Cardio2D Manual
Information in this document is subject to change without notice. No part of this document may be reproduced or transmitted without the express written permission of Multi Channel Systems MCS GmbH. While every precaution has been taken in the preparation of this document, the publisher and the author assume no responsibility for errors or omissions, or for damages resulting from the use of information contained in this document or from the use of programs and source code that may accompany it.
Table of Contents Introduction About this Manual Welcome to Cardio2D and Cardio2D+ 1 1 1 Important Information and Instructions Important Safety Advice Guarantee and Liability Operator's Obligations Terms of Use for the Program Limitation of Liability 3 3 4 4 5 5 Software and Hardware Installation Installing the Cardio2D Software First Use of Cardio2D after Installation Data Acquisition Hardware 7 7 9 10 Cardio 2D Software The Cardio2D Program : Overview Control Window Control View Viability Labbook C
Cardio2D Manual iv Cardio 2D+ Analyzer Software The Cardio2D+ Analyzer Program: Overview Control Window Cardio2D+ MEA Window Cardio2D+ Field Potential Cardio2D+ ROI Selection Heartbeat Cardio2D+ HB Averager Analysis of Field Potential Duration 69 69 82 84 85 93 95 103 106 Troubleshooting Troubleshooting Error Messages 109 109 109 Theoretical Background Cardiac Action Potential 111 111 Appendix Technical Support Test Model Probe Contact Information 113 113 113 114 Index 115
1 Introduction 1.1 About this Manual This manual comprises all important information about the first installation of the hardware and software, and about the daily work with the instrument. It is assumed that you have already a basic understanding of technical and software terms. No special skills are required to read this manual.
2 Important Information and Instructions 2.1 Important Safety Advice Warning: Make sure to read the following advice prior to install or to use the device and the software. If you do not fulfill all requirements stated below, this may lead to malfunctions or breakage of connected hardware, or even fatal injuries. Warning: Obey always the rules of local regulations and laws. Only qualified personnel should be allowed to perform laboratory work.
Cardio2D Manual 2.2 Guarantee and Liability The General conditions of sale and delivery of Multi Channel Systems MCS GmbH always apply. The operator will receive these no later than on conclusion of the contract. Multi Channel Systems MCS GmbH makes no guarantee as to the accuracy of any and all tests and data generated by the use of the device or the software. It is up to the user to use good laboratory practice to establish the validity of his findings.
Important Information and Instructions 2.4 Terms of Use for the Program You are free to use the program for its intended purpose. You agree that you will not decompile, reverse engineer, or otherwise attempt to discover the source code of the software. 2.5 Limitation of Liability Multi Channel Systems MCS GmbH makes no guaranty as to the accuracy of any and all tests and data generated by the use the software.
3 Software and Hardware Installation 3.1 Installing the Cardio2D Software System Requirements Software: One of the following Microsoft Windows ® operating systems is required: XP, Vista, Windows 8.1 or Windows 7 (English and German versions supported) with the NT file system. Other language versions may lead to software errors. Hardware: Please read chapter "Hardware Acquisition Systems".
Cardio2D Manual Installing the Cardio2D Software Make sure to have a valid license for the Cardio2D software! Please ask license@multichannelsystems.com. You do not need a license for the Cardio2D software if you like to test the software for getting an idea of how it works and what is possible. You can test all functions, run the viability test to see your data, but you are not able to start an experiment. Please contact license@multichannelsystems.com to receive a valid license key.
Software and Hardware Installation 3.2 First Use of Cardio2D after Installation Double-click the Cardio2D program icon. The "Data Acquisition Settings" dialog appears. You have to select the data acquisition device, if more than one data acquisition is available. Select the "Sample Rate", the "Range" (when using the MC_Card only), and the gain as an intrinsic property of the connected amplifier. The "Resolution", the number of "Channels" and the "Driver Version" are displayed.
Cardio2D Manual For saving the settings as default, please enter "File" menu "Save Defaults". 3.3 Data Acquisition Hardware Caution: You have acquired a high performance data acquisition and analysis computer. Do not modify the system, do not install new hard- or software, or another operating system without asking MCS or your local retailer for advice. Cardio2D do not install virus scanners or firewalls because these programs are known to interfere with the data transfer to the hard disk.
Software and Hardware Installation Data Acquisition with the MEA2100-Systems The MEA2100 recording system is an all-in one solution consisting of headstage and interface board. The MEA2100-System with integrated amplification, data acquisition, online signal processing, and integrated stimulus generator. You can connect one or two headstages to the interface board. The MEA2100-System uses an USB 2.0 connector per headstage to transfer digitized data to any computer.
Cardio2D Manual The USB-ME128-System and the USB-ME64-System are in principle the same devices as the USB-ME256-System, but with 128 or 64 electrode channels respectively. Data Acquisition with the MC_Card The data acquisition computer with the with the internal data acquisition board MC_Card comes preinstalled and preconfigured by MCS for a flawless operation.
Software and Hardware Installation Data Acquisition with the USB-MEA256-Systems The USB-MEA256-System is a compact and portable stand-alone solution for MEA recordings with integrated amplification, data acquisition, and analog / digital conversion. The system acquires data from up to 252 electrode channels, four additional analog channels, and 16 digital IN / OUT channels. The digitized electrode data is transmitted to the connected computer via universal serial bus (High Speed USB 2.0).
Cardio2D Manual MC_Card 64: MEA_6Well_60MEA_200_30 MEA_8x8_100_10 MEA_8x8_200_30 MEA_8x8_1000_30 MEA_6x10_500_10 MEA_6x10_500_30 FlexMEA72_750_100 FlexMEA_6x6_300_30 Note: It is possible to connect one or two FlexMEAs! EcoFlexMEA_6x6_300_50 Note: It is possible to connect one or two EcoFlexMEAs! MC_Card 128: MEA_6Well_60MEA_200_30 MEA_8x8_100_10 MEA_8x8_200_30 MEA_8x8_1000_30 MEA_6x10_500_10 MEA_6x10_500_30 FlexMEA72_750_100 FlexMEA_6x6_30
Software and Hardware Installation USB-MEA64: MEA_6Well_60MEA_200_30 MEA_8x8_100_10 MEA_8x8_200_30 MEA_8x8_1000_30 MEA_6x10_500_10 MEA_6x10_500_30 USB-MEA256: MEA_16x16_100_30 MEA_9Well_256MEA_300_30 USB-ME32 EcoFlexMEA_6x6_300_50 FlexMEA_6x6_300_30 USB-ME64: MEA_6Well_60MEA_200_30 MEA_8x8_100_10 MEA_8x8_200_30 MEA_8x8_1000_30 MEA_6x10_500_10 MEA_6x10_500_30 FlexMEA72_750_100 FlexMEA_6x6_300_30 Note: It is possible to connect one or two
Cardio2D Manual USB-ME128: MEA_6Well_60MEA_200_30 MEA_8x8_100_10 MEA_8x8_200_30 MEA_8x8_1000_30 MEA_6x10_500_10 MEA_6x10_500_30 FlexMEA72_750_100 FlexMEA_6x6_300_30 Note: It is possible to connect one or two FlexMEAs per 68-pin MCS standard connector! EcoFlexMEA_6x6_300_50 Note: It is possible to connect one or two EcoFlexMEAs per 68-pin MCS standard connector! USB-ME256: MEA_6Well_60MEA_200_30 MEA_8x8_100_10 MEA_8x8_200_30 MEA_8x8_1000_30 MEA_6x10_50
4 Cardio 2D Software 4.1 The Cardio2D Program : Overview Main Window: Overview The main window of Cardio2D consists of two in case of several connected MEAs of three window panes. The left "Control Window" is for controlling and documentation of the experiment. The bigger window on the right side, the "MEA Window" displays heartbeats in different characteristics in real-time monitoring. The "Summary" window is only available, if more than one MEA is connected.
Cardio2D Manual Control Window The experimental protocol is visualized by three icons on the left orange control view. The icons can be clicked for editing the protocol. Below the control view the log file documents the ongoing experimental proceedings. Summary Window The "Summary" window is only available, if more than one MEA is connected. It presents an overview about the layout of the MEAs which are connected and you can select or deselect the recording of connected MEAs.
Cardio 2D Software Main Menu File Menu to open or save templates, to restore and save defaults, and to exit the program. If you design experiments for a certain purpose, and you want to reuse the experimental schedule, please save the file as a template. You can reuse the template file as often as necessary. You can save as much templates as needed. You can also save local and global settings as a template. The inscriptions appear as soon as you open the template and there is no need to fill in again.
Cardio2D Manual Tools Menu to activate the offset correction, if necessary. Enable the option "Offset Correction" and the following dialog appears. An offset correction may be necessary, if you observe disturbing voltage offset on the input channels. Click the check box "Correct Offset" and Cardio2D takes 100 ms of the recorded data in the moment when the check box is selected to calculate the DC offset.
Cardio 2D Software Click "Data Acquisition" in the Settings menu. The "Data Acquisition Settings" dialog opens. This dialog is the same which shows up before starting Cardio2D for the first time. Please see above. Please enter the drop down menus to select the current data acquisition. In dependency of the data acquisition device you can modify the "Sample 'Rate" and the "Range". The "Resolution", "Number of Channel", and the "Driver Version" will be displayed.
Cardio2D Manual Design of the MEA window when connecting three MEAs, and using a MEA with a 6 x 10 layout grid in MEA_3 tab. Design of the MEA window when using one EcoFlexMEA36 with a 6 x 6 layout grid. Design of the MEA window when using a 256MEA with a 16 x16 layout grid.
Cardio 2D Software Click "View Summary Page" to open the "Summary" window. This window is only available, if more than one MEA at a time is connected. The window presents an overview and all connected MEAs are visible. The dark green color code spotlights the MEA which is displayed in the MEA tabbed page. The layout of the MEA is displayed respectively. You can uncouple the "Summary" window to move it to a different place on the monitor.
Cardio2D Manual In this example the "Summary" page shows 60-6wellMEAs with adapted color map. Help Menu for starting the Cardio2D online help, to monitor the dialog "About Cardio2D", and to enter the license key. For a valid license please read chapter "Installing Cardio2D Software". Select "About Cardio2D". The dialog shows basic information about the version of Cardio2D.
Cardio 2D Software 4.2 Control Window The "Control Window" is divided into two window panes. The "Control View" controls the experimental protocol, and the "Log File" documents the experimental proceedings.
Cardio2D Manual 4.3 Control View In the Control window you can enter the settings for setting up and running an experiment. If more than one MEA is connected each MEA is represented via a tabbed page in "MEA Window". In "Control View" it is possible to set different parameters for each MEA and to start and stop all MEAs simultaneously.
Cardio 2D Software 4.4 Viability Click Viability. The "Viability" icon changes into "Stop" icon , and the duration of the viability test is counted up in hours, minutes and seconds. Stop the viability test manually by clicking on "Stop" icon. Running a viability test Before you start recording, a viability test is recommended. The data is not recorded. The viability test monitors whether the MEA electrodes are acceptable for recording, and the cell culture or tissue is fine.
Cardio2D Manual For example: Type the desired settings into the "Study" tabbed page of the global labbook selected by the icon in the "Control View". Confirm the entry with "Enter". During writing the background of the input field will be orange, after confirming the window is white again. Close the global "Labbook Settings" dialog. Open the local labbook via icon in the MEA window. The local settings will be changed.
Cardio 2D Software 4.6 Compound / Basic Compound Click "Compound" in main menu "Experiment". The "Basic" icon is replaced by the "Compound" icon. Click the "Compound" icon . The "Compound Settings" dialog appears. In this dialog you can choose series of compounds which are applied to the cardiac cells or tissue on the MEA. In parallel to the "Basic Settings" dialog the options referring to the recording phases are available. Please see above.
Cardio2D Manual In the control window beneath the "Compound" icon, the currently applied dose of compound is displayed. When clicking on the "Compound" icon the "Set Marker" dialog appears which is called here "Apply Compound" dialog In the header, the next dose to apply is additionally displayed. Type your comments in the "Comment" window. Basic Click "Basic" in main menu "Experiment". The "Compound" icon is replaced by the "Basic" icon. The recordings are counted up and displayed. Click Basic.
Cardio 2D Software There are different ways to influence the recordings. 1. Select "Timer" in the "Switch" drop down menu and define the phase duration (1 s to 30 min) from the "Duration" drop down menu. Start the recording manually. A new recording phase will start automatically after the predefined recording interval is finished. Stop the recording manually whenever it is reasonable. Timer: The recording time span is fixed. 2. Select "Timer" and define a "Duration" of a recording phase.
Cardio2D Manual Apply Phase: The recording time is user defined. The data of the recording phase is saved, the data of the apply phase is excluded from the data file. The log file on the left side of the window shows following entries: 5. Enable the check box "Apply Phase" and the check box "Record". Start the recording manually. The first phase of this experiment is the "Apply" phase and the dialog "Set Marker" show up. Write a comment if necessary and manipulate the experiment.
Cardio 2D Software 4.7 Stimulation of MEA Electrodes Click "Stimulate" icon . The stimulation feature gives the possibility for voltage driven stimulation of a MEA electrode. This is useful, for example, if a cardiomyocyte culture did not form a pacemaker. This dialog allows to connect a stimulus generator STG, and to design the stimulation pulse. You can connect either one stimulus generator STG with more than one channels, or several STGs to stimulate the different MEA amplifiers.
Cardio2D Manual Stimulus Pulse In the "Stimulus" menu you design the stimulus pulse. The pulse shape of a stimulus pulse is usually biphasic symmetric rectangular. The parameters are the negative or positive sign of the first pulse phase, the duration of each phase, and the amplitude of the pulse. Click the check box "Symmetric", and the pulse will automatically be symmetric and biphasic. To design the pulse shapes, please use the numeric up-down controls "Phase 1" and "Phase 2".
Cardio 2D Software When using the blanking feature, please enable the "Sync Out" check box. You have to define the time of "Extension". The TTL pulse for the blanking signal starts synchronal to the stimulus pulse, but it should last longer to avoid stimulus artefacts. This time interval is called extension time. The extension time varies between 0 and 1000 μs, the default is 300 μs. In some cases it may be necessary to see the stimulus artefacts.
Cardio2D Manual MEA_Select MEA_Select is a software program for controlling the blanking circuit of the MEA amplifier. With MEA_Select it is possible to select the MEA electrode you want to stimulate. Start MEA_Select parallel to Cardio2D. The MEA_Select main window appears. When using more than one MEA amplifiers, in "Port" drop down menu, for example, COM1 defines MEA amplifier 1, COM 2 belongs to amplifier 2 and so on.
Cardio 2D Software 4.8 Stimulation with MEA2100-System Click "Stimulate" icon . When working with Cardio2D and any type of MEA2100-Systems with integrated stimulus generator STG the "Stimulate" dialog provides the possibility of stimulating via three different stimulation patterns on user defined electrodes. The dialog mirrors the layout of the currently used MEAs, in this case a MEA2100-2x60-System with two slots for two 60 electrode MEAs. Use the tool bar thereunder to define the stimulation sites.
Cardio2D Manual Second section: Click one of the three color coded "Select Stimulus" icons to select the respective stimulus pattern for further processing. You can define the location of the selected stimulus by clicking on electrodes in the electrode panel or by drawing rectangles over the desired area. Each stimulation electrode accepts one stimulus pattern at a time. It is not possible to overlap patterns, but to overwrite them with a new selection or to clear the sites.
Cardio 2D Software Stimulation Pattern To open the "Define Stimulus 1" dialog, please select the first "Select Stimulus 1" icon. The "Define Stimulus" icon in the "Stimulation" window will be available. Each stimulus can be defined independently, so the dialog corresponds with the previous selected stimulus pattern 1, 2 or 3. The dialog for the settings of the stimulus patterns are build analog, here, for example the "Define Stimulus 1" dialog.
Cardio2D Manual Building Stimulation Patterns On the left side of the upper section you can choose the provided default stimulus pattern. Move the primitive icon via drag and drop into the white field in the middle of the dialog for creating a user defined stimulation pattern. Add as much primitives as needed. It is possible to change the sequence of the primitives by drag and drop. For deleting one of the pattern, please drop it into the bin.
Cardio 2D Software Modulation of the provided Stimulation Patterns Initially the provided stimulation patterns are default. To adjust a primitive to your requirements, please click onto the icon, which will be highlighted in pale blue. In the upper part of the dialog the setting parameters for this pattern appears on the left and a picture of the actual shape of the pattern on the right.
Cardio2D Manual Stimulus Pattern: Sine Wave Modulate the amplitude "PP Amp (mV)", the period "Period (μs), the shift "Shift" and the phase "Phase (') of the sine wave pattern via up-down boxes. Setting the number of cycles to more than one enables the inter stimulus interval "ISI (μs)" up-down box. Additionally the “Arrow” button will be enabled.
Cardio 2D Software Stimulus Pattern: Rectangle Modulate the amplitude "Amplitude (mV)" of the ascending and the descend part of the pulse and the duration "Duration (μs)”, of the rectangle pulse via up-down boxes. Setting the number of cycles to more than one enables the inter stimulus interval "ISI (μs)" up-down box. Additionally the “Arrow” button will be enabled.
Cardio2D Manual Stimulus Pattern: Ramp Modulate the amplitude "Amplitude (mV)" and the duration "Duration (μs)” of the ramp pulse via up-down boxes. To adjust the steepness of the arms of the pulse, please use the three updown boxes for the ascending part, the plateau and the descend arm independent from each other. Setting the number of cycles to more than one enables the inter stimulus interval "ISI (μs)" up-down box. Additionally the “Arrow” button will be enabled.
Cardio 2D Software Stimulus Pattern: Biological Pulse If you like to use, for example, a biological signal as stimulus pulse, you can import signals by clicking the “Import” button. The imported file must mandatory have the following format: Timestamp Voltage Value Timestamp Voltage Value Timestamp Voltage Value ... The unit for the timestamp is μs and for the voltage value nV. The units are not part of the file.
Cardio2D Manual When using more than one cycle you can click onto the arrow symbol for one additional option: A highlighted parameter field appears to modulate the “Amplitude (%)” in relationship to each circle of the signal. The window "Single Pattern" shows the imported pulse as overlay plot. The lower window "Complete Stimulation Pattern" shows the combined sequence of all stimulus pulses. Tool Bar The settings in this tool bar influence the complete sequence of the stimulus patterns.
Cardio 2D Software Analyzer Settings Click "Analyze" icon . The "Analyzer Settings" dialog will open. Click "HB Detection" tab. As in the "Labbook" settings the same is in "Analyzer" settings: You can have local settings on each MEA or each well of a MEA or you control all MEAs at a time via global settings. The global settings (white background) overwrite the local settings (orange background). In this dialog you define the parameter for detecting the waveform of a field potential.
Cardio2D Manual The drop down menu "Min/Max Type" describes the type of the minimum or maximum "Local" or "Global". These options refer to one single field potential signal: "Local" means the first detected minimum or maximum, "global" means the deepest minimum or highest maximum in the field potential within the Min./Max. search interval. Please see the picture.
Cardio 2D Software Click "2D" tab in Analyzer Settings. In this dialog you can define the time span from the drop down menu in a range of 1 to 200 ms in which the "Propagation Time" should be calculated. The action potential runs over the heart cell culture or tissue or whole heart from the pacemaker to the peripheral regions of the syncytium. The excitation propagation is measured in high spatial resolution by the MEA electrodes detecting cardiac field potentials and extracting local activation times.
Cardio2D Manual 4.9 Start / Stop Start Click Start to start recording. The "Start" icon changes into "Stop" icon , and the duration of recording is counted up in seconds. Stop recording manually by clicking on "Stop" icon. Cardio2D generates three types of files, the Raw Data file (*.c2d), the Result file (*.c2r), and the Settings file (*.c2s). Raw data file and the associated settings file always belong together, in the result file the settings file is included.
Cardio 2D Software 4.11 Summary Window Click "View Summary Page" in main menu "Settings" to open or to remove the "Summary" window. This "Summary" window is only available, if more than one MEA at a time is connected. The window presents an overview and all connected MEAs are visible. The dark green color code spotlights the MEA which is displayed in the MEA tabbed page. The layout of the MEA is displayed respectively. The screenshot for the example above was taken with a MEA2100-2x60-System.
Cardio2D Manual When clicking on the "Summary" button the window will be visible again. 4.12 MEA Window The "MEA Window" is divided into four window panes, all showing heartbeats in different characteristics. The settings made in MEA Window influence all four smaller windows respectively. The prominent "MEA Electrode" view displays electrode activity of all electrodes in the layout. The "Single Channel View" displays an user defined single channel to have a closer view.
Cardio 2D Software The "MEA Window" changes depending on the connected hardware. If you connect, for example, an USB-MEA256-System for data acquisition, you can use it together with a 256-9wellMEA and each of the nine wells is displayed and controlled separate in nine MEA window tabs, MEA A to MEA I. If you connect, for example, an USB-ME256-System for data acquisition, it is possible to connect up to four MEAs, MEA_1 to MEA_4.
Cardio2D Manual 4.14 Single Channel View Click field potential "FP" tab in single channel view. The single channel view allows a closer view to one of the MEA electrode channels. You can zoom to any channel by clicking on a channel in the MEA electrodes plate. The zoomed channel is displayed in the larger single channel view. The plot shows the field potential (μV) against the time (s). To scale the axes, please use the drop down menus.
Cardio 2D Software Click heartbeat "HB" tab in single channel view. The HB tab shows a diagram of the duration of the RR interval in ms if you select "RRI (ms)" from the drop down menu. In whole heart experiments the RR interval is defining one complete heartbeat from one ventricular depolarization to the next ventricular depolarization. 1/RR is the beating frequency of the heart. Any modulation of the beating frequency is an indicator for possible arrhythmia.
Cardio2D Manual Click average "Ave" tab in single channel view. The recordings of one user defined electrode channel are displayed in a color coded overlay plot. Each recording represents, for example one concentration indicated by a different color. The number of the recording is displayed beside the plot and the number of averaged segments are denoted in the brackets behind. Please specify the length of the averaged segments in "Analyzer Settings" "HB Average" tab.
Cardio 2D Software The diagram of Latency window shows the latency (μs) of the excitation calculated between the first and the last detected electrode as a function of the time (s) whereupon the time is the duration of the experiment from starting the recording. The actual value of the latency is displayed above the plot in red letters. The different recording phases are displayed color coded in overlay.
Cardio2D Manual 4.16 Color Map The color map displays the propagation of each heartbeat across the MEA. The isochrones are indicated by black lines in this screenshot, but the user can change their color in “Contour”. The colors map is represented as false color. Below the map the actual timestamp is displayed. In the screenshot above one well of a 256-9wellMEA with a 6 x 5 electrode grid is displayed. Different layout grids are possible, please see chapter “Data Acquisition Settings”.
Cardio 2D Software Click the "Propagation Path" icon to set the propagation path. The electrodes on the MEA are symbolized by circles: Black circles indicate a detection of a heartbeat on this electrode in the currently displayed cluster, white circles indicate no detection of a heartbeat in the currently displayed cluster, but the color map is completed by interpolation of data. Click the "Map Settings" icon . The dialog "Map Settings" appears. Select "Map" tab.
Cardio2D Manual Color Map: The usual used color map for cardiac issues is the "Jet Invers" map, but you can also choose "Hot, Cool or Grey" maps. To remove the colors from the map, click "Off". The isochrones will remain only. Contour: The drop down menu is for changing the color of the contour for the isochrones into "Black, Jet, Hot, Cool and Grey". Or you can remove the lines with "Off". The time step is defined in the dialog. Arrows: The feature is not yet implemented in this version of Cardio2D.
Cardio 2D Software Select "Data" tab in "Map Settings" dialog. Interpolation: In the "Map" tab of the "Map Settings" dialog the interpolation parameter refers to points between measured data. In the "Data" tab the interpolation parameter refers to the data, generating the map. Default is the interpolation method "Nearest Neighbor". Please see above. It is possible to interpolate "Missing Data" or "Missing Channel".
Cardio2D Manual 4.17 General Software Features Tracking Axes For tracking the axis manually, click on the axis and the mouse pointer will change into a hand To scroll the axes, please move the mouse with the hand pointer. For zooming the axes, press the CTRL key and keep it pressed while moving the mouse or the wheel of the mouse. The mouse pointer turns into a double arrow symbol . A right mouse click on the axes gives four options: "Edit", "Tracking Enabled", "Update Rescue Values", and "Zoom to Fit".
Cardio 2D Software Click the "Plot" icon . The following toolbar appears.
Cardio2D Manual Click "Edit" icon . The following dialog appears. Now it is possible to format the plot.
5 Experimental Proceeding 5.1 General Performance / Noise Level Please use the provided model test probe to test the amplifier immediately after installation. The test model probe is already mounted on the amplifier. It simulates a MEA with a resistor of 220 k and a 1 nF capacitor between bath and electrode. Use the Cardio2D program to record from the test model probe and to check the amplifier. This also presents an opportunity for you to learn about the program before starting a real experiment.
Cardio2D Manual 5.2 Detection Parameter of the Under the tab "HB Detection" in "Analyzer Settings" of the MEA electrodes plate Cardio2D program you can adjust the parameters for the field potential detection. Sometimes it will be necessary to play with this parameters until you find the best configuration for your experiment. Click "Analyze Raw Data" in menu "Tools" in Cardio2D+ program.
Experimental Proceeding The sodium current is detected by the rapid change in the field potential, indicated by the rise time. The rise time refers to the time required for a signal to change from a specified low value to a specified high value. Typically, these values are 10 % and 90 % of the step height. The output signal of a system is characterized also by fall time. If the rise time is chosen very short, the slope of the peak is very steep, if the rise time is longer, the slope will be smooth.
Cardio2D Manual Slope Calculation The calculation of the slope of each data point (P) is achieved by linear regression. This linear regression is performed within a time interval around each data point. Increasing the interval will help to reduce noise on the slope. The time interval around a data point P can be defined by the user in the "Analyze" dialog of the "QT-related Analysis" tab in Cardio2D+. Please read also chapter "QT-related Analysis".
6 Cardio 2D+ Analyzer Software 6.1 The Cardio2D+ Analyzer Program: Overview Main Window: Overview The main window of Cardio2D+ is divided in two main windows. On the left side is the "Control Window", and on the right side the "MEA Window" with the "Field Potential", "ROI Selection", "Heartbeat", "HB Averager", and the "QT-related Analysis" tabbed page.
Cardio2D Manual MEA Window The window on the right side is divided into four separate window panes and has five tabs: "Field Potential", "ROI Selection", "Heartbeat", "HB Averager" and "QT-related Analysis". The setting of the tabs influences each of the separate windows in the tab. Select "Field Potential" tab. Please see the upper picture. The single channel view on top of the right window panes shows field potentials: Recorded data traces are displayed in the MEA electrodes pane.
Cardio 2D+ Analyzer Software Select "ROI Selection" tab. Please see the picture below. This tabbed page lists the single recording sequences from one electrode. Select the electrode in the "Channel control" field of the "Control Window". Via ROI you can discard one part or several parts of the recording sequence which are now excluded from the analysis. Additionally it is possible to discard a complete recording sequence by deselecting the respective check box.
Cardio2D Manual The color map corresponds with the single channel view, and shows the excitation patterns of that heartbeat marked with the blue arrow in single channel view. Select "HB Averager" tab. Please see the picture below. Important: Please pay attention to the "Analyze" dialog. To use the average tool, you have to enable the check box in the "HB Average" tab of the "Analyze" dialog in main menu "Tools".
Cardio 2D+ Analyzer Software The "MEA electrode field" window corresponds directly with the "Channel overlay" view. The field potential of one selected channel or of several selected channels are shown in overlay in the "Channel overlay" window. Select the channel(s) via mouse click in the MEA electrode field. Select one channel from the "Heartbeat" window pane of the "Control Window" and the desired channel will be displayed with its different recordings in the "Recording Overlay" window.
Cardio2D Manual Select one channel from the "Heartbeat" window pane of the "Control Window" and the desired channel will be displayed with its different recording phases in the "QT-related Analysis" window on the left side. If possible, a dose responds curve of this channel will be calculated and displayed in the "QT-like Interval" window of the right side. You can choose whether you want to display a normalized or an absolute curve from the drop down menu.
Cardio 2D+ Analyzer Software Main Menu File Menu to open or save result files, to open raw data files, and to exit the program. Cardio2D generates different types of files, the Raw Data file (*.c2d), the Result file (*.c2r), and the Settings file (*.c2s). Raw data file and the associated settings file always belong together, in the result file the settings file is included. The log file is not automatically generated, but by users command.
Cardio2D Manual Click "HB Detection" tab in the "Analyze" dialog. In this dialog you define the parameter for detecting the waveform of a field potential. Please see also chapter "Automated Waveform Detection". Because you have to define these settings before recording data, the same dialog appears in Cardio2D (MEA electrode window, Settings"). "Analyzer The first parameter for detection is the "Rise Time". Select the minimum and the maximum of the rise time from the drop down menus.
Cardio 2D+ Analyzer Software The drop down menu "Min/Max Type" describes the type of the minimum or maximum "Local" or "Global". These options refer to one single field potential signal: "Local" means the first detected minimum or maximum, "global" means the deepest minimum or highest maximum in the field potential. Please see the picture.
Cardio2D Manual Click "HB Average" tab in "Analyze" dialog. If the heartbeat average feature is welcome, please enable the check box "On". If you do not use this feature, do not enable the check box to save computer performance. Select the "Recording" for the heartbeat average from the drop down menu. Define the time interval before a heartbeat "Pre HB Interval" and the interval after a heartbeat "Post HB Interval" from the up down boxes. During these time spans, no additional heartbeat should be detected.
Cardio 2D+ Analyzer Software Click "2D" tab in "Analyze" dialog. The first up down box in this tab of the dialog defines the minimal number of cluster channels. The "Min Cluster Channels" gives the minimum number of channels at which heartbeats have to be detected to form a cluster. For example, for a MEA electrode layout with 60 electrodes it could be 10, but if less than 60 electrodes are involved, it could be 5 or 2.
Cardio2D Manual Click "QT" tab in "Analyze" dialog. This dialog defines the parameters for the QT-related Analysis. QT-related Analysis is possible only if the heartbeat average is enabled. QT-related Analysis will always be performed on the interval from the heartbeat time stamp to the last data point of the average heartbeat.
Cardio 2D+ Analyzer Software Help Menu for activating the online help for Cardio2D+, and for displaying the dialog "About Cardio2D+". The dialog shows basic information about Cardio2D+.
Cardio2D Manual 6.2 Control Window Cardio2D+ The control window of Cardio2D+ is divided in three separate windows: The "Recording control" on top, the "Selection control" view and the "Settings file" below.
Cardio 2D+ Analyzer Software Recording Control of Cardio2D+ In the recording control window the name(s) of the currently analyzed Cardio2D raw data and / or result file is displayed. Additional the information about the recordings saved in this experiment are listed in a table. To select one recording phase to be analyzed, please click onto the desired row, which will be highlighted in dark blue and displayed in the single channel view of the MEA window.
Cardio2D Manual In this screenshot a MEA electrode grid 8 x 8 is displayed. Different layout grids are possible, depending on the selected raw data file. The numbers of the electrodes refer to their position in the grid, to rows and columns. Using a different type of a MEA, for example, the layout will be adapted. If you have different clusters of cells with different pacemakers on the MEA, you have to analyze each cluster separate.
Cardio 2D+ Analyzer Software 6.4 Field Potential Cardio2D+ Click "Field Potential" tab. The "Field Potential" tab is divided in four separate windows: The "Single channel view", the "MEA electrode field", the "Channel overlay" and the "Color map". Single Channel View The single channel view displays the field potentials of one recording phase of the MEA electrode selected in the "Channel control". With the "Zoom Box" icon Click the "Reset Axes" icon you can zoom to any region of the data trace.
Cardio2D Manual Click "Edit Plot" icon . A plot menu appears. With the help of the plot menu it is possible to modify a lot of plot parameters. For example, you can adapt the figure to a special layout wanted for a special publication. Please see also chapter "General Software Features". The two red bars in the heartbeat plot of the single channel view represent the region of interest ROI. Click on the middle of one red bar near to the zero line.
Cardio 2D+ Analyzer Software MEA Electrode Field Recorded data traces are displayed in the MEA electrode field. To adapt the y-axis, please use the drop down menu. The MEA electrode field corresponds with the overlay plot window. The data traces of the MEA electrodes, selected in the MEA electrode field, and highlighted in white are displayed in the overlay plot window in different colors. The MEA electrode field refers also to the single channel view.
Cardio2D Manual Channel Overlay If you select one or more than one MEA electrodes in the MEA electrode field, they are displayed in an overlay plot in the Channel overlay window. To adjust the y-axis, please use the drop down menu. There is a prominent red bar in the diagram. The function is different compared to the two red bars in single channel view, limiting the ROI. This red bar here defines the timestamp, belonging to the currently shown excitation patterns of the color map.
Cardio 2D+ Analyzer Software Color Map The color map displays the field potentials on the MEA electrodes at a time stamp, defined by the channel overlay display. It shows the excitation of a heartbeat from the pacemaker (or the earliest time point detected, if the pacemaker is not on the MEA) to the outer regions of the cardiac cell culture or tissue. The isochrones are indicated by black lines. The colors indicates the time distance between the first signal and outer regions of excitation.
Cardio2D Manual Click the "Map Settings" icon . The dialog "Map Settings" appears. Click "Map" tab. In this dialog you have to define the settings for the color map. Color Map: The usual used color map for cardiac issues is the "Jet Invers" map, but you can also choose "Hot, Cool or Grey" maps. To remove the colors from the map, click "Off". The isochrones will remain only. Contour: The drop down menu is for changing the color of the contour for the isochrones into "Black, Jet, Hot, Cool and Grey".
Cardio 2D+ Analyzer Software Auto: Click the check box "Auto" in "Maximum" option. The "Minimum" is zero, you are not allowed to modify it in Cardio2D.The Cardio2D program automatically finds for the maximum of each heartbeat, and adapts the scaling of the color map. It is recommended to use the auto scaling function. Size (W x H): You can modify the size of the color map. Interpolation: Interpolation is a mathematic method to calculate missing information between two points of a line.
Cardio2D Manual Click the "Record Movie" icon . The dialog "Movie Recorder" appears. The movie of the color map will be recorded in *.AVI format. The *.AVI format is a common format. You just need a Media Player to replay the video. You can choose the "Frame Rate" between one and hundred frames per second. Select "Compression" of the movie with "On", or deselect the compression with "Off". Save the movie by clicking the "Save" icon you want to store it.
Cardio 2D+ Analyzer Software 6.5 ROI Selection Click "ROI Selection" tab.
Cardio2D Manual The "ROI Selection" tabbed page lists the single recording sequences from one electrode. Select the electrode in the "Channel control" field of the "Control Window". Via ROI you can discard one part or several parts of the recording sequence which are now excluded from the analysis. Additionally it is possible to discard a complete recording sequence by deselecting the respective check box. The data in the ROI will not be included in the analysis.
Cardio 2D+ Analyzer Software 6.6 Heartbeat Cardio2D+ Click "Heartbeat" tab. The "Field Potential" tab is divided in four separate windows: The "Single channel view", the "MEA electrode", the "Latency" and the "Color map". Single Channel View The single channel view displays the amplitude and the beating rate of all heartbeats of one recording phase. Select the number of the displayed MEA electrode in the "Selection control" window of the "Control" window on the left side.
Cardio2D Manual Single channel view and the color map of the "Heartbeat" tab correspond with each other: The excitation pattern of that heartbeat marked with a blue arrow is exactly shown in the color map. Selecting a new heartbeat with the blue arrow, the color map will be adapted immediately. The user has four possibilities to analyse the heartbeats: Click near to any heartbeat, and it will be marked with the blue arrow. This way you can jump to any heartbeat of that recording phase.
Cardio 2D+ Analyzer Software Color Map Click the "Copy to Clipboard" icon to copy the color map to the clipboard. Click the "Save Picture" icon to save the color map as a picture in *.png format. Note: Stopping the real-time monitoring saves computer performance! The color map displays the spreading of the excitation of a heartbeat from the pacemaker to the outer regions of the cell culture or tissue. If the pacemaker is not on the MEA, the earliest point of detection is used instead of the pacemaker.
Cardio2D Manual When changing the propagation path from the minimum maximum default to manual, both displays in the "Latency / Velocity" window are immediately re-calculated and adapted. The electrodes on the MEA are symbolized by circles: Black circles indicate a detection of a heartbeat on this electrode in the currently displayed cluster, white circles indicate no detection of a heartbeat in the currently displayed cluster, but the color map is completed by interpolation of data.
Cardio 2D+ Analyzer Software Color Map: The usual used color map for cardiac issues is the "Jet Invers" map, but you can also choose "Hot, Cool or Grey" maps. To remove the colors from the map, click "Off". The isochrones will remain only. Contour: The drop down menu is for changing the color of the contour for the isochrones into "Black, Jet, Hot, Cool and Grey". Or you can remove the lines with "Off".
Cardio2D Manual Click the "Map Settings" icon Click "Data" tab. . The dialog "Propagation Map Settings" appears. In the "Map" tab of the "Map Settings" dialog the interpolation parameter refers to the color of the map. In the "Data" tab the interpolation parameter refers to the data, generating the map. Default is the interpolation method "Nearest Neighbor". Please see above. With the method of interpolation you can substitute missing channels also.
Cardio 2D+ Analyzer Software Click "2D" tab in Analyzer Settings. The setting for the "Propagation Path" is from minimum to maximum by default. If you like to define a propagation path manually, for example, when propagation is not linear but circular, please select the option "Manual Path". This option enables the option "Set Propagation Path" in the color map. Latency / Velocity Select "Latency" from the drop down menu.
Cardio2D Manual Select "Velocity" from the drop down menu. The diagram shows the velocity (cm/s) of the excitation in dependency of the time (s) between the minimum and the maximum color coded in overlay for each recording separately. This "Latency" and "Velocity" windows are directly linked to the "Single channel view", the "Color Map" and the file information table in the "Control Window". Click on one of the data points and the mouse pointer turns into a hand.
Cardio 2D+ Analyzer Software 6.7 HB Averager Click the "HB Averager" tab. The "HB Averager" tab is divided in three separate windows: The "MEA electrode field", the "Recording overlay " and the "Channel overlay" window.
Cardio2D Manual Important: Please pay attention to the "Analyze" dialog. To use the heartbeat average tool, you have to enable the check box in the "HB Averager" tab of the "Analyze" dialog in main menu "Tools". MEA Electrode Field Recorded data traces are displayed in the MEA electrode field. To adapt the y-axis, please use the drop down menu. The MEA electrode field shows the averaged heartbeats of all channels of the currently selected recording.
Cardio 2D+ Analyzer Software If you like to zoom the Natrium peak of the heartbeats for QT-like analysis, please click the icon "Na-Peak" . From the same recordings displayed before now the Natrium peak only is automatically displayed in this window. Channel Overlay In the "Channel Overlay" window the heartbeats of the different recording phases of one single channel are displayed in a overlay plot.
Cardio2D Manual 6.8 Analysis of Field Potential Duration The "Analysis of Field Potential Duration" is also called "QT-related Interval". The field potential duration corresponds to the action potential duration, which can be correlated to a QT-like Interval in an electrocardiogram. It is measured from minimum of the Na+ peak to the maximum / minimum of the IKr current peak. The "QT-related Interval" tabbed page until now divided in two separate windows.
Cardio 2D+ Analyzer Software Enable the check box "On" to use the QT-related Analysis tool. Select the "Detection Deadtime" in ms in the first up down box to define the time span in which no detection is performed. Detecting extremal values for QT-related Analysis is achieved by analysing the slope of the signal. Select the "Slope Calculation" in ms in the second up down box to define the time interval in which the slope of one point of the signal is calculated.
Cardio2D Manual Dose Response Curve The right side shows a diagram of the dose response curve, if the recorded data allow to calculate it. The curve is available in "Normalized" or in "Absolute" form, the x-axis is logarithmic. In "Normalized" plots the reference is not integrated in the plot. In "Absolute" plots of the dose response curve the standard reference of 0 μM is set to 1 μM and displayed in the logarithmic scale. Data Export to ASCII Click the "Plot" icon . The following toolbar appears.
7 Troubleshooting 7.1 Troubleshooting Most problems occur seldom and only under specific circumstances. In most cases, it is only a minor problem that can be easily avoided or solved. If the problem persists, please contact your local retailer. The highly qualified staff will be glad to help you. Please inform your local retailer as well, if other problems that are not mentioned in this documentation occur, even if you have solved the problem on your own.
Cardio2D Manual Your license key is not valid. Please contact Multi Channels Systems MCS GmbH. The minimum rise time has to be shorter than the maximum rise time. The maximum rise time has to be longer than the minimum rise time. The detection deadtime must be shorter than the shortest post heartbeat interval of the averager. The post heartbeat interval must be greater than the detection deadtime of the QT-related Analysis.
8 Theoretical Background 8.1 Cardiac Action Potential Over the last 30 years, non-invasive extracellular recording has developed into a widely used standard method. The semi-permeable lipid bilayer cell membrane separates different ion concentrations (charges) on the inner and outer side of the membrane. Conventional methods measure the membrane potential that results from the electrochemical gradient directly with an intracellular electrode.
Cardio2D Manual Electrocardiogram (Reference: "Estimation of Action Potential Changes from Field Potential Recordings in Multicellular Mouse Cardiac Myocyte Cultures", Marcel D. Halbach et al.
9 Appendix 9.1 Technical Support Please read the "Troubleshooting" part of the user manual or Help first. Most problems are caused by minor handling errors. Contact your local retailer immediately if the cause of the trouble remains unclear. Please understand that information on your hardware and software configuration is necessary to analyze and finally solve the problem you encounter.
Cardio2D Manual 9.3 Contact Information Local retailer Please see the list of official MCS distributors on the MCS web site. User forum The Multi Channel Systems MCS GmbH User Forum provides the opportunity for you to exchange your experience or thoughts with other users worldwide. Mailing list If you have subscribed to the Mailing List, you will be automatically informed about new software releases, upcoming events, and other news on the product line.
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Cardio2D Manual 116
Control Window Cardio2D+ A Automated Waveform Detection 73 Basic 31 Blanking Circuit BC 36 Blanking Pulse ...................................36 Extension ...........................................36 Sync Out .............................................36 TTL Pulse ............................................36 C Cardio2D 1 Cardio2D Program Overview 17 Cardio2D+ Analyzer 1 Cardio2D+ Analyzer Program Overview............................................75 Cardio2D+ Control window ................
Cardio2D Manual General .......................................................56 Study ...........................................................56 MEA Types 10 120MEA200/30 ..................................10 256-9wellMEA ...................................10 60-6wellMEA .....................................10 60MEA100/10 ....................................10 60MEA200/30 ....................................10 60MEA4Q-1000 .................................10 60MEA500/10 .................................
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