User Manual for

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Ana is a program for the analysis of electrophysiological data. The emphasis is on pulsed data, in particular of voltage-dependent currents. But also continuous data can be analyzed. In particular single-channel analysis is possible.
Ana can read various file-formats .
Data can be edited extensively (i.e. traces or parts of traces can be deleted, copied, pasted, ...).
The main purpose is fitting of traces, extracting of IV´s (and other things like that), leak subtraction , scaling, (non-stationary) noise analysis, single-channel analysis, and spectral analysis.
Ana is not able to save data in any of the formats used by the standard acquisition programs but ONLY in its own format! In this format, however, it is possible to store a lot of data analysis.

The program is for free but no responsibility whatsoever is assumed by the author. For the latest version, comments and suggestions contact:

Michael Pusch, Istituto di Biofisica, Genova, email: pusch@ge.ibf.cnr.it.

Known limitations and bugs:

- The complete data file is loaded into memory. Its size must therefore not exceed the memory of the computer.
- Practically no internal checks are done for sufficient memory. This may lead to crashes if more memory is needed than available.
- Sometimes the program may crash if it is attempted to read a data file in a format that is not supported.

Index

1.1. Installation

No special installation or registration is needed. Ana is contained in the single ana.exe file that can be launched from where you want. It might be convenient to have a pool of setting files (*.set) in a dedicated folder.

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2. Subwindows

More than 1 file can be loaded at the same time. The window for each file is divided into 3 subwindows. The left subwindow is mainly for a tree-like view of all traces of the file allowing their simple selection. The right part of each window is divided into 2 parts. In the lower part the currently selected data traces are plotted. In the upper half various results of previous analysis or other information is displayed.

Index	Up to '2. Subwindows'

2.1. Tree window

The tree window shows in the upper part an expandable tree structure of the data. The hirarchy is: Root->Series->Entry.
Each series is composed of several "entries". A file may contain many series'. The series' are numbered sequentially, starting from 1. Also the entries are numbered sequentially, and the entry numbers DO NOT START WITH "1" in each series. You can move within the tree using the arrow keys or using the mouse.
For displaying a single entry or a whole series, the following rules apply:
If you have selected Update on moving in tree in the Settings-dialog SINGLE ENTRIES are displayed in the trace window whenever the cursor is on them. Otherwise, single entries, are displayed only if you hit return or if you click the RIGHT mouse button.
The latter rule applies ALWAYS for whole series'.

The controls below the tree are explained in separate sections (i.e. the sliders and the relevant segment).

Index Up to '2. Subwindows'

2.2. Traces window

Here the actual, currently selected , data traces are normally displayed. In addition five vertical and two horizontal cursors and a zero line are displayed. The time- and y-range that are displayed are changed as described in 6. Scaling the display of the traces.
The display and/or subtraction of leak-records ("P/N") is controlled as described in the 7. Parameter settings section.

If you have performed noise analysis you can display the mean and the variance in the trace window by selecting View->LowerWindow->Noise.

If you have performed spectral analysis you can display the spectrum in the trace window by selecting View->LowerWindow->Spectrum.

Index Up to '2. Subwindows'

2.2.1. Filtering traces

A Gaussian filter can be activated by inserting the desired frequency value (in Hz) in the corresponding field in the parameter settings dialog. Filtering does NOT change the data, but is only for display.
If you want to filter and change the data permanently, see 8.3. Permanent filtering.
For undoing such filtering, see 8.1. Undoing an erroneous operation : Exchange.

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2.3. Results window

The upper right window can display various kind of information. The actual viewtype is selected in the menu point View (or via the toolbar).

If View->General is selected the following general information is displayed:
If the root in the tree window is selected, general information like filename, number of series and entries etc. are shown.
If a series in the tree window is selected, information about this series is displayed, including sample time, number of entries, gain, and a list in which for each entry is shown:
i) index
ii) the actual "x-value" of this segment. This depends on the relevant segment and is either a voltage, a duration of the segment, the time-point of acquisition of this entry or its 0-based index. The "x-value" is used for export of analysis results and for the series fit
iii) the mean current calculated between the 2 green cursors in the "trace" window

If View->SeriesFit is selected the results of the series fit are displayed. A common example of this is an 'I-V'.

If View->StimWave is selected the pulse program that generated the data is shown as a table. The time and voltage values of the pulse program are used by some procedures to locate cursor positions and they are used by some leak subtraction procedures. The pulse program is also very important for the definition of the relevant segment.

If View->Analysis Results Table is selected, a table of analysis results of the currently selected series is displayed. The first column contains the respective "x-value" that is determined by the relevant segment and is either a voltage, a duration of the segment, the time-point of acquisition of this entry or its 0-based index. The other columns are speciefied in the settings dialog and are values like initial value, mean, time constants etc. Importantly, the text of the table can be selected with the mouse and copied into the windows clipboard with a right button mouse click. Note that selecting Select All in the popup menu that pops up with a right button click AUTOMATICALLY ALSO COPIES EVERYTHING INTO THE WINDOWS CLIPBOARD.

If View->Stimwave is selected, the pulse protocol of the currently selected series is displayed. If you wish you can change it with the Edit->Stimwave command. See 17.1. Changing the pulse protocol ("Stimwave")

If View->ShowSomePoints is selected, data points around the left green cursor (left column) and the right green cursor (right column)are shown of the currently selected entry are shown. The values are the zero-based index and the current in pA. See 4. Cursors in the "trace" window how to move the cursors. The arrows indicate the position of the respective cursor.

If View->AmplitudeHistogram is selected, the all-points histogram is displayed if this has been constructed previously (see 14.1. Amplitude Histogram and Gaussian Fit)

If View->BetaDistributionFit is selected,...

If View->LorenzFit is selected, a log-log plot of the power spectrum together with a fit with the sum of Lorentzians is shown, if it has been previously constructed (see 15. Spectral analysis).

If View->DwellTimeHistos is selected, log-plots of cumulative dwell-time histograms of the various single channel conductance levels are shown if these have been previously constructed (see 14.3. Dwell time analysis).

If View->VarMean is selected, the variance-mean plot of the non-stationary noise analysis together with a parabolic fit is shown if it has been previously constructed (see 13. Noise analysis).

Index Up to '2. Subwindows'

3. The relevant segment

In an IV-plot you plot current versus voltage. The relevant segment serves to "tell" the program which voltage shall be used for the IV-plot. It is taken from the voltage-protocol that was used to generate the series (see 2.3. Results window to get info on the voltage-protocol).
Note that counting starts with zero, i.e. the first segment has index 0.
The relevant segment is selected either in the control in the lower part of the Tree window or in the Parameter settings dialog.
If you do not want to use the voltage of a segment as the "x-value" but rather its duration as would be the case in "recovery from inactivation" protocols, you have to uncheck the Segment Voltage (else: segment duration) box in the Parameter settings.
If you want to analyze the series data not as a function of voltage or segment duration but rather as a function of the absolute time of the acquisition of the entries or as a function of the index within the series, you have to select "-2" or "-1" respectively as the relevant segment.

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4. Cursors in the "trace" window

There are 5 vertical (2 green, 2 red and one blue) and two (red) horizontal cursors in the Traces window. One of each group (i.e. one vertical and one horizontal) cursor is SELECTED, implying that its color is gray and that its movement can be controlled by the keyboard (see below). You can toggle with the SPACE bar the currently SELECTED cursor.
Vertical cursors are moved by the mouse (LEFT BUTTON!) and the selected (gray) cursor can additionally be moved with the LEFT and RIGHT arrow keys in combination with either the SHIFT key (fine movement) or the CONTROL key (coarse movement) (see 5. Short-cut keys for an overview of frequently used key-commands).
Attention: if you try to move a cursor by dragging with the left mouse button it may happen that you don't grasp it correctly and that you instead select a small display range (or vice-versa) (see 6. Scaling the display of the traces). The best thing to do in that case is to press Ctrl+Z in order to undo the display scale setting (see 7.4. Undoing a parameter settings change).
Horizontal cursors are moved with the right mouse button and the SELECTED (gray) horizontal cursor can additionally be moved by SHIFT+ARROWUP(DOWN) (fine movement) and PAGE-UP(DOWN) (coarse movement)(see 5. Short-cut keys for an overview of frequently used key-commands).

The horizontal cursors serve (so far) for only one purpose: to measure amplitudes of any kind "by eye". The difference between the two corresponding values of the horizontal cursors is displayed as "cursors" if View->General is currently selected and if a single entry is displayed. (see 2.3. Results window).

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5. Short-cut keys

Many shortcuts are found in the menus. Since Sept. 2005 Ctrl+C or Edit->Copy (Copy) has a new behavior in certain situations: If in the upper right window (the results-window) text in a gray results-field is selected, and if the results-window is active (i.e. the selection is visible), the short-cut "Ctrl+C" (or Edit->Copy) copies this text into the general Windows clipboard. It will not copy the selected tree item into the buffer as is instead normally done by the Copy command.

Some shortcuts are listed below:

Cursor movement: With space the active (gray) cursor is toggled
Move the selected (gray) vertical cursor with Shift+Left/right arrow for a fine movement and with Ctrl+left/right arrow for a coarse movement.
Alt+LeftArrow(RightArrow): Move ALL cursors corresponding to the time increment of the relevant stimulation segment
Shift+Arrowup(Arrowdown): Move selected horizontal cursor by about one pixel
PgUp(PgDown): Move selected horizontal cursor by about 50 pixel

Data Editing: Arrow keys: move in the tree (if tree sub-window is active);
Right mouse click or return in the data tree: activate the selected item for display and analysis;

Misc.: Plus (minus) key on keypad: Double (reduce to half) display gain
Ctrl+PgUp(Down): Shift the traces up/down
With del the currently selected item of the tree window is deleted; see 8.2. Copying/pasting/cutting/deleting/merging traces and series

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6. Scaling the display of the traces

The traces can be scaled up/down by the +/- key of the keypad, or by the corresponding toolbar buttons. The display gain can be defined also in the Parameter settings dialog. Traces can be shifted up or down by check Ctrl+PgUp/PgDown or by the arrow buttons in the toolbar or by defining the value of the y-offset in the Parameter settings dialog.
The visualized time-range can be selected by the slider controls of the Tree window. It can be scrolled through also with these controls. The time-range can also be defined in the Parameter settings dialog.

An easy way to select a rectangle for enlargement is to define it directly with the mouse (keeping the left mouse button pressed). Since also the vertical cursors are moved with the left mouse button pressed you have to make sure that the starting edge of the rectangle does not overlap with a vertical cursor.
It may happen that you erroneously selected a too small region and you would like to return to the previous display settings: just hit Ctrl+Z (or the undo button of the toolbar) . A circular buffer keeps the last 20 display (and all other) settings in memory. With Shift+Z you can redo the changes in the settings. See also the menu items Edit->Undo and Edit->Redo.

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7. Parameter settings
Several parameters that concern display settings , cursor positions , definition of fit functions, many switches and other parameters are kept in the Parameter Settings structure that can be saved in a settings file (.set) and reloaded in several ways. The last settings file that has been stored by ana is automatically loaded at the next execution.

7.1. Editing parameters

The parameters are accessible in various ways:
Display parameters are most commonly changed as described in display settings .
Cursor positions are determined as described in cursor positions .
The "General Parameters" dialog is recalled by Settings->General, or by Ctrl+A or by the corresponding toolbar button. Here you can change:

- the display gain, Offset x (left and right) (for the time range), Offset Y (see also display settings );

- the relevant segment;

- the "noise margin" (times SD): this value is used in the selection of records in the noise analysis. The higher the value, the more tolerant is the automatic procedure for the rejection of "bad" traces;

- the "Scale Var Trace": if you display in the trace window the result of a noise analysis this parameter is used to scale the display gain of the variance trace;

- "SD Gaussian noise / pA": with the command Edit->rarely used->Add white noise gaussian noise can be added to the traces. The SD of this added noise (in pA) is defined here;

- "number of components": this value determines the number of terms for various kinds of fits: For exponential fits it determines the number of exponential components. For polynomial fits it determines the number of coefficients;

- "Filter (Hz)": this value defines the value used for the digital Gaussian filter. A value of 0 dictates that no filtering is to be done;

- "ABF gain factor": When reading Axon Binary Format files, there was sometimes a problem with the gain. The value defined in this field is used to multiply the gain found in the ABF file, and serves to get correct current values. This parameter is now obsolete. See also 18.1. Supported data file types;

- "Line Frequency": If you use the manual line frequency (50/60 Hz) subtraction at this point is the initial value of the used line-frequency defined.

In the column of check boxes on the right you define which values are written on a file when you export analysis results (mean, tau, peak, etc). The same values are diplayed when you select View->Analysis Results Table in the results window.

The remaining check boxes define the following behavior:

- "Use SD for idealize": if this checkbox is NOT checked, the algorithm for single channel idealization uses a simple 50% threshold criterion. If the checkbox IS checked, the idealization algorithm uses the width (SD) of the peaks of the gaussians of the amplitude histogram in order to decide to which of the peaks a given data point most likely corresponds.

- "Update on moving in tree": If this checkbox is checked and you select an ENTRY in the tree window by moving the arrow keys or by selecting with the mouse, the display in the trace window is updated. If not checked, the display is ONLY updated if you hit return or click the right mouse button within the tree window.

- "Leak subtract": If a leak trace is stored in your data file together with the data trace (as is e.g. the case in HEKA-Pulse files when you use P/N) you can decide with this checkbox if you want to subtract the leak or not.

- "Show Leak": If a leak trace is stored in your data file together with the data trace (as is e.g. the case in HEKA-Pulse files when you use P/N) you can decide with this checkbox if you want to show it as a green trace or not.

- "Draw lines": if checked, traces are drawn by connecting the individual data points by a line, otherwise they are shown as dots.

- "Segment Voltage (else: segment duration)": if checked, the voltage of the the relevant segment is used as the "x-value" for series fits and for exporting analysis results (mean, tau, peak, etc). Otherwise its duration is used as the x-value (see also 3. The relevant segment).

- "Use 2nd trace (reading pulse-files)": if checked and if you read a HEKA-Pulse data file, and if you have recorded TWO input channels, the second channel is read into Ana. Otherwise, the first channel is used.

- "Show Time": if checked in the general view the "Unix" time in seconds is displayed for series and entries.

-"Show Stop Button": Sometimes you would like to be able to interrupt lengthy erroneously evoked operations by a mouse click. For example you are fitting too many traces with too many exponentials. If this option is checked, a "Stop-Button" appears for several actions. It does not work, however, perfectly and in particluar it seems to be quite incompatible with Windows2000.

- "Log scale in dwell time histo": if checked the vertical scale in the dwell time histograms is in log scale, otherwise it's linear.

- "Dimmed overlay": if checked the traces of a series are drawn in grey, except the last one that remains black Otherwise all traces are black.

-"pick events with right button": normally the right mouse button is used in the trace-window to move the horizontal cursors. If, however, this check-box is clicked, idealized single-channel events can be manually edited with the right mouse button.

Parameters that are relevant for a series fit are accessible in a dialog that is activated by Edit->SeriesFit Settings or Ctrl+G or by the corresponding toolbar button. The meaning of the various parameters is described in the series fit chapter.

Other settings are:

-"Settings->Hide/Show Cursors (Ctrl+H)" that toggles on/off the display of the cursors in the "trace" window.

-"Settings->Hide/Show Zero line" that toggles on/off the display of the zero line.

-"Settings->Dwell Time Settings (Ctrl+W)" are described in 14.3. Dwell time analysis.

Index Up to '7. Parameter Settings'

7.2. Loading/Saving

The parameter settings can be saved on a binary file (Ctrl+S, Settings->Save Settings, toolbar) and loaded from these files (Ctrl+L, Settings->Load Settings, toolbar). The last settings file that was stored is automatically loaded at the next execution. (The filename is saved in the ana.ini file that is in the windows directory). You can prepare several standard setting files for various purposes.

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7.3. Context sensitive parameters

Sometimes you might want to save a set of parameters (that partially include also results of analysis) in association with a specific entry or series. Having selected the desired series/entry and pressing X (or Settings->Save Context Set) opens a file dialog for saving a set-file with a name derived uniquely from the data file name and the index of the series or entry. For reloading such a setfile you just press Y (orSettings->Load Context Set) and a file dialog with the same set-filename as default appears and hitting return will load it. You can of course also load any other set file thru this command.

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7.4. Undoing a parameter settings change

If you have e.g. erroneously selected an unfavorable display setting, or you just want to return to some previous cursor settings: just hit Ctrl+Z (or the undo button of the toolbar) . A circular buffer keeps the last 20 display (and all other) settings in memory. With Shift+Z you can redo the changes in the settings.

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8. Editing commands - The internal buffer

An internal buffer keeps in memory a single entry or a whole series. It works somewhat similar as the windows clipboard but is completely distinct. In particular it is defined only within a single instance of Ana and it exists in parallel to the windows clipboard. You can use it to transfer data from one file to another, to delete traces, series and so on. An important application is that in most cases when a data-changing operation is performed, like e.g. permanent filtering or deleting of a stretch, or scaling, the original data are copied into the buffer and the operation can be undone using the "exchange" mechanism (see 8.1. Undoing an erroneous operation : Exchange).
Important Note: Only the raw data and no analysis results (like series fit, noise, fits, ...) are saved in the internal buffer. Any of such information is lost!

In order to highlight entries in the tree (single entries or whole sweeps) that have been altered by a data changing operation, such entries are displayed in the tree with a differently colored icon. This "altered" status can be explicitly reset with Changed->Remove "Changed" Status. Saving the file will also reset this flag.

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8.1. Undoing an erroneous operation : Exchange

Many operations permanently change the data (scaling, permanent filtering, cutting, undersampling, deleting a stretch, subtracting leak, exchanging data with fit, subtracting capacitance, subtracting line-frequency, and some others) and sometimes you realize (hopefully immediately) that you would like to undo an operation. Fortunately, Ana makes a copy of the original data and stores it in the internal buffer. Doing so, it overwrites (without asking) the previous contents of the internal buffer. So you can undo only the last operation. For undoing you have to select within the data tree the corresponding item (series or entry) and then click Edit->Exchange or type Z or click on the double-arrow in the toolbar.

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8.2. Copying/pasting/cutting/deleting/merging traces and series

With Ctrl+C (or Edit->Copy ) you copy the currently selected item of the data tree into the internal buffer, overwriting (without confirming) the current contents of the buffer. This applies only to single entries or single series (Note: under certain circumstances Ctrl+C or Edit->Copy, will copy selected text in gray windows as described here). The internal buffer does not take more than one series, i.e. you can not copy the root.

Important Note: Only the raw data and no analysis results (like series fit, noise, fits, ...) are saved in the internal buffer. All this information is lost passing through the buffer!

With del the currently selected item is deleted without making a copy and thus without overriding the contents of the buffer while with Ctrl+X, Edit->Cut the item is deleted and copied into the buffer. The contents of the buffer can be pasted before (Ctrl+V, Edit->Paste Before) or after (Ctrl+T, Edit->Paste after) the currently selected item. If a series with several entries is currently stored in the buffer these can be merged after the currently selected entry of the tree window by Edit->Merge entries after.

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8.3. Permanent filtering

By specifying a filter frequency (larger than 0 and smaller than the sampling frequency) in the parameter settings the data are filtered only for display each time before display without changing the original data. You can perform a permanent, data-changing filtering by F12 or Edit->Filter. The original data are copied into the internal buffer and as long as nothing else is copied into the internal buffer the filtering can be undone by the "Undoing an erroneous operation : Exchange" mechanism.

You can also filter the whole file (i.e. all series') by Edit->Filter whole file (Ctrl+F12) but as the internal buffer contains at most 1 series, this command cannot be undone.

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8.4. Undersampling

Undersampling by a factor of "n" means to take only every n'th point and to throw away the others. This reduces the number of points by a factor of n. This can be done by Edit->Undersample or F10. A filtering effect can be achieved if you use Edit->Undersample And Average. The original data are copied into the internal buffer and as long as nothing else is copied into the internal buffer the undersampling can be undone by the "Undoing an erroneous operation : Exchange" mechanism.

You can also undersample the whole file (i.e. all series') by Edit->Undersample whole file (Ctrl+F10) but as the internal buffer contains at most 1 series, this command cannot be undone.

Index Up to '8. Editing commands - The internal buffer'

8.5. Calculating the average

With Edit->Calculate Mean (or F3) the average of the selected entries is calculated and placed in the internal buffer from where it can be pasted.

Index	Up to '8. Editing commands - The internal buffer'

8.6. Copying selected records

With Edit->Copy (Ctrl+C) the currently selected entry (if an entry is selected in the Tree window) or the currently selected series (if a series is selected in the Tree window) is copied into the internal buffer from where it can be pasted.

Index	Up to '8. Editing commands - The internal buffer'

8.7. Concatenate entries

Several entries can be concatenated by this command (Edit->Concatenate) into a single entry that is placed into the internal buffer from where it can be pasted.

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8.8. Deleting a stretch

The data stretch between the green cursors can be deleted by F8 or Edit->Delete piece between green cursors. The original trace(s) is(are) copied into the internal buffer allwoing to undo the deletion.

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8.9. Extracting a stretch

The data stretch between the green cursors can be extracted (F9 or Edit->Extract piece between green cursors ) and copied into the internal buffer from where it can be pasted.

Index	Up to '8. Editing commands - The internal buffer'

8.10. Overlay envelope

Sometimes time increments or decrements are used in the pulse protocols, e.g. in recovery from inactivation protocols. The "relevant" or interesting segment then may not stay "within" the cursors for the analysis when viewing the traces. Data recorded by HEKA-pulse (I think not pclamp-data) have a variable number of samples for each record in such cases. This allows the following trick: applying the Even more analysis->overlay envelope command Ana adds zeros to the beginning of each trace such that all traces will have the same number of samples resulting in a perfect superposition of the segments after the variable-length period.

Index	Up to '8. Editing commands - The internal buffer'

8.11. Swapping data and fit

If a trace fit has been performed, that is shown in blue, the blue, fitted, stretch can be swapped with the corresponding data stretch by the command Even more analysis->Swap data-fit.

Index	Up to '8. Editing commands - The internal buffer'

8.12. Reverse time

With this command (shortcut = Shift+T) you can reverse the time of one or more entries. This could be useful to test if your (single-channel) analysis depends on the direction of time or, more profanely, you can use this to extrapolate a fit not to a previous time point but to later times. If more than one entry is subjected to the time-reversal you can chose to reverse also the order of the entries to get a complete time reversal, or not. In the former case the pulse-protocol (if varying voltages or times are involved) WILL NOT BE VALID AFTER THE REVERSAL!

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8.13. Paste as fit

With the command Edit->Paste as fit (shortcut = F5) the internal buffer content is pasted as a trace fitting (i.e. a blue line) overriding any fit present, starting at the beginning of the trace! With Edit->Paste as fit between green cursors (shortcut = Ctrl+F5) the internal buffer content is pasted as a fit only between the green cursors.

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9. Trace fitting

The data traces can be fitted by a variety of functions, in particular exponential functions with various constraints, polynomials, and, using an equation-parser, any user-defined function .

The following general rules apply:

The fit is displayed on top of the data in blue color.

Fitting is performed using the Simplex algorithm (except for the polynomials that are calculated directly). The number of iterations and the stepsize are defined in the Series Fit parameters dialog .

The fit is applied to a single entry if a single entry is selected in the tree window or to a group of entries within a series if a series is selected in the tree window.

The trace is fitted between the two green cursors, i.e. the parameters of the fitted function depend on the data values between the green cursors.

If the blue cursor is located to the left of the left green cursor, the fit is extrapolated up to the blue cursor. This is for example used to estimate the initial current value after a voltage jump that is disturbed by a capacitive artifact.

A single entry can "contain" only a single fit. A new trace fit overrides the old one. This implies that all results values that depend on the fit are also overwritten (e.g. the "initial value").

The fits are saved on the file if it is saved.

If entries are copied into the internal buffer , the fit is not copied.

The data trace and the fit can be "exchanged". This does, however, not lead to a recalculation of the initial value or other values that depend on the parameters of the fit.

The entries of the internal buffer can be "pasted as fit", i.e. any old fit trace is overwritten and the pasted trace is shown in blue. Pasting starts from the first data point (i.e. the position of the cursors have no influence). This is used mainly for display purposes. It does not lead to a recalculation of the initial value or other values that depend on the parameters of the fit.

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9.1. Exponential fits

In all exponential fits traces are fitted by functions of the form

a0 + a1×exp(-t/tau1) + a2×exp(-t/tau2) + ... + an×exp(-t/taun)

n, the number of exponentials, must be at least 1. It is determined in the general parameter settings dialog as "number of components". The time point 0 is determined by the left green cursor if the blue cursor is located to the right of it and otherwise by the position of the blue cursor (used for backextrapolation, see 4. Cursors). In a simple full free exponential fit (Ctrl+E, toolbar) all parameters (ai, taui) are freely fitted, while several specially constrained exponential fits are described below. Of particular importance is the common exp fit which is used to fit the same time constant(s) for all entries in a constant-voltage tail protocol to extract the steady state popen from tail currents. The parameters (see "initial value (from fit)", "fast tau", "slow tau", "very slow tau", "coefficients", in the general parameter settings dialog) of exponential fits can be exported on a file or displayed as a table and selected with the mouse. The initial values for the time constants are guessed based on the time interval. Sometimes these guesses are not appropriate and manual intervention is necessary. In this case you can try to play with the stepsize and the number of iterations (see tracefitting). If this does not help you can manually enter starting values by hitting "i" on the keyboard while the fit is running (this works only if the checkbox allow keyboard input in fit (see Series Fit)) in the series fit dialog is checked). If also this does not help you can try a user-defined function where the initial values have to be manually set. Note that the coefficients, ai, are not dependent on any initial value assumption as they are optimally calculated for a given set of time-constants.

Index Up to '9. Trace fitting' Up to '9.1. Exponential Fits'

9.1.1. Simple exponential fit

In a simple exponential fit all coefficients and time constants are freely fitted. See '9.1. Exponential Fits' for a general description.

Index	Up to '9. Trace fitting'	Up to '9.1. Exponential Fits'

9.1.2. Fit several traces simultaneously with the same time-constants for all

In this fit several entries are fitted simultaneously with the SAME time constant(s) for all entries. The coefficients are calculated individually for all entries. A frequently used protocol to estimate the steady-state voltage-dependence is to apply a pulse to a fixed voltage after prepulses to varying voltages. The initial current at the constant "tail" pulse is proportional to the open-probability at the end of the prepulse. The "common exp" fit is generally used to estimate the initial value by exponential fitting and backextrapolation. It yields also a good estimate of the time constant(s) at the fixed tail voltage.

Index	Up to '9. Trace fitting'	Up to '9.1. Exponential Fits'

9.1.3. Other exponentials

Somewhat differently treated from the normal sum of exponentials is the product of two exponentials (Analysis->Special Exps->fit product of two exps) because the coefficients have to be fitted too. A variant of this is Analysis->Special Exps-> product of two exps, fix one in which one of the time constants is fixed.

In the normal "sum-of-exponentials fit", there is also the option to keep one time constant fixed, either for each trace singly (Analysis->Special Exps->fit exps, fix one) or keeping the same taus for all traces (Analysis->Special Exps->fit common exps, fix one; see 9.1.2. Fit several traces simultaneously with the same time-constants for all).

Another option is to constrain the coefficients in a way to keep the initial derivative equal to zero:
dY/dt( t=0) = 0
Try Analysis->Special Exps->fit exps with DI(0)=0 (Ctrl+D) and Analysis->Special Exps->fit common exps with DI(0)=0.

Index Up to '9. Trace fitting' Up to '9.1. Exponential Fits'

9.1.4. Envelope

Not yet documented. But see: 8.10. Overlay envelope.

Index	Up to '9. Trace fitting'	Up to '9.1. Exponential Fits'

9.2. Polynomial

The "number of components" in the parameter settings dialog determines the number of coefficients of the polynomial to be fitted, i.e. 1 corresponds to a horizontal line, 2 is a straight line, 3 is a parabola, etc. The coefficients can be visualized in the Results window from where they can be copied and pasted.

Index	Up to '9. Trace fitting'

9.3. User defined

With More Analysis->Fit trace with user function or Ctrl+U the fit of the trace(s) by an arbitrary user-defined function is invoked. The function is defined in the series-fit dialog. The time-variable can be written as "t" or as "v". The parameters of such a fit are written as a[0], a[1], etc. and the initial values are defined in the dialog invoked by clicking Settings->Edit Params or typing E. Here you also specify for each parameter if you want it to be adjusted (checking the corresponding "DoFit" box) or if it should remain fixed.
As an example you can compare the intrinsic exponential fit with the following user defined function:

(a[0]+a[1]*exp(-t/a[2]))*1e-12
The factor 1e-12 here is just for convenience to keep the parameters a[0] and a[1] in pA. Time is always in SECONDS!
See 18.3. Syntactic rules for the user-defined function for the syntactic rules of the user-function.

Index Up to '9. Trace fitting'

10. Series Fit

Series fit means that you display and fit a particular value (e.g. the mean calculated between the 2 green cursors) for all the entries of a series. The x-value can be either

- the voltage of the relevant segment (if the checkbox "Segment Voltage (else: segment duration)" in the parameter settings dialog is checked).

- the duration of the relevant segment (if the checkbox "Segment Voltage (else: segment duration)" in the parameter settings dialog is NOT checked).

- the entry-index (starting with 0 for the first entry) defined if you set the relevant segment to -1

- the absolute time of acquisition of the entry (starting with 0 for the first entry) defined if you set the relevant segment to -2

The type of function to fit is selected in the Series Fit settingsdialog (Ctrl+G) . The radio buttons on the top left of the dialog decide what function you want to fit. (see below for a precise definition). In the column of radio buttons on the right you select which value you want to fit. The other fields mean:

- # components in poly fit: The number of coefficients for a Polynomial fit.

- Maxiter: the number of iterations in the fit.

- Stepsize: the stepsize for the simplex search in the fit.

- User function: here the user function is defined. It is used either by the user defined trace fit or by the User defined series fit.

- Bin width ampl histo (in pA) : The bin-width for the Amplitude Histogram

- Bin width dwellt. histo (in ms) : The bin-width for the Dwell time analysis

- min dwelltime (ms): The minimal dwell-time for the Dwell time analysis

- Invert Y-data : if checked the values to be analyzed are multiplied by -1.

- Equidistant gaussians : if checked the peaks of the gaussians are equidistant in the Amplitude Histogram

- Allow keyboard input in fit: only if checked has keyboard input an influence during trace- and series-fit. With "s" the fit is interrupted. With "i" new initial values of the various parameters can be entered during fitting.

The data values and the fit are shown in the upper window. Clicking with the right mouse button on the individual data points you can display the corresponding x and y value.

Index Up to '10. Series Fit'

10.1. Boltzmann

In a Boltzmann-fit the following function is fitted:

offset + (Max-offset)/(1+exp( (v-V_1/2)/k ))

with the four parameters, offset, Max, V_1/2, and k. If you want to fit a different kind of function (e.g. fix the offset to 0) you have to use the User defined series fit.

Index Up to '10. Series Fit'

10.2. Polynomial

A polynomial is fitted with the number of coefficients, Ncoeff, as described above (see 10. Series Fit). With NCoeff=1 the mean is calculated (and displayed). With NCoeff=2 a straight line is fitted and assuming that the SeriesFit is an IV also the extrapolated reversal potential is calculated and displayed. With NCoeff=3 a parabola is fitted and assuming that the SeriesFit is an IV also the two possible extrapolated reversal potentials are calculated and displayed. For Ncoeff>3 only the coefficients are displayed.

Index	Up to '10. Series Fit'

10.3. User defined function

The function is defined in the series-fit dialog. The independent variable can be written as "v" or as "t". The parameters of such a fit are written as a[0], a[1], etc. and the initial values are defined in the dialog invoked by clicking Settings->Edit Params or typing E. Here you also specify for each parameter if you want it to be adjusted (checking the corresponding "DoFit" box) or if it should remain fixed.
As an example you can compare the intrinsic Boltzmann fit with the following user defined function:

a[0]*(a[1]+(1-a[1])/(1 + exp((v-a[2])/a[3]))*1e-12

The factor 1e-12 is to keep the parameter a[0] in pA. You can fix e.g. the "offset" to zero by entering a[1]=0 in the dialog and not fitting the value.
See 18.3. Syntactic rules for the user-defined function for the syntactic rules of the user-function.

Index Up to '10. Series Fit'

11. Leak, capacity, and line frequency (50/60 Hz) subtraction

Before these various subtractions, the original data are copied to the internal buffer and the operations can be undone using the "exchange" mechanism (see 8.1. Undoing an erroneous operation : Exchange).

Index

11.1. Baseline subtraction

With Leak Subtraction->subtract baseline or Ctrl+B the data between the two red cursors is averaged and subtracted from the trace(s).

Index	Up to '11. Leak, capacity, and line frequency (50/60 Hz) subtraction'

11.2. Leak subtraction

Ana offers 2 possibilities to perform offline leak subtraction. The first is two subtract a scaled series of leak records, the second is based on a baseline segment ...
no help yet, sorry.

Index	Up to '11. Leak, capacity, and line frequency (50/60 Hz) subtraction'

11.2.1. Subtract a scaled series

no help yet, sorry.

Index	Up to '11. Leak, capacity, and line frequency (50/60 Hz) subtraction'	Up to '11.2. Leak subtraction'

11.2.2. Subtract using a baseline segment where channels are supposed to be closed

no help yet, sorry.

Index	Up to '11. Leak, capacity, and line frequency (50/60 Hz) subtraction'	Up to '11.2. Leak subtraction'

11.3. Capacity correction

Suppose one segment of the stimulation pulse goes to a voltage where no current flows because the driving force is 0 (i.e. a step to the reversal potential). You can use this segment to subtract the capacity from other segments as in the following example. Suppose your protocol is like this:

segment 0: VHold, 10 ms
segment 1: VTest1, 10 ms
segment 2: VTest2, 5 ms
segment 3: VHOLD=EREV, 10 ms

You can subtract the capacitive artefact from segments 1 and 2 (if VTest1 and VTest2 are different from EREV). Let's do it for segment 1. Select the sweep (or series) where you want to subtract the capacity. Put the "relevant segment" to 1. Select "View->Put green cursors on segment" (Ctrl+-) to put the green cursors on segment 1. Select "View->Put red cursors on segment" (Ctrl+<) to put the red cursors on segment 3!
Select "Leak Subtraction->Separated segments capacity correction" (Ctrl+F11) to subtract the capacity. Select "3" as the "leak" segment.
The original data are copied into the internal buffer and as long as nothing else is copied into the internal buffer the subtraction can be undone by the "Undoing an erroneous operation : Exchange" mechanism.
You can of course also manually put the cursors to fit exactly the start of the segment if there is an incongruence with the stimulation protocol.

Index Up to '11. Leak, capacity, and line frequency (50/60 Hz) subtraction'

11.4. line frequency (50/60 Hz) subtraction

no help yet, sorry.

Index	Up to '11. Leak, capacity, and line frequency (50/60 Hz) subtraction'	Up to '11.4. line frequency (50/60 Hz) subtraction'

11.4.1 Automatic line frequency (50/60 Hz) subtraction

no help yet, sorry.

Index	Up to '11. Leak, capacity, and line frequency (50/60 Hz) subtraction'	Up to '11.4. line frequency (50/60 Hz) subtraction'

11.4.2 Manual line frequency (50/60 Hz) subtraction

no help yet, sorry.

Index	Up to '11. Leak, capacity, and line frequency (50/60 Hz) subtraction'	Up to '11.4. line frequency (50/60 Hz) subtraction'

12. Scaling traces

12.1 Scaling by a factor and add a constant

With the command MoreAnalysis->ScaleTrace (or via the toolbar) the selected entries are transformed by
x -> m*x + b
with a factor m and a constant b. Note that b must be given in units of Ampere. The original data are copied into the internal buffer and as long as nothing else is copied into the internal buffer the scaling can be undone by the "Undoing an erroneous operation : Exchange" mechanism.

Index	Up to '12. Scaling traces'

12.2 Scaling traces of a series to have a common value in a certain segment

With the command MoreAnalysis->ScaleTails (or hitting "J") the entries are scaled in a way that the mean of the stretch delimited by the green cursors is the same for all and corresponds to one that can be selected. This is e.g. useful if you are recording an instantaneous IV and there is run-down and you want to scale the traces to have the same prepulse amplitude. The original data are copied into the internal buffer and as long as nothing else is copied into the internal buffer the scaling can be undone by the "Undoing an erroneous operation : Exchange" mechanism.

Index	Up to '12. Scaling traces'

13. Noise analysis (non-stationary!)

Non-stationary noise analysis serves to estimate the single-channel conductance and the number of channels. For a theoretical background please refer to

1. Sigworth , 1980, The variance of sodium current fluctuations at the node of Ranvier. J Physiol. 307:97-129.
2. Heinemann and Conti , 1992, Nonstationary noise analysis and application to patch clamp recordings. Meth. Enzymol. 207:131-48.

Briefly, a fixed pulse during which the channels undergo a time-dependent relaxation is applied repeatedly, e.g. 50 times. The analysis is divided into three steps:
1. Selection of the time stretch and the background stretch to be used (if any)
2. Selection of records
3. Variance-mean analysis with a parabolic fit

Index Up to '13. Noise analysis'

13.1. Selection of records and cursor settings

The stretch of the record that is used for the noise analysis is defined by the green cursors. If you want to subtract a baseline variance you have to position the red cursors over the corresponding stretch.

It is often advisable to eliminate "bad" records that contain e.g. disturbances or unwanted electrical activity from the set of recorded traces to be used for the noise analysis. One possibility is to go manually through the records and throw out those that appear to be bad. Ana offers also a somewhat more objective possibility to detect bad records. As a criterion Ana identifies a record as potentially bad if it deviates at a single data point from the crude mean by a certain number of S.D.'s. This factor is defined in the general parameter settings dialog.

With Analysis->Noise etc->NoiseAnalysis or Ctrl+N the selection of the records is started. First you have to select the range of records for which the noise-analysis is to be performed. If you want to use all records, no further selection is necessary. Also, if you have done previously a selection (that is saved on a "lst"-file; see below) that you want to use, no further selection is necessary; just confirm the corresponding questions. If you want to select the records manually, follow the instructions: In the trace window the single records are now displayed overlayed with the cumulative mean of the selected traces (apart from the first trace, that is shown without mean). If a trace has been identified as potentially bad by Ana, this will be written on top. The selection has now to be done with the keyboard:
- to accept a trace as good: hit "Y" (yes)
- to not accept a trace: hit "N" (no)
- to stop the selection process and proceed the analysis with the traces selected sofar: hit "S" (stop)
- to completely interrupt the noise-analysis: hit "Q" (quit)
- to go back one record: hit "B" (back)
- to rush, i.e. to accept automatically all good records and to ask explicitely only for the potentially "bad" records: hit "R" (rush)

After finishing the selection it is written on a file with the same name as the data-file but with the extension ".lst". If such a file exists already you are asked if you want to overwrite the old selection. Thus, the selection of the good records is saved and you don't have to redo it. You just answer "yes" to the question "Use old selection(lst)?", the next time.
Because the selection file is simply a list based on the indexes of the traces it is not any more applicable if you change the data file by deleting or adding records. To avoid such a possible error you can also "extract" the selected records from a series by Edit->Extract records, that will put only the selected records as a series into the internal buffer that you can paste for example in an empty file.

After the selection - using either all records, or using an old selection, or by going through the selection process as described above - the user is asked for "the number of segments". This has the following meaning: for the variance-mean plot one often does not want to display all data points but to have a more condensed representation. For this purpose, the current range of the calculated mean (within the selected stretch (green cursors)) is horizontally sliced into segments (this is the "number of segments"). This cuts the current trace into several segments of equal vertical extension but possibly variable length. Rapidly changing parts are thus shorter, while a long plateau could be fit into a few segments. Within each segment of the mean current trace the current and the corresponding variance values are averaged and yield one point in the variance-mean plot (see also Sigworth , 1980, J Physiol. 307:97-129 and Heinemann and Conti , 1992, Meth. Enzymol. 207:131-48). If you don't want to do this kind of averaging you can enter a very high number as the number of segments (e.g. larger than the number of points between the green cursors).

The result of the noise analysis so far is that the mean current and the variance has been calculated (and stored; you can display them as described in 2.2. Traces window) for the whole trace. Furthermore, for the part between the green cursors the current and variance values that come from the slicing procedure described above, are stored in an extra array that can be further analyzed by the variance-mean parabolic fit as described below. Also the (average) variance between the red cursors has been calculated as background variance, that can be subtracted (or not) for the variance-mean analysis.

A remark regarding the sign of the mean currents: Ana automatically converts a negative current range (between the green cursors) into a positive range by multiplying with -1!

Index Up to '13. Noise analysis'

13.2. Variance-mean analysis

By Analysis->Noise etc->Var Mean, or Ctrl+J the variance-mean parabolic fit dialog is invoked. This does not work if you haven't previously calculated the mean and variance as described above (if you have loaded a data file that was previously saved with Ana after having calculated the variance, the variance is also loaded; you don't have to redo the calculation; see 18.2. The internal data-format of ana: what is saved on a file?). In the dialog you specify the initial values for the parameters, the number of iterations, which parameters are to be fit and how the background variance and the leak has to be treated. The fit is according to the following formula

sigma² = sigma₀² + i*(1+o^2)*(I-leak) - (I-leak)²/N,

where sigma₀² is the background variance, leak is the leak current, i is the single channel current, N the number of channels, o is the "fraction open channel noise" (normally = 0; see below), and I the independent variable, the mean current. It is, of course, not advisable to have both, leak and background variance, as free parameters. Instead it is better to have as most as possible independent measurements for these two parameters, and to keep them fixed. After clicking OK in the dialog, the parabolic fit is performed and the results are displayed. You can export the results of the variance analysis by File->Export Analysis->Export Var-Mean. The results are written on a plain white-spaced ASCII file as columns. If you answer "YES" to the "Write trace?" question, the first three columns of the file contain the time, mean current (in pA), and mean variance values (in pA^2) of the whole trace. Note that the background variance has NOT been subtracted yet from the variance values. The next three columns contain: mean current of the segments, mean variance of the segments, and standard deviation of the variance. The background variance is written in the last (single-valued) column.
Note on the parameter "o" (fraction of open channel noise): The "profane" user should just ignore this value (setting it to 0). It is not a real parameter to fit as it is absolutely not independent from the parameter i. However, for the calculation of the maximal open probability it is sometimes useful to have an idea about how an "open channel noise" would influence the estimate of pmax. The idea is that with all channels open the variance is not zero, but equal to N*sigma_open², where sigma_open² is the variance of an open channel that is parameterized as sigma_open²=(o*i)², hence the meaning of the parameter o. If not all channels are open, the open channel noise is assumed to behave as N*p*sigma_open², and putting this into the normal variance-mean-equation results in the above formula.

Index Up to '13. Noise analysis'

14. Single channel analysis

Two basic features are offered for single channel analysis: amplitude histogram analysis and dwell-time analysis. The dwell-time analysis can only be done after having constructed and fitted the amplitude histogram by a sum of Gaussians, because in this way the levels are defined that are used for the automatic idealization. Note that the idealization is COMPLETELY AUTOMATIC; it is not possible to change the single transitions. This possibility might be incorporated in future versions.

Index	Up to '14. Single channel analysis'

14.1. Amplitude Histogram and Gaussian Fit

To construct the amplitude histogram select Analysis->Construct Ampl. histogram (or Shift+H or via the toolbar). A dialog appears where you select
- if you want to restrict the analysis to the region between the green cursors
- the first and last entry to analyze
- the bin width used for the histogram
- the current-range

Then you fit the amplitude histogram with a sum of Gaussians by selecting Analysis->Fit Ampl. histogram (or Ctrl+M or via the toolbar). A dialog appears where you select

- the number of gaussians (identical with the "number of components" of the general settings)
- if the gaussians shall be equidistant
- how the initial parameters of the Gaussians shall be chosen
- if the fitted parameters should be copied in the "parameter array" after the fit
- the stepsize and the number of iterations of the fit
- if keyboard input is allowed during the fit (this option turns on e.g. the possibility to interrupt the fit by hitting "s" (stop); however as the keyboard input is checked asynchronously any "s" typed during the fit, also if in another program, like e.g. a text-writing program, will interrupt the fit and you might want to disable this feature such that you can write during a lengthy fit).

The program will likely NOT find automatically the correct parameters for a multi-Gaussian fit but they have to be selected manually in a simple graphical fashion described below, requiring thus at least two rounds of the Gaussian fit. The first time you better select "Automatic re-calculation" for the start params that leads to a practically random placement of the mid-points of the Gaussians. Each Gaussian component has a vertical green line at the midpoint, a horizontal green line at the peak value and circles at the ends of the horizontal line indicating the sigma-value of the Gaussian. The midpoint (vertical lines) can be moved with the left mouse button. The amplitude (vertical line) of the Gaussians can be moved with the right mouse button and the sigma value can be changed by moving one of the circles with the left mouse button. You should thus adjust manually all Gaussians in an approximate manner and then select again Analysis->Fit Ampl. histogram (or Ctrl+M or via the toolbar) but now selecting the option "Use last values" for the start parameters. Also the step size should not be too big ( a value of 0.1 is normally ok together with about 1000-10000 iterations, depending of course on the number of Gaussians and how well they are defined by the data).

If you select the option "equidistant means", the LEFTMOST midpoint and the next one are used for determining the position of the others!

From the Parameters of the Gaussians the relative areas under each component are calculated (in %) and displayed together with the other parameters in the grey selectable and copyable text box on the right. Clicking on the button "Copy params to array" copies the parameters into the data array that is reachable by hitting "E" (or via the menu: Settings->EditParams). This array is part of the settings. It can be used here to define precise starting values and/or to save the gaussian parameters in a setting file from where they can be reloaded as starting values as described above.

Once the Gaussians are fitted you can proceed with the Idealization.

Index Up to '14. Single channel analysis'

14.2. Idealization

Once the amplitude histograms has been fitted with a sum of Gaussians, the midpoints of the Gaussians are used as the levels for the idealized current traces. Two different kinds of citeria can be applied for the idealization:
- 1. a simple 50% threshold criterion or
- 2. a criterion based on the width (SD) of the respective Gaussians (as explained below).
Which of these criteria is used is selected in the General Settings dialog in the check box "Use SD for idealize". If checked, criterion 2, otherwise criterion 1 is applied. Criterion 2 works as follows:
Suppose there are n levels, mi is midpoint of level i, vari is the SD^2 of level i, and y is a data point that has to be assigned to a level.
For each level the quantity pi=exp( -(mi-y)^2/ 2 / vari) is calculated and the level with the largest pi is chosen as the most likely one.
(Actually the pi's are calculated as (mi-y)^2/vari and the smallest pi is selected. The result is the same).
Idealization is dependent on a further parameter that is asked for when "Analysis->Idealize" (or Ctrl+I) is selected: the minimal number of data points that an acceptable event must be long.

The idealized trace is shown in blue and represents as such a "fit" of the data trace, implying that any other fit, like fitting an exponential will override and replace the idealization. Saving a data file in this status will save also the idealization, as any other fit.

Index Up to '14. Single channel analysis'

14.3. Manual editing of the idealized records

It may happen that some events are not acceptable (e.g. too short) after the idealization. These can be eliminated in the following way: first, manually editing has to be enabled by a check-box-flag in the Settings dialog: make sure that the check box "pick events with right button" is checked in the settings dialog. This makes sure that the right button in the trace-window does not move the horizontal cursors but picks the events. Then place the mouse over the event that you want to change and press the right button. A popup-menu appears with various choices. The last action (elimination) can be undone if, and only if, the corresponding stretch is at least partially visible on the screen.

Index	Up to '14. Single channel analysis'

14.4. Dwell time analysis

Like the amplitude-histogram, the dwell times are delt with in 2 steps: 1st construction of dwell time histograms, then 2nd. fit by a sum of exponentials. Several important general features apply, however, to the dwell time analysis:

- The fit by the sum of exponentials is completely independent of the representation in a histogram (bin width etc.). All events of a given level are explicitely taken into account (larger than a minimum dwell time to be specified) and the fit is done using a Maximum Likelihood criterion as detailed below.
- The dwell time histograms are cumulative histograms with a linear time scale. The vertical scale can be either linear or log dependent on the setting in the general parameters dialog(also selectable in the fitting dialog described below).

Selecting Analysis->Construct Dwell Times (or just hitting O, or toolbar) the dwell time histograms for all levels are constructed and displayed. Here, the only important parameter is the bin width of the histograms, to be selected in the series fit settings.
Selecting Analysis->Fit Dwell Times (or just hitting Q, or toolbar) the dwell times of the specified level are fitted by the sum of exponentials as specified in the dialog that appears. Here you select

- the number of exponentials
- start values for each time constant and coefficient to be fitted
- if you want to fit or otherwise fix the time constant and/or coefficient
- some other parameters

Note that the LAST COEFFICIENT WILL ALWAYS BE VARIABLE because the sum of the coefficients is constrained to be 1, such that the number of effective parameters is reduced by 1, excluding the last coefficient as a fittable parameter.

The fit is done according to the Max.Likelihood criterion i.e. for each event, e, of duration te, the likelihood is calculated as

Le= sum over i (a[i]*exp(-te/tau[i])) / LMINMAX
where LMINMAX is calculated as
LMINMAX=sum(a[i]*(exp(-MinDwell/tau[i]) - exp(-MaxDwell/tau[i]))
and is the probability that an event has a duration between MinDwell and MaxDwell. The parameter MinDwell has to be specified in the above mentioned dialog. The parameter MaxDwell is determined from the shortest length of the records used for the construction of the dwell time histogram.

Index	Up to '14. Single channel analysis'

14.5. Binomial Peak analysis

Sometimes, for example if several channels are in a patch, one would like to perform a binomial analysis of the areas of the amplitude histogram. One reason is to determine the open-probability assuming the presence of N independent channels. Or, the independence of the channels shall be tested. For these purposes, the "Binomial Peak Fit" has been implemented. It can be assessed via "Analysis->Fit Binomial Distribution" that invokes a start-dialog. Data can be entered in the white window. The format is "n A F", for each data point, where n is the associated current level, A the experimental probability (in %) and F is the fitted probability. The latter can be set to any value for input and can be used to read out the fitted value after the fit.

Let's take an example. Assume two channels, each with popen=p=0.5. The "ideal" experimental data would be

0 25 0
1 50 0
2 25 0

I.e. Prob=0.25 to have no channel open, 1/2 that one channel is open and 0.25 that both are open.

You can enter the data either manually, or, better, paste them from another program.

NOTE THAT TO MAKE A NEW LINE IN THE DATA INPUT WINDOW YOU TO PRESS CTRL+ENTER!

If (and only if) you have previously fitted an amplitude histogram, the corresponding area values are shown initially as experimental values in the input window. You don't need to use these, however. By clicking on the "Copy Data from amplitude histogram" button, the data from the amplitude histogram (if that is present) will be copied into the data window and overwrite any other data there.

Three options are offered for fit.
1. The simple binomial fit. This is the usual case. Each level, k, has probability

Note that the parameter N can be selected as a free parameter. However, for numerical reasons, the Simplex algorithm might not find the best value. Try to do the fit from several starting values for N (and reasonable values for p=p1) and check the resulting SD value shown after the fit. The best fit has the lowest SD value.
For this simple binomial fit the parameters p2 and N2 are irrelevant.

2. The second case assumes two "modes" of the channel, one mode with p=p1 and another mode with p=p₂. N is the total number of channels, while N₂ is the number of channels in mode 2. This N₁=N-N₂ is the number of channels in mode 1. Again, the N's can be chosen as free parameters but the Simplex algorithm has difficulties finding the true minimum!!
The formula for calculating the probability for each level, k, in this case is

Two modes

with the definition that (n over j) = 0 for j>n.

3. The third option assumes a double-barreled channel with a common gate with popen=ps=p1 and a protopore gate with popen=pf=p1. The two gates are assumed to be independent.

After performing the fit, experimental values are shown as bars, fitted values are shown as symbols.

Index Up to '14. Single channel analysis'

15. Spectral analysis

no help yet, sorry.

Index	Up to '15. Spectral analysis'

16. Exporting data

16.1. Export raw data

Raw data can be exported in ASCII format. The format is in columns with column 1 containing the time, column 2 containing the data of the first entry, column 3 the data of the 2nd entry, ... The columns are separated by "white space". You can chose to export either all data or only between the green cursors. The default time/current units are seconds/Amperes but there is also the possibility to scale these to export either in ms/pA or ms/microA. The Biopatch ASCII format can be read by an analysis program of Biologic. The other options (autocovariance, average between cursors) are quite special and not further documented at this time.
Since Jan. 2006, you can export data also using cut and paste. Selecting View->Numbers the upper right window displays the selected traces as columns of numbers that can be copied into the Windows clipboard (right mouse button!) and pasted into Excel ot other programs. The data format of the numbers (i.e. time in seconds or milliseconds, current in A or pA or microA, or raw data) is defined in the Settings->Export/Table scalings dialog.

Index	Up to '16. Exporting data'

16.2. Export analysis results (mean, tau, peak, etc)

no help yet, sorry.

Index	Up to '16. Exporting data'

16.3. Export noise analysis

You can export the results of the noise analysis by File->Export Analysis->Export Var-Mean. The results are written on a plain white-spaced ASCII file as columns. If you answer "YES" to the "Write trace?" question, the first three columns of the file contain the time, mean current (in pA), and mean variance values (in pA^2) of the whole trace. Note that the background variance has NOT been subtracted yet from the variance values. The next three columns contain: mean current of the segments, mean variance of the segments, and standard deviation of the variance. The background variance is written in the last (single-valued) column.

Index	Up to '16. Exporting data'

16.4. Export spectrum

no help yet, sorry.

Index	Up to '16. Exporting data'

16.5. Export amplitude histogram

You can export the results of the amplitude histograms analysis by File->Export Analysis->Amplitude Histogram. The results are written on a plain ASCII file as columns with "white space" separating the columns. Column 1 contains the current bins. Column 2 contains the data. Column 3 contains the Gaussian fit. Column 4 contains the parameters of the Gaussian fit in the order: mean, sigma, amplitude, area (in %).

Index	Up to '16. Exporting data'

16.5. Export dwell time histogram

You can export the results of the dwell time histogram by File->Export Analysis->ExportDwellTimeHisto. The results are written on a plain ASCII file as columns with "white space" separating the columns. The data for the various levels are written on top of each other, separated by a blank line. Column 1 contains the time bins. Column 2 contains the data and column 3 the fit (if present) with the sum of exponentials.

Index	Up to '16. Exporting data'

17. Misc.

Index	Up to '17. Misc.'

17.1. Changing the pulse protocol ("Stimwave")

no help yet, sorry.

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18. Appendix

Index	Up to '18. Appendix'

18.1. Supported data file types

Below is a list of files that can be read by Ana. For Axon binary files (abf-files) it was sometimes difficult to get the correct gain-settings. For this reason, the "abf-gain-factor" has been introduced in the Parameter Settings that allows the user to multiply the default-read gain by a factor in order to get the correct amplitude, but this should not be necessary anymore. For all files you can a posteriori change the gain in the Edit->SeriesParameters for each series individually (maybe tedious).

- HEKA Pulse Version 7: Select the ".dat" file (the pul and pgf must of course be present)
- HEKA Pulse Version 8: Select the ".dat" file (the pul and pgf must of course be present)
- Axon abf
- pclamp Clampex 5.5.
- pclamp Fetchex 5.5.
- Biologic DTR file (digital tape recorder)
- Atari Pulsed (Affolter), dataversion 200
- Pulse++ of Uli Rexhausen (only version 1.2)
- GePulse: data files generated by the free acquistion program written by M. Pusch (for National Instruments PCI-6035E and PCI-6052 and now also many other NI interfaces and Instrutech ITC-16 and ITC-18 interfaces). Download here.

If you are interested in reading YOUR favorite data file, send me a documentation and an example and I will try my best.

Index Up to '18. Appendix'

18.2. The internal data-format of ana: what is saved on a file?

All the original data and the performed analysis is saved on the file, but not the parameter settings. For a rough description of the file format, see below. If you are interested in a precise description, see Data-Format (detailed). If you select File->Save As Ana proposes as default a name that is distinct from the original data file name in order to avoid that you overwrite it. The "standard" but not required extension is "EAT".

The data file (version 6.0) is written by the following procedure: The 2 procedures used are:

WriteBytes(CFile * fp, UINT pos /* file positions */, UINT NumberOfBytes, BYTE * variable); and

"WriBy" is a macro that writes a variable in binary format using the size of the type as the number of bytes.

WriteBytes(fp, pos, 64, Version);
WriteBytes(fp, pos, 12, MainUnits);
WriBy(ADCConversion)
WriBy(NumberOfChannels)
WriBy(Temperature)
WriteBytes(fp, pos, 256, buff);
if (versionnumber>4){
RootText.WriteOnFile(pos, fp); // Writes the text assigned to the root. The size depends on the string-length (if any).
}
WriBy(NEntries)
WriBy(NSeries)
for (__int32 i=0; i<NEntries; i++){
WriBy(entries[i].NData)
WriBy(entries[i].FilePos)
WriBy(entries[i].P4Present)
WriBy(entries[i].Temperature)
WriBy(entries[i].Time)
WriBy(entries[i].IsBold)
WriBy(entries[i].s1) // unused variables
WriBy(entries[i].s2)
WriBy(entries[i].s3)
WriBy(entries[i].s4)
if (VersionNumber>4){
entries[i].EntryText.WriteOnFile(pos, fp);
}
} // end for entries
for ( i=0; i<NSeries; i++){
WriBy(series[i].VHold)
WriBy(series[i].Filter)
WriBy(series[i].StimInterval)
WriBy(series[i].SampleTime)
WriBy(series[i].Gain)
WriteBytes(fp, pos, SERIESCOMMENTSIZE, series[i].Comment);
WriBy(series[i].firstrec)
WriBy(series[i].NPulses)
WriBy(series[i].NumAveraged)
WriBy(series[i].P4Present)
WriBy(series[i].P4VHold)
if (VersionNumber>4){
series[i].SeriesText.WriteOnFile(pos, fp);
}
WriBy(series[i].NSegs)
for (int k=0; k<series[i].NSegs; k++){ //MaxSegs; k++) {
WriBy(series[i].Pulses[k].A)
WriBy(series[i].Pulses[k].T)
WriBy(series[i].Pulses[k].SV)
WriBy(series[i].Pulses[k].ST)
WriBy(series[i].Pulses[k].FV)
WriBy(series[i].Pulses[k].FT)
WriBy(series[i].Pulses[k].SK)
WriBy(series[i].Pulses[k].s1)
WriBy(series[i].Pulses[k].s2)
WriBy(series[i].Pulses[k].s3)
WriBy(series[i].Pulses[k].s4)
}
WriBy(series[i].IsBold)
WriBy(series[i].s1)
WriBy(series[i].s2)
WriBy(series[i].s3)
WriBy(series[i].s4)
WriteNoise(fp, pos, i);
WriteSpectrum(fp, pos, i);
series[i].Histo.Write(fp, pos);
series[i].VM.Write(fp, pos);
series[i].Lore.Write(fp, pos);
series[i].SF.Write(fp, pos);
series[i].DwellTimes.Write(fp, pos);
for (int kk=0; kk<19; kk++){
BOOL dummy=FALSE;
WriBy(dummy)
}
}
for (i=0; i<NEntries; i++){
WriteBytes(fp, pos, entries[i].NData*2, entries[i].IntData);
if (entries[i].P4Present) {
WriteBytes(fp, pos, entries[i].NData*2,entries[i].P4Data);
}
if (versionnumber>2){ // Write Fit and Results
WriteFit(fp, pos, i);
results[i].Write(fp, pos);
for (int kk=0; kk<20; kk++){
BOOL dummy=FALSE;
WriBy(dummy)
}
}
}

Index Up to '18. Appendix'

18.3. Syntactic rules for the user-defined function

The independent variable can be written as "v" or as "t".
Parameters are a[0], a[1], etc. The index, i, in a[i] must be a constant number (i.e. not an expression).
Arbitrary nested expressions (with parenthesis) are allowed.
Spaces are ignored. No distinction between upper- and lowercase is made.
Allowed functions are:
a+b
a-b
a*b
a/b
a^b
exp(a)
exp10(a)=10^a
inv(a)=1/a
sqrt(a)=square root
log(a)=natural log
log10(a)=log base 10
sin(a)
cos(a)
fabs(a)=absolute value
step(a) = 0 for a<0; 1 else; (this can also be used to calc. the maximum and minimum: Max(a,b)=a*step(a-b)+b*step(b-a) ; Min(a,b)=a*step(b-a)+b*step(a-b) )

Comments can be added after the function definition after a '. Examples:

((a[0]+a[1]*exp(-t/[a[2]))^2)*1e-12 ' square of a single exponential

step(1-t)*a[0] + step(t-1)*a[1] ' = a[0] for t<1 and a[1] for t>=1

exp(a[0]+log(a[1]*sqrt(fabs(t-a[8]))))^(1-step(a[3]-t)) ' absurd example

Index Up to '18. Appendix'

User Manual for

Ana

Michael Pusch, Istituto di Biofisica, Genova, email: pusch@ge.ibf.cnr.it Last change: 23.08.2007

1. Introduction

1.1. Installation

2. Subwindows

2.1. Tree window

2.2. Traces window

2.2.1. Filtering traces

2.3. Results window

3. The relevant segment

4. Cursors in the "trace" window

5. Short-cut keys

6. Scaling the display of the traces

7.1. Editing parameters

7.2. Loading/Saving

7.3. Context sensitive parameters

7.4. Undoing a parameter settings change

8. Editing commands - The internal buffer

8.1. Undoing an erroneous operation : Exchange

8.2. Copying/pasting/cutting/deleting/merging traces and series

8.3. Permanent filtering

8.4. Undersampling

8.5. Calculating the average

8.6. Copying selected records

8.7. Concatenate entries

8.8. Deleting a stretch

8.9. Extracting a stretch

8.10. Overlay envelope

8.11. Swapping data and fit

8.12. Reverse time

8.13. Paste as fit

9. Trace fitting

9.1. Exponential fits

9.1.1. Simple exponential fit

9.1.2. Fit several traces simultaneously with the same time-constants for all

9.1.3. Other exponentials

9.1.4. Envelope

9.2. Polynomial

9.3. User defined

10. Series Fit

10.1. Boltzmann

10.2. Polynomial

10.3. User defined function

11. Leak, capacity, and line frequency (50/60 Hz) subtraction

11.1. Baseline subtraction

11.2. Leak subtraction

11.2.1. Subtract a scaled series

11.2.2. Subtract using a baseline segment where channels are supposed to be closed

11.3. Capacity correction

11.4. line frequency (50/60 Hz) subtraction

11.4.1 Automatic line frequency (50/60 Hz) subtraction

11.4.2 Manual line frequency (50/60 Hz) subtraction

12. Scaling traces

12.1 Scaling by a factor and add a constant

12.2 Scaling traces of a series to have a common value in a certain segment

13. Noise analysis (non-stationary!)

13.1. Selection of records and cursor settings

13.2. Variance-mean analysis

14. Single channel analysis

14.1. Amplitude Histogram and Gaussian Fit

14.2. Idealization

14.3. Manual editing of the idealized records

14.4. Dwell time analysis

15. Spectral analysis

16. Exporting data

16.1. Export raw data

16.2. Export analysis results (mean, tau, peak, etc)

16.3. Export noise analysis

16.4. Export spectrum

16.5. Export amplitude histogram

16.5. Export dwell time histogram

17. Misc.

17.1. Changing the pulse protocol ("Stimwave")

18. Appendix

18.1. Supported data file types

18.2. The internal data-format of ana: what is saved on a file?

18.3. Syntactic rules for the user-defined function

Michael Pusch, Istituto di Biofisica, Genova, email: pusch@ge.ibf.cnr.it
Last change: 23.08.2007