Bar Data Col
123. n the subsequent dialog Click OK If the Guess starting values check box is off AxoGraph attempts to guess the starting values of the free parameters This slows the fit and may not work If the Guess starting values check box was on the Guess dialog appears a exp x b 1 Enter starting guesses for the free parameters a b etc then click the Test button The starting values are substituted into the general equation and the resulting curve is drawn to the screen If no curve is seen then the starting guesses are so bad that the curve is above or below the visible region of the graph Try several starting guesses until the resulting curve loosely approximates the data then click the Done button The Range Selector dialog appears Use it to select the range of data values to be fitted See section 11 2 for details on how to select the range Click OK and the general equation is fitted to each trace in the front group The fitted curve s are drawn on the screen The parameters that produced the best fit and the goodness of fit measure SSE are sent to the log window The Fitted Function dialog appears that specifies how to handle the fitted curve By default the curve is not saved To print or export the graph together with the fitted curve the curve must first be incorporated into the graph Turn on the Append to current file radio button and click OK The fitted curve is appended to the graph window and the new tra
124. n under System 7 8 and 9 At least 32 MBytes of RAM is recommended for optimal performance with System 8 or 9 At least 64 MBytes of RAM is recommended when using the data acquisition package see Chapter 3 1 for more information AxoGraph is compatible with virtual memory but virtual memory is not recommended when using the data acquisition package 1 6 Bug Reports Axon Instruments provides technical support for AxoGraph via e mail or Fax Whenever possible send a file that demonstrates the problem as an e mail attachment It is very helpful if you can supply a step by step description of the conditions that demonstrate the problem It is often difficult for us to duplicate your problem without an example file Send bug reports to AxoGraph Support webone com au e mail 1 510 675 6200 Fax Visit the AxoGraph web page for upgrade information by clicking on the following link http www axon com CN_AxoGraph4 html 2 Terminology 2 1 Digitized Graph and Text Files 2 2 Continuous and Episodic Data 2 3 Traces and Groups 2 1 Digitized Graph and Text Files AxoGraph can open three types of files Digitized Files binary digitized integer data Graph Files binary floating point data Text Files words and numbers A graph or digitized file opens into a graph window A text file opens into a text editor window or a graph window AxoGraph attempts to blur the differences between digitized files and graph
125. nd the amplitudes of each trace at the measurement location are displayed on the graph and written to the log as a short cut double click the mouse button at the desired location Click OK again in the Cursor Measurements Complete dialog and the amplitude measurements are cleared from the graph Range Selector If the Measure between two cursors radio button is selected Select Measurement Region 4 All Groups m 1 two range delimiters vertical From To s lines are drawn and the Range Selector dialog is presented The measurement range can be entered by typing minimum and maximum values or the range delimiters can be positioned using the mouse Point to one end of the range in the graph window and click the mouse button once To switch to the other range delimiter hit the tab key or double click the mouse button By default all traces in the front group are measured To measure all visible traces turn on the All Groups check box Click OK and the specified measurements are made for each trace over the selected range and are displayed on the graph and written to the log Click OK again in the Cursor Measurements Complete dialog and the amplitude measurements are cleared from the graph When a series of files is to be analysed the repeated presentation of the Cursor Measurement dialog will slow the measurement process To bypass this dialog turn on the Shift Cmd N bypasses dialog check box and initiate cursor measurements
126. ne Selected Graphs A dialog with a list of currently open graph windows is presented Traces from the selected windows are combined into a single new graph as above Program gt Graph Utilities gt Concatenate Open Graphs All currently open graph and digitized files are combined into a single new graph Traces are merged end to end in the combined graph This option is useful for combining data sets that were generated sequentially The time sequence of the data points is preserved in the combined graph The onset of each trace is aligned with the termination of the preceding trace Program gt Graph Utilities gt Overlay Open Graphs All currently open graph and digitized files are combined into a single new graph Traces are merged and overlaid in the combined graph This option is useful for combining several scatter plots into a single plot Program gt Graph Utilities gt Decimate Front Graph The number of data points in the front open graph is reduced by decimating the x and y axis data columns This consists of replacing groups of N data points with either the last point in the group or the average of the group Program gt Graph Utilities gt Decimate with Error Bars The number of data points in the front open graph is reduced as above An additional data column is generated containing the Standard Error of each group of N data points This column is used to generate error bars for each data point of the decimated graph Program gt
127. ng minimum and maximum values or the range delimiters can be positioned using the mouse Point to one end of the range in the graph window and click the mouse button This sets the first range delimiter To set the second range delimiter hit the tab key or double click the mouse button with the mouse still at the first range delimiter then point the other end of the range and click the mouse button again Linear Fit Click OK and a line is fitted to each trace in the front group The line is drawn on the screen together with the equation describing the line Fitted Function Next the Fitted Function dialog appears This specifies how to handle the fitted Don t save Append Limit range line By default the line is not saved Turn on the Don t save radio button and click OK The fitted line is erased To incorporate the fitted line into the graph turn on the Append radio button and click OK The fitted line is appended to the graph window and added to the front group The graph including the fitted line can now be printed or exported The appended line can be limited to a selected range In the Fitted Function dialog turn on the Limit range check box and click OK A Range Selector dialog appears Range Selector Use this dialog to specify the Append fitted curve range limits for the appended line and click OK The fitted line is From To appended to the graph window only over the selected range The slope y ax
128. nning the program Load the program by selecting highlighting it in the text editor window then hitting the enter key program Example Program 0 Print Print Calculate factorial numbers from 1 to 10 f 1 For i 1 to 10 f f i Print Factorial i f Next i end If the program loads successfully Example Program should now be the last item under the Program menu To run the program select Program _ Example Program 16 3 A Programming Example in Fortran Here is an example of how to create a simple program using the Fortran language First select Program _ Preferences and turn on the Fortran radio button then click OK A program always begins with the word program This word is followed by a string enclosed in quotes The string is appended to the Program menu when the program is loaded If the second last character of the string is a slash then the last character becomes a command key equivalent for running the program Load the program by selecting highlighting it in the text editor window then hitting the enter key program Example Program O write Calculate factorial numbers from 1 to 10 s newLine f 1 doi 1 10 f f i write Factorial i f newLine endDo end If the program loads successfully Example Program should now be the last item under the Program menu To run the program select Program gt Example Program 69 16 4 A Programming Example in Pascal Here is an
129. nt group turn off the All Groups check box 9 5 Digital Filter Digital Filter Analyse 2 Digital Filter A digital filter will be applied to all visible traces Filter cutoff Hz EJ All Groups Filtering is performed by convolving a Gaussian with the data The Filter width Filter width 5 ms can be specified directly the width equals 4 standard deviations of the Gaussian envelope or it can be specified in more familiar terms as the Filter cutoff frequency The default filter cutoff frequency is 1 Sth the sampling frequency To limit application of the digital filter to traces in the front group turn off the All Groups check box 31 The filter algorithm is provided in Chapter 19 Technical Information Filtering a large data file may take several seconds or tens of seconds Type the esc key or the Cmd period keys to interrupt a slow filter operation Select File gt Revert to undo the interrupted filter operation 9 6 Mouse Measurements Mouse Measurement Analyse gt Mouse Measure The dialog specifies the type of mouse measurement that will follow Measure Select one of the radio buttons to measure amp Position OH Distance Bon By default both x and y components of the mouse Oo measurement are sent to the log window If only one Amplitude of these components is required turn on either the X Cancel or Y radio buttons Amplitude Difference O 23M bypasses dialog
130. omatic amp X vs Y 4 4 Click on the Options button in the File Open dialog and the text import Skip Lines Y only dialog appears X Column Number fis If the Automatic check box is on mapa AxoGraph scans the text file and 2 i attempts to guess the text data format Comma ok number of columns column delimiter etc This will only work for Tab or Comma delimited text data To manually specify the text data format turn off the Automatic check box Turn on the Read column titles check box if column titles are present Specify how many lines to skip at the start of the file there is often several lines of header information to skip Select the column delimiter Tab Comma single Space or multiple Spaces Read column titles Caluunm Delimiter To import a text data file in a non standard format e g some other delimiter the text will need to be pre processed using a text editor 11 4 9 Append a Graph File to an Open Graph Window Open a graph file 2 Pulses for example File gt Append then select another graph file or select the same file again for this demonstration New traces are added in the bottom half of the graph window but are partially overlaid The x axis data from the appended file is also appended to the combined graph The x axis data columns can be viewed or edited by selecting Display gt X vs Y and Error Bars After appending a graph select Trace gt Tile to tidy up t
131. onverts aStr to a number Function that converts aNum to a string Function that converts aStr into an C string array Function that converts anArr into a Pascal string Length of string Find subStr in aStr and returns position Concatenate strings and numeric variables Return a sub string of aStr starting from character start with length len Delete len characters starting from start Insert iStr into aStr before start 18 4 Byte and Bit Manipulation GetBit aNum Nth Bit SetBit aNum bit Bit GetByte aNum Nth Byte SetByte aNum bit Byte GetWord aNum Nth Word SetWord aNum bit Word 18 5 Array Functions arraySize arr sum arr from to av arr from to mean arr from to stdDev arr from to sd arr from to stdErr arr from to sem arr from to variance arr from to min arr from to max arr from to Procedure gets the Nth bit of aNum and passes it to the variable Bit Procedure that sets the Nth bit of aNum to the first bit of the variable Bit Procedure gets the Nth byte of aNum and passes it to the variable Byte Procedure that sets the Nth byte of aNum to the first byte of the varible Byte Procedure gets the Nth word of aNum and passes it to the variable Word Procedure that sets the Nth word of aNum to the first word of the varible Word Total number of elements in array Sum of array elements
132. op of a graph turn on the Show d Show Comment W Show Document Title Conmant chadk boxand enter the immediately below To display the Notes can be displayed anywhere on the graph name of the graph file turn on the dl Show Notes Show Document Title check box The comment and title are added at he ES the top of the graph window and cannot be moved To add multi line text to a graph turn on the Show Notes check box Notes can be typed or pasted into the text ba edit area below this check box 9 Nota Notes are displayed in the graph window using a small font size A small black square at MM the top left of the notes can be used to drag them anywhere in the graph window 6 3 Symbol and Line Display Options Display gt Symbols and Lines The resulting dialog can change the symbols and or lines used to display each trace Selected Trace The symbol and line options for all traces can be edited without leaving the dialog by using the Selected Trace popup menu to move between traces sid Each trace can be displayed as a line a set of symbols Biting Symbols L Histugram circles squares triangles etc as a histogram or as any combination The color of each trace can be specified by clicking on the Color button Line thickness and dash style can be controlled by clicking on the desired setting or typing into the Other text edit box Fractional thickness settings cannot be accurately represented on the screen but will affect the
133. ore than one column of data The only requirement is that the vertical lists are separated by one or more blank lines 39 10 5 Transpose a Tab Delimited Table It is sometimes necessary to transpose a table of results In the following example Simple Stats has been applied to four data columns and the results are 40 112 150 190 Average 8 165 21 23 34 66 43 21 S E M To generate a bar graph of the four Average and S E M values the text table must first be rearranged so that the two rows become two columns that is the table must be transposed Bring a text editor window to the front and select a tab delimited table Text gt Transpose Selection and the table is reorganized as follows 40 8 165 212 21 23 150 34 66 190 43 21 Average S E M The first row has been converted to the first column and the second row to the second column The transposed data values can now be selected and graphed see section 10 2 For information on how to display the S E M column as error bars see section 6 10 10 6 Load Vertical Lists into Arrays If one or more tab delimited columns of numbers are highlighted in the front window and Text gt Selection to Array is selected each column is loaded into a data array within AxoGraph By default the arrays are named arrl arr2 If the selected columns have titles at the top the array names are taken from the titles instead The arrays can then be manipulated and graph
134. procedures convert their parameter list to a line of text and send it to the front window Write does not add a return at the end of the output line WriteLn Print and Printf are interchangeable Valid parameters are numerical logical or string variables and expressions Any number of parameters may be specified including none and they may appear in any order Note Pascal and Fortran use single quotes to enclose character strings but Basic and C use double quotes To include a quote tab or new line in a character string use V or V for a quote X for a tab and r or n for a new line As an alternative the built in strings tab and newline can be added to the parameter list of an output statement FlushOutput Text output from Write WriteLn and Print is normally accumulated in a buffer and is output all at once when a program finishes running Use FlushOutput to force the output to occur while the program is still running 19 5 Read and Write Binary Data Files Binary read and write routines can only be used with a file that already exists These routines were designed for importing foreign data files into AxoGraph See Import Modules folder for examples of their use CreateBinary fileName fileCreator fileType This procedure creates a new empty binary file with the specified name creator and type The three parameters are all string variables Only the first 4 characte
135. r programs The log window is indicated in the zd Window menu with a black diamond beside its 12 name see figure on next page Programs To execute a program highlight the code then press enter for i 1 to 3 do begin writeln Ci tab i 2 end 3 3 The Window Menu Pile Tile Offset Next 36 Previous S86 Show Log SSL Get Graph Info S3F i o Gra p hal 2 2 Pulses v 3 Dose Response 3 4 The Program Menu Load or Evaluate enter List gt Programming Prefs Reload Drop ins Copy and Paste Document Utilities Graph Utilities More Utilities Select Events Trace Appearance Trace Manipulations Trace Transforms Electrophys Tools Statistics vwvrrowevrvrvrvrvvy 36 I Programming Help Online User Manual The names of all open windows are listed at the end of the Window menu Select a window s name to bring that window to the front The front window has a check mark next to it The windows are numbered in the order they were opened These numbers are important when running AxoGraph custom analysis programs Text editor window names are displayed in outline style The log window is indicated with a black diamond The names of all available analysis programs are listed under the Program menu or in sub menus of the Program menu Selecting a program s name executes that program To reconfigure this menu quit AxoGraph move text files or sub folders t
136. r analysing data are given below One exception is the fast Fourier transform FFT algorithm used to calculate the power spectrum This is a standard algorithm that can be found in many numerical recipe books and is too long to be included here Standard Deviation and Standard Error Standard deviation SD and standard error of the mean SEM are calculated by several different analysis routines The following formulas are used For a list of N data points Yi i 1 to N let SY Sum over i Yi SYY Sum over i Yi Yi SD Square Root SYY SY SY N N 1 SEM Square Root SYY SY SY N N N 1 Digital Filter The digital filter uses a Gaussian filter algorithm to smooth data traces It numerically convolves a Gaussian envelope with the data trace The user selects a Filter cutoff or a Filter width in the Filter dialog box and the selected parameter is used to calculate the standard deviation of the Gaussian envelope Sigma as follows Sigma Filter width 4 or Sigma 0 132505 Filter cutoff Sigma is then converted from real units to number of data points N by dividing by the Sample Interval assumed to be constant N Sigma Sample Interval A set of N Gaussian filter coefficients Fi is calculated from Sigma as follows Note is multiplication and is exponentiation If Sigma lt 0 62 then N 3 b Sigma Sigma 2 Fl b F2 1 2 b F3 b If Sigma 2 0 62 then N
137. r by Trace The 1st trace is displayed in blue the 2nd trace in red and the 3rd trace in green Up to 8 traces will automatically be set to different colors Program gt Trace Manipulations gt Set Color by Group The Ist group is displayed in blue the 2nd group in red and the 3rd group in green Up to 8 groups will automatically be set to different colors Program gt Trace Manipulations gt All Blue All traces and symbols are displayed in dark blue Program gt Trace Manipulations gt All Black All traces and symbols are displayed in black This options is useful when preparing a graph for publication Program gt Trace Manipulations gt All White All traces and symbols are displayed in white This options is useful when using a dark graph background 60 Program gt Trace Manipulations gt Set Symbols by Trace The 1st trace is displayed using open circles the 2nd trace using open squares and the 3rd trace using open diamonds Up to 12 traces will automatically be set to use different symbols If more than 12 traces are present the symbol sequence will repeat Program gt Trace Manipulations gt Set Symbols by Group The 1st group of traces is displayed using open circles the 2nd group using open squares and the 3rd group using open diamonds Up to 12 groups will automatically be set to use different symbols If more than 12 groups are present the symbol sequence will repeat Program gt Trace Manipulations gt Se
138. rating principles Chapter 8 describes how to obtain the best graphical output quality Navigate this document using bookmarks see Adobe Acrobat Reader documentation To search for a key word click on the binoculars icon in the toolbar or select Find under the Edit menu 1 2 About This Manual A convention for specifying menu selections is employed throughout the manual File gt Open indicates that the Open item under the File menu is to be selected Worked examples in this manual rely on files provided in the Example Data folder 1 3 Supported File Formats A variety of import and export options are provided so that AxoGraph can be used in co operation with other graphics and analysis programs Supported file formats include Import and Export Igor Pro 3 0 binary wave KaleidaGraph 3 0 CricketGraph III and tab delimited text Import Only Clampex 6 Fetchex 6 pClamp 7 amp 8 AxoScope AxoTape 2 AxoData 1 2 Chart 3 6 Setup2 Neurocom C Lab space or comma delimited text Export Only MacDraw PICT can be read by ClarisDraw Canvas PowerPoint etc Copy to the clipboard in PICT format can be pasted into Canvas ClarisDraw etc Any single run binary digitized data file recorded on any computer IBM Mac etc can be imported into AxoGraph acquisition parameters are entered via a dialog More complex data files in any format can be imported via a program written in AxoGraph s development environment Modules
139. reflect 2 f Xmax then reflection was a complete failure and contraction is tried without first replacing Xmax If f Xcontract f Xmax the contraction was successful and Xmax is replaced by Xcontract and the procedure returns to step 2 But if f Xcontract 2 f Xmax the contraction was a failure and the last simplex is shrunk around the minimum vertex as follows Xi 0 5 Xi Xmin for i 1 to n 1 The function is evaluated at each of the new vertices and the procedure returns to step 2 Precision The search continues until the convergence criterion is satisfied This occurs when an estimate of the relative error for each search parameter drops below the specified Precision value This value is expressed as a percentage in the Curve Fit dialog If there are N search parameters then the relative error is checked following every 10 x N calls to the function being optimized The relative error Err in each parameter is estimated as Err 10 x change in parameter since last check initial step size The initial step size for a parameter is typically 10 of the starting guess for that parameter but will be a small arbitrary value if the starting guess was zero If Err is set to zero an alternative convergence criterion is used Empirical observations suggest that contraction is unsuccessful only when the search has almost converged to the limits of computational accuracy A tally is kept of the number of unsuccessful contract
140. rr and returns the result in the same arrays 86 18 11 Advanced Procedures e Evaluate aStr bStr Execute the contents of the string parameter s as if the user had selected the string s and typed enter A string can be built and executed within a program or procedure It can access variables local to that procedure The following example uses the Evaluate command to perform a user specified analysis on a newly opened file The name of the analysis procedure is contained in the string AnalysisProcedure and the procedure takes a window number as its parameter Open a graph file and analyse it new Window openGraph Evaluate AnalysisProcedure newWindow Minimize precision UserFunction a b c Find the parameters a b c that minimize a user defined function precision is optional and controls the convergence criterion for the simplex optimization algorithm The function can have up to 25 free parameters The value of the parameters at the time Minimize is called are used as the starting guess for the optimization This curve fitting approach is much more flexible than Fit General under the Analyse menu but it is slower The following example uses the Minimize command to find the minimum point of a parabola Find the minimum of a parabola Function Parabola x begin Parabola 5 x x 5 x 5 end xMin 1 The starting guess Minimize Parabola
141. rs See Sections 6 10 and 6 11 for information on how to display the S E M values as error bars on the average trace The Residual Variance calculation corrects for amplitude fluctuations in the signal by subtracting the optimally scaled average from each episode The Trial to Trial Variance calculation corrects for gradual drift or run down in the signal amplitude For additional details on the algorithms used to calculate Ensemble Variance see Chapter 20 Technical Information 48 13 Analysis Case Studies 13 1 How to Use the Case Studies 13 2 Construct a Current Voltage Curve from Voltage Clamp Step Data 13 3 Calculate an Amplitude Histogram and Open Time Histogram from Single Channel Data 13 1 How to Use the Case Studies A convention for specifying menu selections is employed throughout the case studies File gt Open indicates that the Open item under the File menu is to be selected The case studies rely on files provided in the Example Data folder 13 2 Construct a Current Voltage Curve from Voltage Clamp Step Data Note a program that automates current voltage analysis is shipped with AxoGraph It is described in the next chapter section 14 3 under the sub heading I V Analysis The following manual method provides more control over intermediate steps in the analysis File gt Open Digitized Select the file VoltageClamp DAT This is a Clampex binary data file containing 10 episodes in two A D channels or
142. rs of the fileCreator and fileType parameters are used OpenBinary fileName This function opens the file with the specified name for binary input read only It returns a file ID number that is used by the binary read and close routines If the file is not found return 1 OpenBinary Write fileName This function opens the file with the specified name for binary input or output It returns a file ID number that is used by the binary read write and close routines If the file is not found return 1 IndexOpenBinary index Open a binary file by index position in the current folder s file list IndexOpen returns a file ID number that is used by the binary read write and close routines 92 If the file is not found return 1 CloseBinary importFileID This procedure closes the specified binary data file BinaryTitle importFileID This function returns the name or title of the specified binary file BinaryLength importFileID This function returns the length in bytes of the specified binary file e ReadBinary importFileID offset length kDataType dataArray WriteBinary importFileID offset length kDataType dataArray These procedures read or write binary data from the specified file The operation starts at offset bytes from the start of the file and continues for length bytes The offset parameter is automatically incremented by length bytes Data
143. s Add aStr to the end of a window s notes Show a window s notes when display True Add a tag to a window at the specified sample point To add a tag at a specified time tagTime calculate tagPoint tagTime sampleRate Show a window s tags when display True Returns the number of interval bars GetIntervalBar w BarNumber aStr x Min x Max offset SetIntervalBar w BarNumber aStr xMin xMax offset Get or set the display parameters of an interval bar BarNumber is the number of the bar to modify If the bar does not exist a new bar is created aStr is the bar label xMin is the start of the interval xMax is the end of the interval offset is the y location of the bar Should be set in the range 0 1 bottom to top 76 ShowIntervalBars w showBars titlesBelowBars showBars whether to display interval bars titlesBelowBars whether the titles are displayed above or below the interval bars GetFront w t Returns the front trace and front window number SetFront w t Move the specified window and trace to the front Update w t Redraw the specified window and trace NTraces w Returns the number of traces in a window NGroups w Returns the number of groups in a window NEpisodes w Returns the number of episodes in a window YNPoints w t Returns the number of data points in a trace NPoints w Returns the maximum number of data points across all traces in a window deltaX w Returns the x axis
144. s very similar to the Hide dialog above Click the Cancel button to avoid deleting any traces 26 7 8 Manipulating Graph Windows Open several graph windows The names of the open windows are listed at Pile the end of the Window menu This menu can be used to move between the Tile various windows Offset Next 36 Previous x The windows can be rearranged on the screen using the first three items at Show Log REL the top of the Window menu Get Graph Info SEF Window gt Pile Expand and overlay all graph windows kAnin Window gt Tile Resize and rearrange the graph windows 2 2 Pulses so that all graphs can be seen 3 Dose Response Window gt Offset Resize and rearrange the graph windows v 4 Current DAT Copy so that the title bars of all windows can be seen To include text editor windows when using Pile Tile or Offset hold down the shift key while selecting the menu item The remaining Window menu items are used to cycle through the open windows get the log window or list file format information Window 2 Next Send the front window to the back Window 2 Previous Bring the back window to the front Window gt Show Log Bring the log window to the front Window gt Get Graph Info List information about the front graph window into a new text window 27 8 Print or Export a Graph 8 1 Use a Page Setup Scaling of 25
145. s The data points in a scatter plot are re ordered so that the x axis data values are in ascending order This may improve the appearance of a graph when using line display format Program gt Trace Manipulations gt Duplicate Each Group Each group of traces is duplicated into a new graph window This program permits each group to be analysed or manipulated independently of the original data Program gt Trace Manipulations gt Duplicate Section of Trace A selected region of the front group of traces is duplicated into a new graph window This program permits the selected sub section of the displayed data to be analysed or manipulated independently of the original data Program gt Trace Manipulations gt Sloping Baseline A dialog appears which is used to select a region over which to calculate a sloping baseline For each trace in the front group a line is fitted to data points in the selected region and the fitted line is subtracted from the trace 61 Program gt Trace Manipulations gt Two Region Baseline A dialog appears which is used to select two regions over which to calculate a sloping baseline For each trace in the front group a line is fitted through all the data points in the two selected regions and the fitted line is subtracted from the trace 14 15 Trace Transforms The commands in the Trace Transforms sub menu perform simple arithmetic transforms on traces in a graph window Program gt Trace Transforms gt Inver
146. s created before afterTime return 3 OpenAIIText afterTime OpenAllGraphs afterTime OpenAllDigitized afterTime Opens all text graph or digitized files in the current folder modified since afterTime The time parameter is in seconds since Jan 1st 1904 Mac standard A reference time can be obtained using the GetTime function If no time parameter is supplied then all files in the current folder are opened Use the nWindow function before and after OpenAIl to check whether any new windows were opened SetGraphType window theType Set the graph file format for a subsequent save The format is specified by the value of theType as follows AxoGraph when theType 1 KaleidaGraph when theType 2 CricketGraph when theType 3 Tab Text when theType 4 SetGraphCreator window theType Set the creator for a subsequent graph file save The creator is specified by the value of theType as follows AxoGraph when theType 1 KaleidaGraph when theType 2 CricketGraph when theType 3 Tab Text when theType 4 89 Save window Save the contents of the specified window to disk SaveAs window fileName Save the contents of the specified window to a new file SaveViaDialog window fileName Save the contents of the specified window to a new file Always present a standard save file dialog SaveACopy window fileName Make a copy of the specified window and save to a new file
147. s in the selected text that contain only spaces and letters of the alphabet Lines that contain numerals tabs brackets etc will not be deleted This deletes file names and description of analysis generated by AxoGraph analysis routines L Delete column titles non numeric text with tabs and or brackets Turn on the Delete column titles check box to delete all lines in the selected text that contain non numeric characters Lines with tabs and brackets will be deleted Lines containing numeric data values will not be deleted This deletes the column titles generated by AxoGraph analysis routines 10 4 Convert Vertical Lists into a Tab Delimited Table Analysis results often accumulate as a series of lists one below the other For example consider the following 9 lines of text X s 0 1584 0 2988 0 4437 Y nA 2152 570 4 124 3 To calculate Simple Stats for the two lists the data must first be reorganized as two tab delimited columns a two column table Bring a text editor window to the front and select two or more lists that are organized one below the other as shown above The selected text should include the list titles Text gt Selection to Table and the two vertical lists are reorganized into two tab delimited columns side by side X s Y nA 0 1584 2152 0 2988 570 4 0 4437 124 3 This feature also works when there are more than two vertical lists and when the vertical lists contain m
148. s is a standard data file format that can be read by most graphics and spreadsheet programs Open the file 2 Pulses in the Example Data folder File gt Save as then change the File Format setting to Text Data and change the file name to 2 Pulses Text Now click on the Options button By default the first row of the new file contains the column titles Bd Include Column Titles followed by the numerical data in subsequent rows If the Include 4l Created by AxoGraph Column Titles check box is turned off the column titles row is not ox written 4 15 Export Only Part of a Graph Window A selected section of a graph can be saved to a new file Open the file 2 Pulses in the Example Data folder Drag trace 2 to the Hide area then zoom in on the stimulus artifact of trace 1 see section 5 2 File gt Save a Copy then change the File 2 Pulses Artifact Format setting to AxoGraph and change the file name to 2 Pulses Artifact Now click File Format AxoGraph T on the Options button If the All Data radio button is on the copied file will contain all the Copy data from the original file Q All data amp Only Displayed Data If the Only Displayed Data radio button is on the copied file will d Open Copied File only contain the currently visible data the artifact from trace 1 in this example v Include Column Titles EJ Created by AxoGraph If the Open Copied File check box is turned on the copie
149. should be listed 2 Pulses Current Voltage AxoGraph Current Voltage Comma Text Current Voltage CricketGraph Current Voltage KaleidaGraph Current Voltage Tab Text Dose Response Operational Model Data Program Document Utilities Open Selected Documents The selected highlighted documents are opened For example select the names Dose Response and Operational Model Data in the above list then run the above program The two selected graphs will be opened This feature is useful when a large number of data files are collected in a single folder Selected subsets of files can be conveniently opened for review and analysis Program gt Document Utilities gt Open All Documents All documents in the selected folder are opened A dialog appears asking which class of documents to open Graph Digitized Episodic Text or All documents This is followed by a standard dialog that indicates the folder containing the files to be opened Program Document Utilities Open Next Digitized Open the next file in the alphabetic list of digitized data files the current folder It is assumed that the first digitized data file in the folder has been manually opened Program Document Utilities Open Prev Digitized Same as the above command but open the previous digitized data file in the alphabetic list 55 Program gt Document Utilities gt Close Graph Windows Close all currently open graph windows A dialog appears asking whether to sa
150. ste a Graph to a Word Processor File gt Page Setup Set Reduce or Enlarge to 100 and Orientation to vertical File gt Page Layout To place a graph in the top 1 3rd of a page set Vertical to 30 and Horizontal to 100 Edit gt Copy Copies the graph to the clipboard Switch to a word processor and paste the graph into the document The resolution of the pasted graph is the same as the screen 72 dpi When printed it will not use the full resolution of the laser printer This problem is in part due to the word processor s limited graphics handling ability 8 6 Export to a PICT File File gt Save a Copy Change the File Format setting to MacDraw PICT and change the file name to avoid over writing the original graph file for example append PICT to the file name The exported PICT file can be imported by ClarisDraw Canvas PowerPoint or any other drawing or page layout program 9 Data Analysis Graph 9 1 9 2 9 3 9 4 Scale and Offset X and Y Data Change X Axis Zero Normalize Peak Amplitude Subtract Baseline Digital Filter Mouse Measurements Cursors Measurements Peak Amplitude Measurement Peak Shape Measurement Peak Detection Convert to Histogram Power Spectrum 9 1 Scale and Offset X and Y Data 29 All sections in this chapter assume that you have a graph window open and that it is the front window File gt Open Scale and Offset Y Data For the front trace or group of traces
151. t Front Group Each trace in the front group is inverted multiplied by 1 Program gt Trace Transforms Rectify Front Group Each trace in the front group is rectified absolute value of all data points Program gt Trace Transforms gt Integrate Front Group Each trace in the front group is numerically integrated The integrated traces are displayed in a new graph window Program gt Trace Transforms gt Differentiate Front Group Each trace in the front group is numerically differentiated The differentiated traces are displayed in a new graph window Program gt Trace Transforms Delta Y Front Group Each data point in each trace in the front group is replaced by difference between that point and the preceding point The result is similar to numerical differentiation The resulting traces are displayed in a new graph window Program gt Trace Transforms gt Raise Front Group to Power Each trace in the front group is raised to a power that is specified via a dialog The Y axis units are not adjusted Program gt Trace Transforms gt Perfect Filter Every trace in the front window is filtered FFT s are used to implement a perfect filter i e sharp cutoff above the selected frequency Program gt Trace Transforms gt Notch Filter Every trace in the front window is filtered FFT s are used to implement a perfect notch filter i e remove a selected frequency Program gt Trace Transforms gt Box Car Filter E
152. t Line Width by Trace The Ist trace is displayed using a line width of 1 the 2nd trace using a line width of 2 etc Program gt Trace Manipulations gt Set Line Width to 1 All traces are displayed using a line width of 1 Program gt Trace Manipulations gt Remove All Symbols All symbols are removed Traces will be displayed using only lines Program gt Trace Manipulations gt Remove All Lines All lines are removed Traces will be displayed using only symbols 14 14 Trace Manipulations The commands in the Trace Manipulations sub menu perform basic mathematical manipulations on traces in a graph window Program gt Trace Manipulations gt Group Math A dialog appears requesting the math operation to perform on two groups of traces Select Subtract Add Multiply or Divide and click OK A second dialog appears requesting the window and group numbers of groups to process The selected math operation is applied to episode 1 of the first and second group then episode 2 of the first and second group etc A new window is created that contains the resulting traces Program gt Trace Manipulations gt Trace Math A dialog appears requesting the math operation to perform on two traces Select Subtract Add Multiply or Divide and click OK A second dialog appears requesting the window and trace numbers of traces to process A new window is created that contains the resulting trace Program gt Trace Manipulations gt Sort Data Point
153. t an x axis data column Xdata w t is an x axis data column Ydata w t is a y axis data column or trace The parameters are w window number shown in the Window menu t trace number shown in the Group dialog under the Trace menu The parameters w and t can be numbers variables or expressions Floating point values are rounded to the nearest integer If w is set to zero the front window is assumed If t is set to zero the front trace is assumed If t is omitted from xData w the default xData column is assumed Traces can be combined into groups For episodic digitized data e g Clampex or AxoData traces are automatically grouped by A D channel number Individual traces can be accessed by group number and trace number within the group Edata w g e is a trace or episode of data g group number or A D channel number for episodic data e trace number within group or episode number Some episodic digitized data files e g AxoData have a hierarchical structure and episodes can be organized into runs Individual traces can be accessed by A D channel number run number and episode number REdata w g r e is an episode of data Multi run digitized file r run number for a multi run data file 71 Xdata Ydata Edata and REdata act like ordinary array variables and can be used to manipulate graph data For example Ydata 2 1 Ydata 2 1 2 This command multiplies the 1st
154. t cannot be interrupted In general a larger group size e g 4096 will result in faster processing and provide more low frequency information but the spectrum will be noisier because fewer data segments will be averaged 36 10 Data Analysis Text 10 1 Create a Graph From the Text Editor 10 2 Simple Statistics in the Text Editor 10 3 Tidy Selected Text 10 4 Convert Vertical Lists into a Tab Delimited Table 10 5 Transpose a Tab Delimited Table 10 6 Load Vertical Lists into Arrays 10 7 Text Editor Display Options All sections in this chapter assume that you have a text editor window open and that it is the front window File Open Text Select and open a file containing text data Current Voltage Tab Text in the Example Data folder for example A text editor window is opened and the text data displayed The data should be organized in a single column of numbers or in several tab delimited columns of numbers 10 1 Create a Graph From the Text Editor Select a list of data values in a text editor window by holding down the mouse button and dragging the cursor until the list is highlighted Voltage mV Current pR 98 6 88 2 70 9 50 29 50 47 40 64 Text gt Graph Selection This is the Text Format Data Organization Import Options dialog for text data also 2 Automatic amp Xvs Y described in section 4 8 If the Q Y only Automatic check box is on the selected Skiz Les text data is scanne
155. tains a whole cell current trace which includes the response to a square voltage clamp test pulse The pulse parameters are entered in a dialog The series resistance of the patch electrode is then calculated and written to the log 58 Program gt Electrophys Tools gt Correct for Rs This program assumes that the front window contains a whole cell current response containing large amplitude synaptic transients The synaptic current is corrected for any systematic errors due to electrode series resistance This is a simple linear correction that assumes all membrane conductances have a reversal potential of zero Program gt Electrophys Tools gt Measure Synaptic Peaks This program assumes that the front window contains evoked synaptic currents It measure the peak amplitudes of synaptic responses by averaging over a region around the peak of each response then scaling these measured amplitudes to match the peak amplitude of the ensemble average synaptic response Program gt Electrophys Tools gt Measure Two Peaks This program measures the peak amplitudes of two separate synaptic responses using the same technique as above Program gt Electrophys Tools gt Calculate Variance Mean This program assumes that the front window contains evoked synaptic amplitudes plotted against stimulus number It requests an analysis range then measures the variance and mean of the synaptic amplitudes within that range The program optionally corrects for r
156. that the signal rises to a peak from a baseline with approximately zero amplitude then falls away again towards zero If this is not the case then subtract a baseline from the signal by selection Analyse gt Baseline before using the following options Analyse gt Peak Measure The largest amplitude peak in each trace is detected and the amplitude and shape parameters are measured The dialog controls which of these parameters are reported Switch on the Location of peak check box to measure the x axis location of the detected peak Switch on the Rise check box to measure the rise time of the peak The default settings measure the rise time from 10 to 90 of the peak Switch on the Width check box to measure the width of the peak The default setting measures the width at 50 of the peak Switch on the Onset check box to measure the onset time of the peak The default setting measures the onset at 5 of the peak If the signal is noisy the performance of the shape measurement options may be improved by adjusting the settings For example the rise could be measured from 20 to 80 and the onset could be measured at 20 9 10 Peak Detection Peak Detection Options Detect Peaks amp Maximum First Direction Q Positive Q Negative Q All M Relative to first point in trace Auto Select the Positive radio button to detect only peaks gt 0 Select the Negative radio button to dete
157. tit b RASEN O Aio EEE ETTOL EEEE ES Re S Pee De tint 50 14 1 Overview of the Program Menu ote De RHODE RR ER REE MERI cate Pens 50 14 2 Customize the Program Menu sssssesseeeseeeeeeeeneeneneeetn nennen eetn eene trennen teene nennen 51 14 3 Commands Under the Program Menu seen enne nennen trennen nete 51 14 4 The Default Plug In Programs sees ener trennen nennen ener enitn eene nenne 52 14 5 Copy and Past Display Styles ene et t RED EE RR ER Oe ehe erus 53 14 6 Copy and Paste Graph Data eterne theme EEEE EEE EESE EEES EEES nenne 53 14 7 Document Utilities 4 oue rU ER t te the rei ep edeete 54 14 8 Graph Utilities ce eere n EE ERR BERN EE ESTEE XH EISE He Ee Edge 55 14 9 More Utilities ode pe OR RERO ROTE EO RE HERREN Dre UR CHER ETRE EE 56 14 10 Select Event P 57 14 11 Electrophys Tools o riore Cet eter ode e o Bd e pie pt do ende 57 14 12 Statistics eceeesete e e dude d eee e ee et te e ler eue ERRER 59 14 13 Trace Appearance tee TR BO I POET BER RP RE ERE REEL De PCR uns 59 14 14 Trace Manipulations sitet teret nenet Esenta ESEE EKER KERERE EEEE EEEk 60 14 15 Trace Transforms cccccceccccccsssccessssccsssscsccesscsccsssssccesusesecsssssecessssececsssssececsesecesesecesseseceesesecessess 61 14 16 Online Help C 62 14 17 Add Custom Programs to the Menu ce ees eseeseecs
158. tne tn NTE KoSo ero ST aE ESEA T E SS 87 19 1 Change Folders opto HEU TEC e ERO EROR E HERE IRE stt 87 19 2 Create and Open Files 4 euet deir he eH ERI eek ee ege ape etae eroe se 2s 87 19 3 Edit Texten tele their EBORE Phe teet tle re tee ri ee Pu Ete Dre E inen 89 19 4 Read and Write Text Files sees eene tenete eene nenne nnnm nenne trementes inet tn eene tenente 90 19 5 Read and Write Binary Data Files oed eee ene pe ten retire pietate 91 20 Technical Information scccsccssssscsssscssssssssesssssscsssssssssseseessesssssssesessssesssssessesessessseessscessssessesesseseseesess 93 20 1 AxoGraph File Form t tete rte ie He re HR RE vevesseussususevsssesestescesecsess 93 20 2 Floating Point Data nsn e eO ERREUR o o e Fi OR EE HERR e ETE SS 94 20 3 Formulas and Algorithms seeseseeseeeeee tenente ennemi treten nenne nrene teens treten nenne nennen 95 20 4 Simplex Optimization Algorithm eeeeseeseeeeeeeeeeenee ener nennen enne tenne nennen 98 20 5 Chebyshev Algorithm 5 uin tereti E e pre E ee Pee eee 99 1 Introduction and Overview 1 1 Time Saving Information 1 2 About This Manual 1 3 Supported File Formats 1 4 Axon Instruments PC and Windows Data Acquisition Software 1 5 System Requirements 1 6 Bug Reports 1 1 Time Saving Information A few minutes going over Chapters 2 and 3 is recommended These sections introduce fundamental concepts and ope
159. trace of the 2nd window by 2 Passing an array to a non existent trace will cause a new trace to be appended to a graph For example Ydata 1 999 Ydata 1 2 2 This command multiplies the 2nd trace of the 1st window by 2 and graphs the result in a new trace 999 is an arbitrary large number However this will not work with digitized data files The trace commands can subtract two columns or episodes of data Data from more than one window can be manipulated For example Edata 2 1 1 Edata 2 1 1 Edata 3 1 1 This command subtracts the 1st episode in the 3rd window from the 2nd episode in the 2nd window Data can be passed to a temporary array variable created on the fly For example bufferArr Ydata 2 1 This command copies the data column from the Ist trace of the 2nd window and passes it to the array variable bufferArr Modifying the array variable does not affect the original data For example bufferArr bufferArr 2 17 2 Graphs with Multiple X Axis Data Columns Typically a graph has only a single x axis data column However some graphs will have different x axis data columns for different traces for example two traces with different sample intervals Two numbered lists of data columns are maintained for each graph the list of X data columns and the list of all data columns including X data columns The following commands apply to the list of X data columns The parameter xc r
160. uage Q Pascal Q Fortran Q Basic c Trig functions amp Degrees Q Radians Output Significant figures S o Program gt Reload Plug Ins All global variables programs procedures and functions are deleted from memory then all programs in the Plug In Programs folder are reloaded When programs or folders containing programs are dragged into or out of the Plug In Programs folder selecting Reload Plug Ins dynamically reconfigures the Program menu This feature is also useful when writing or debugging a plug in program 14 4 The Default Plug In Programs All the menu items that appear below Reload Plug Ins under the Program menu are plug in programs and folders that were automatically loaded into memory when AxoGraph was launched Selecting a Program menu item runs the associated program Section 14 2 describes how to customize the Program menu The ten default hierarchical items under the Program menu correspond to sub folders of the Plug In Programs folder Functionally related programs are grouped under hierarchical menu items The ten default hierarchical items are Copy and Paste copy and paste display styles and graph data between windows Document Utilities open and close multiple graph digitized or text files Graph Utilities combine overlay decimate or interpolate graphs More Utilities blank artifacts normalize align events average with error bar Select Events select events based on shape
161. umn numbers set to dashes 6 11 Error Bar Options Display gt Symbols and Lines The symbols dialog also includes the error bar display options These will be dimmed unless an error bar column has been assigned to the selected trace see section 6 10 above Error bars will be drawn above check box and or below check box each data point The Width of the horizontal line at the end of each bar is given in pixels 21 6 12 Zero Crossing Axes 0 current Voltage AxoGraph FI BY Open the file Current Voltage Episode 2 2 AxoGraph in the Example Data Current nA folder and turn on the Default Display Settings check box The graph shows a simulated current voltage graph Display gt Type of Axes gt Zero Crossing This moves the axes so they cross at 0 0 This is the standard style for a current voltage graph The axis range should span zero for both x and y axes lelo lelo ME Lt Pars l 17 leki 6 13 Log Axes 5 2 s pri Ej Make the 2 Pulses graph file or a similar file the front window Display gt Type of Axes gt Linear vs Log changes the x axis from linear to log Both x and y axes can be switched between linear and log by selecting items in the Type of Axes sub menu The Tick Interval settings in the Change Axis Range dialog are not effective when a log axis is displayed Current nA lt c c o k k 2 o 0 1 T
162. umns 6 11 Error Bar Options 6 12 Zero Crossing Axes 6 13 Log Axes 6 14 Raster Display 6 15 Split Clock Support 6 16 Display Tags 6 1 Axis Range and Title Y Axis Range Auto Manual From nA Selected Axis All sections in this chapter assume that a graph window is open File gt Open then select a graph file 2 Pulses in the Example Data folder for example Double click on the x or y axis in a graph window A dialog appears that controls the axis range and style Display gt Change Axis Range opens the same dialog All the x and y axes can be modified without leaving this dialog by using the Selected Axis popup menu to move between axes To select the default axis range turn on the Auto radio button in the Axis Range box To manually set the range enter values in the From and To text edit boxes Title Units Enter the axis title and units in the Title and pxje 2a Units text edit boxes Tic Intervals Auto Manual Minor Tics nR MajorTrics 1 nR By default major and minor tick intervals are selected automatically To set these manually click on the Manual radio button in the Tick Intervals box and enter values in the Major Ticks and Minor Ticks text edit boxes 17 6 2 Comment Title and Notes Options Comment Title and Notes Display gt Comment and Notes To Comment and Document Title are displayed at top of graph add a short one line comment tothe t
163. undown of the response and eliminates outlier amplitudes before calculating the mean and variance The results are directed to the log When variance and mean have been measured under several different release probability conditions the results accumulated in the log can be used to construct a variance mean V M plot This can be done by tidying the results into two tab delimited columns the plotting them via Graph Selection under the Text menu A V M plot typically has a parabolic form Program gt Electrophys Tools gt Fit Var Mean Parabola This program assumes that the front window contains a V M plot see above It fits a parabola to the V M plot thereby providing estimates of the average quantal parameters for the synapse These parameters are the average amplitude of the response to a single vesicle of transmitter Q the average probability of transmitter release from a terminal P the number of synaptic terminals N An article introducing variance mean analysis was published in AxoBits recently To access this article click the following hyperlink http www axon com pub axobits AxoBits28 pdf For a complete description of the variance mean technique see John D Clements and R Angus Silver Unveiling synaptic plasticity a new graphical and analytical approach Trends in Neurosciences 2000 23 105 113 To access this article click the following hyperlink http www biomednet com library abstract TINS
164. ve changes before closing the windows Program gt Document Utilities gt Save amp Close Graphs Save changes then close all currently open graph windows Program gt Document Utilities gt Copy Graph Data to Log The data in the front graph window is copied to the log window in tab delimited format Program gt Document Utilities gt Export Graphs to Text All currently open graph windows are exported to tab delimited text data files The export file names are constructed from the current file name plus the extension txt Program gt Document Utilities gt Compare Text Windows Compare the contents of two text windows and list the non matching lines to the log window Program gt Document Utilities gt Tidy AxoData Log The AxoData acquisition program generates a log file that is difficult to read because it contains a lot of extraneous detail This command automatically deletes the extraneous information making the log file more usable The command assumes that the front window is an AxoData log file 14 8 Graph Utilities Program gt Graph Utilities gt Combine Open Graphs All currently open graph and digitized files are combined into a single new graph Traces with the same group number are overlaid in a single group in the combined graph This command is useful for combining and overlying data traces from different data sets The onset of each trace is aligned in the combined graph Program gt Graph Utilities gt Combi
165. very trace in the front window is filtered using a box car filter The advantage of this filter is that it is very fast It replaces each point in a trace with the average of the surrounding points The number of points to average is entered via a dialog Program gt Trace Transforms gt Three Point Filter Every trace in the front window is filtered using a three point filter This filter operation is very fast to calculate It replaces each point in a trace with the center weighted average of itself and its two neighboring points Program gt Trace Transforms High Pass Filter 62 Every trace in the front window is filtered using a high pass filter FFT s are used to implement a perfect high pass filter i e sharp cutoff below the selected frequency Program gt Trace Transforms Sub region Spectrum The power spectrum of the selected sub region of each trace in the front group is calculated Either the average spectrum or the individual spectra are displayed in a new graph window 14 16 Online Help This section describes the last 2 items on the Program menu Program gt Programming Help A dialog appears with a list of help topics related to AxoGraph s programming languages and its built in commands and functions Select one or more topic and click OK An online documentation window is opened with information on the selected topic The documentation is stored in text files in the Documentation Help Menu Files folder Pro
166. w window Xaxis newWindow Xaxis oldWindow Yaxis newWindow Yaxis oldWindow 1 The following commands provide more detailed control over axis appearance xStr and yStr are string variables GetXTitle w xStr GetYTitle w t yStr GetGroupTitle w g yStr SetXTitle w xStr SetYTitle w t yStr SetGroupTitle w g yStr GetXUnits w xStr Get YUnits w t yStr GetGroupUnits w g yStr SetXUnits w xStr SetYUnits w t yStr SetGroupUnits w g yStr GetXRange w xMin xMax GetYRange w t yMin yMax GetGroupRange w g yMin yMax SetXRange w xMin xMax SetYRange w t yMin yMax SetGroupRange w g yMin yMax Scroll w dx DefaultAxes w copy the x axis title copy the trace s y axis title copy the group s y axis title set the x axis title set the trace s y axis title set the group s y axis title copy the x axis units copy the trace s y axis units copy the group s y axis units set the x axis units set the trace s y axis units set the group s y axis units copy the x axis range copy the trace s y axis range copy the group s y axis range set the x axis range set the trace s y axis range set the group s y axis range scroll the x axis dx units to the left set x axis and y axis ranges to default values 75 When Automatic SI Unit Conversion is turned on all the above Get procedures return range values or title units in absolute
167. width is 10 the bin boundaries will be at 715 5 5 15 Select the Zero Aligned radio button to construct bins with boundaries that fall on whole bin widths If the bin width is 10 then bin boundaries will be at 10 0 10 20 Select the Simple Histogram radio button to construct a conventional amplitude histogram bin count plotted against amplitude Select the Probability Density Function Estimate radio button to construct an amplitude histogram that is normalized to have an area of one This format has the advantage that its form is independent of the number of data points used to construct the histogram It is also an estimate of the probability density function underlying the data After clicking OK a range selector dialog appears which permits the analysis range to be restricted 9 12 Power Spectrum Analyse gt Power Spectrum The data trace is divided into segments containing the selected number of data points The power spectrum of each segment is calculated using a fast Fourier transform FFT To calculate the power spectrum the data is divided into groups of points and the spectra of these groups are averaged The fastest calculation will result when the number of points in a group is a power of 2 eg 512 1024 2048 algorithm The average power spectrum is then calculated and displayed This processing is mathematically intensive and may take a few seconds to complete Once started i
168. yed episodes will be masked When an episode masked it will be skipped during subsequent analysis and display Don t display or analyse Episodes Q RII amp Select 8 0 0 Ef Display gt Mask Selected The dialog permits the list of currently masked episodes to be edited Additional episodes can be masked by adding them to the list Masked episodes can be unmasked by deleting them from the list The list consists of numbers separated be spaces or commas A range of episodes can be included by entering a dash between the lowest and highest numbers in the range For example 1 3 5 8 would mask episodes 1 3 5 6 7 and 8 47 12 2 Average Multi Episode Data The ensemble average and ensemble variance of a group of traces episodes can be calculated The ensemble average is a trace with a waveform that is the average waveform of the selected episodes The ensemble variance is a waveform that reflects the amount of episode to episode variability See Chapter 19 for additional details Analyse gt Average Episodes The dialog selects which episodes to average Turn on the All radio button if all episodes are to be included in the average Turn on the Select radio button if only selected episodes are to be included To select episodes enter a list of episode numbers in the Episodes text edit box The list consists of numbers separated by spaces or commas A range of episodes can be included by entering

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