ACQ is run from within MATLAB. To run ACQ, first start MATLAB and type acq at the prompt. The program initializes its internal variables, sets paths to various files from a default configuration file (config/default.mat), allows the user to select a profile (user- dependent directories and parameters) and registers the MATLAB functions (\*.m files) present in the ./source directory as valid commands (these are the only commands recognized). The program may be exited with the command bye; this should close all windows associated with the program. Alternatively, use File - Quit from the menu.
The program window is designed to take the full area of a 1024x768 display. It should not be necessary to view any other windows. (When using the Multiclamp Commander from Axon/MDS, it is convenient to position it over the parameter display area as described below). The display is divided into a four functionally distinct areas. First, down the left hand side there is a margin that holds several control buttons (described below). Second, the top of the next column holds the command line area (white box), the message area (grey box), and the status area (labeled blue boxes). Third, below this is the parameter display area, which shows the current settings for the selected parameter sets. Fourth, the right hand side of the display contains the graphics, and is divided into 3 areas. The top area has 2 vertically stacked graphs that display the output data waveforms for the two digital-to-analog convertor (DAC) channels. The default waveform for a stimulus generation (the one that is played out in scope mode) is shown in red, and the remainder are shown in white. Below this are two graphs that are used for on-line analysis. Currently, they are not programmable, and some stimulus generation routines may use these graphs to summarize the stimulus. Below these are two (or more) large graphs, which display the response data and the recorded stimulus waveforms. When the data is collected in scope mode, or is otherwise not being stored to disk, it is displayed with green lines and the axes are shown in gray. When data is being collected and stored on disk, the display lines are white, and the background axes and grid are blue.
Commands may be given to the program either through the keyboard, or through selection of menu items and graphical interface elements. This section describes how to use the keyboard input. Commands entered at the keyboard are echoed in the field in the upper left hand corner of the screen (white area); when the window has focus, any characters entered at the keyboard should appear in that area. Currently, there is no text editor per se, but a limited editing function is provided by the backspace and delete keys. Commands are entered one to a line, and terminated with the Enter key. A command buffer history is provided, which is accessed through the Control-U (up or back in the history) and Control-D (down, or forward in the history) keys. Commands may be entered while the program is acquiring, but this will stop the current acquisition. Stopping the acquisition with the space bar is recommended, as the first character entered at the keyboard is sometimes, but not always, lost during acquisition (this appears to be a problem with the relative execution times of various routines). There are no editable text fields on the screen that can be accessed with the mouse; all fields are display only. Thus, if the window has focus, it should accept commands.
There are several buttons on the screen, appearing on the left hand margin. The PV button executes the preview function, which computes the stimulus waveforms and displays the results in the stimulus graphs. The scope button starts the program presenting the current stimulus waveform (the one indicated in red in the stimulus waveform window), but data is not stored. This is useful for setting up to patch or search for a cell, or exploring changes in stimulus parameters. The stop button ends scope mode, and also ends any data acquisition, including acquisition initiated by macros. The next button is take 1, which causes the program to present one stimulus and collect the data.
Below these are 4 buttons that are used mainly during initial patching and when switching amplifier modes. The first is the VC-S button, which loads the s.mat protocol. This protocol, designed to be used in voltage-clamp, holds the electrode at 0 mV, with a brief step to -10 mV. The second is the VC-I button, which loads the i.mat protocol. The only difference here is that the pipette is held at -60 mV, and stepped to -70 mV. This is useful just prior to breaking into a cell, so that the break-in doesn’t cause a large shift in membrane potential. Holding the pipette at a negative potential prior to break-in also sometimes helps improve the seal resistance. The next button is the CCI button. This is used in current-clamp mode to provide a short hyperpolarizing current pulse, to check input resistance and time constants once the cell has been ruptured and you are in whole-cell mode. Note that you have to switch the amplifier to current clamp before using this mode. The SW button located below the CCI button tries to coordinate a series of steps (turning off current injection, switching amplifier modes, loading the new protocol, and then enabling current or voltage commands) that minimize disruption of the cell during the transition between current and voltage clamp, as well as from voltage to current clamp.
In this section, the operations of a typical acquisition session is described. Further details on commands are described below. A menu- driven mode is also incorporated, and is described later. First, it is necessary to open a data acquisition file. In the white command entry box, enter aopen. This opens a file to store the results of the acquired data. The file name is determined the same way as in DATAC: the name is formatted as ddmmmyyl.mat, where dd is the current date (leading zero), mmm is the 3-letter abbreviation for the current month, yy is a 2-digit year, and l is a-z, corresponding to the sequence of the file within the current date. It is recommended that this structure be adhered to. The program will display a new window with several fields that should be filled in to describe the overall experiment. The last field is a line for the user (experimenter) to sign. If this is the first acquisition of the day, then the fields will be filled with default values. However, if it is not the first file of the day, then the fields will be filled with the values from the previous file in the sequence and you should modify these as necessary. Click on OK to enter the dialog fields to the data file and close the box. You are now read to begin acquisition. Before proceeding further, you should make sure that the amplifier and the acquisition program are synchronized with respect to the operation mode - either voltage-clamp (VC) or current-clamp (CC). If the mode needs to be switched, use the sw command to detect the amplifier configuration and set the mode switch to the correct mode. Note that at present, this is only important for the Axopatch 200 amplifier. Other amplifiers cannot be read.
While searching for a cell, or beginning the formation of a patch, you will need to operate in a search mode. Typically, this will utilize a short current pulse (or voltage step). Load the stimulus configuration with the g s (this Gets the search file which is called s.mat and resides in the StmPar subdirectory; it also retrieves the acquisition parameters, which are contained in the same the file). Using the mouse, hit the scope button in the upper right hand corner of the display, or type sco in the command window. This begins a cycle of stimulus generation and display in an oscilloscope-style mode that can be used to monitor the electrode resistance or the formation of a patch. Once a seal is obtained, hit the stop button, and get the stimulus file to begin the intracellular acquisition (typically, g i (intracellular)). Once intracellular access is obtained as indicated by the capacitative charging transients and a change in the input resistance and holding current, one is ready for data acquisition.
A stimulus file/acquisition combination file can now be loaded with the g filename command. For example, a current-voltage relationship might be generated using a standard IV protocol, which is loaded with g iv. The command seq then will compute the stimuli and collect the data for an IV, using the parameters in the stimulus display - for an IV, cycling through the voltage steps. During the acquisition, the data will be displayed on the screen (assuming that the acquisition parameters are set to permit this), and the message box will show information about the progress of the acquisition. You may then get other protocols with the g command. Note that stimulus files may include acquisition information internally, or may externally reference a separate acquisition file.
You may also execute several stimulus protocols in sequence, using the do xyz abc command. The do command loads each protocol in order, makes sure the waveforms are current with the parameters, executes a seq, and when the acquisition is done, reloads the protocol that was in effect when the do command was entered, and goes into scope mode. Notes regarding the experiment may be added with the note command. This brings up an editing window, in which you may type a note of arbitrary length. The notes are meant to provide supplemental information, such as when valves are manually changed to apply drugs to a cell, or to indicate information about the status of the cell, that are either necessary or helpful for subsequent analysis. It is recommended that the notes be used generously; they form an important part of the experimental record and can really help untangle the acquisition session in data analysis that make take place years after the data was originally collected. Notes should also point out other files associated with this one, such as imaging data.
The data file is closed with the aclose command. Typically, there is only one cell to a data file (appropriate notes should be added if this is not true), and most cells will have only one data file, although exceptionally long protocols, program crashes, or lengthy recordings may result in several files holding data for one cell.
Because the amplifier gains for current and voltage clamp commands are often different, the stimulus files are keyed to the acquisition mode. The telegraph outputs of the AxoPatch (but not the Multiclamp) series of amplifiers are interpreted by the program to insure that the modes of the amplifier and the intended stimuli are synchronized. If the modes were not synchronized, it would be possible to load a voltage- clamp stimulus file while doing current-clamp recording (or vice- versa), and this very likely would damage the cell. The next two paragraphs provide explicit instructions on how to change modes for the two different sets of amplifiers. You cannot change modes for the AxoProbe amplifier, since it is current-clamp only.
To change the acquisition modes between voltage and current clamp, use the sw (switch) command; this command will wait for you to change the mode switch, and when it detects the change, it will load a default protocol for that mode (specified in the config structure). When setting the default modes, make sure that they have appropriate holding and step sizes for the configuration of the cell/patch that you want. After switching, which should leave you in a safe configuration, you will nearly always load new stimulus protocols associated with the new mode to collect additional data. To do this properly, you should switch to the I=0 setting and wait for the program to recognize the change (there will be an informative display message), before switching the the target mode.
These amplifiers can communicate with the computer via a USB/serial interface. We have an intermediate module that talks to the Multiclamp (the “Multiclamp Server”) and is accessible from MATLAB via a TCP connection. This module allows us to read the amplifier configuration, and update internal parameters, as well as control the amplifier settings. Switching modes in this case involves changing to a protocol with the new mode. No other user interaction is required.
With other amplifiers, we cannot read the mode of the amplifier. Therefore, switching must be done differently. The following sequence of commands will ensure that you are presenting stimuli that are safe to the cell at all times:
Simply reverse this sequence in order to go back to the other mode.
Access to the data in the structures is accomplished via commands from the ACQ command window (the white box in the upper left corner of the main window). To be successful, this command must form the minimum unique sequence of letters that accesses the desired field, and does not collide with MATLAB command names that are registered with the program. The appropriate names are listed to the left of the data fields, and are case-insensitive. When these names have spaces, only the first part of the field is used.
Structure elements that take strings are simply entered (e.g., Name ivx). Structure elements that take single numbers can be entered in the same way. However elements that take more than one argument can be entered in two ways. One is to specify the index, followed by the value (e.g., level 2 -100, which changes the level of step number 2 to -100). The other is to specify the whole array, with a MATLAB-like syntax (e.g., level [-60 0 -60 -120], which changes all of the levels and creates a 4-level step). Some care must be used when entering these numbers in either format, to ensure that valid numbers are entered; there is minimal checking in the program at present. It is also important to be sure that the number of events matches. For example, in steps, the number of levels and durations must be the same. In acquisition, the number of channels, gains, filter settings, scale factors, etc., must all match.
One exception the the entry format just described is that used to specify sequences.
Sequences can be specified in several ways: as a MATLAB array; as a DATAC-style sequence; or as an m-file that returns appropriate values. Furthermore, the sequence may consist of several specified sequences (which in the case of the stimulus structure will be applied in a specific way to different control parameters), each using any of the above formats. Multiple sequences using the MATLAB array format in any of the individual cases must use an & sign between the sequences; this is not necessary if the sequence use only the DATAC-style. The resulting output represents the nested computation of the first sequence, cycled element by element against the second sequence, etc. The command seq permits the rapid collection of parametric data once basic timing has been set. The use of this command can reduce the number of macros that one might maintain on disk as well as permits some flexibility during the experiment as to how the data will be collected. The syntax for sequence is as follows: valuelist is a sequence of the form a;b/xc that lists the values that the variable will take. The argument c is optional. a;b/x will step from a to b with increments of x; it is equivalent to theMATLAB statement [a:a+x:b]. The statement a;b/xn makes xsteps from a to b. a;b/xl makes x steps with logarithmic spacing. a;b/xr is the same as a;b/xn except that the results are in a randomized order. a;b/xs is the same as a;b/xl except that the results are in a randomized order. Floating point values are permitted. When random presentation is done, the sequence is always the same (the same seed is always used). Similar sequences can be generated with MATLAB commands, for example linspace or logspace.
Sequences can be nested in another way also. Furthermore, if a file is specified for addchannel (e.g., for the second DAC output) or superimpose (summed with the primary DAC output), then the primary sequence will be repeated for each element of the sequences present in these stimulus blocks.