Step-by-Step: Analyzing Experiments in DMA

Aurora Scientific’s Dynamic Muscle Analysis (DMA) software allows users to analyze data obtained with ASI muscle contractility systems and Dynamic Muscle Control (DMC) software. DMA and DMA High Throughput (DMA-HT) allow users to automate the analysis of large data sets, such as those collected when performing force-frequency, force-time, position-time or fatigue analysis experiments. This technical blog will provide an overview on how to load data files for analysis in DMA, how to analyze multiple files at once, and the definitions of each of the analysis parameters.

Single File Analysis

There are a couple of different ways to analyze a single data file on DMA, depending on the location of your data.

Loading a Data File from LabBook Study

Open up the DMA software. From the main window, click “Get Data from LabBook Study”. This will open up the Select Study dialog box, which will list the names of each of your LabBook studies. Click on the study you would like to analyze, and then click “Select”. This will open up the Find Study Data dialog box, from which you can select the date that you collected the data. Animals with data on the selected date will appear in the bottom left of the dialog box. Once you select the animal of interest, each of the data files for the animal will appear on the right side of the dialog box. After selecting your data file of interest, click “Select Data”. The data file will populate in the main window.

Loading a Data File from a Folder

If you instead want to get data from a specific folder on your device, you can go back to the main window, and click “File”, then “Open ASI Data file”. You can then select a file from the location or folder of your choosing. Once selected, the data will populate in the main window.

Analyzing a Single Data File

Once your data file is loaded onto the main window, click “Muscle Analysis”. This will open the Muscle Analysis parameter box, listing all the parameters for both force and length analysis.

Parameter	Definition
½ Relaxation Time	The time it takes to go from peak force (for a twitch contraction) or from force at the end of stimulation (for a tetanus contraction) to 50% of that value.
Integration	The area under the curve. This relates to how much work is being done.
Maximum	The maximum force produced.
Time to Maximum	The time at which the maximum force occurs within the data file.
Minimum	The lowest force value within the graph. It is a single point in the data file where force is the least.
Time to Minimum	The time at which the minimum force occurs within the data file.
Maximum Derivative	Represents the slope of the ascending force trace at an instantaneous point.
Time to Maximum Derivative	The time at which the ‘Maximum Derivative’ occurs, or the time point the maximum rate occurs at.
Minimum Derivative	Represents the slope of the descending force trace, therefore representing relaxation rate at a single, instantaneous point.
Time to Minimum Derivative	The time at which the ‘Minimum Derivative’ occurs, or the time point the minimum rate occurs at.

High Throughput Analysis

DMA High Throughput Settings

To analyze multiple files simultaneously in DMA, open the program and click “High Throughput”. There are multiple drop down options in this menu. The “Force-Frequency Analysis”, “Force-Time Analysis” and “Fatigue Analysis” options are based on force profiles, whereas the “Position-Time Analysis” option is based on the length profile.

Once clicking on the high throughput option of your choice, for example, “Force-Frequency Analysis”, the DMA High Throughput window will open up.

Ensure that you select “Enable Filter”. This setting encompasses “Cutoff Frequency” and “Falloff Order” which filter out any signals in the data files higher than a certain frequency. Note, that the lower the Cutoff Frequency is, the smoother the contraction becomes.

In most instances, the following values are ideal:

Cutoff Frequency: 70 Hz
Falloff Order: 2

Select “Remove Baseline” to perform baseline correction. This means that the software will take the peak force, subtract the baseline, and get the amplitude.

Next, ensure that you select “Enable Automatic Inversion” for instances in which the force or torque would be negative, such as dorsiflexion in-vivo. This allows the software to automatically invert contractions into a positive orientation for proper analysis to be done. For all other instances, this can be left unchecked.

There are two options for Cursor Placement in DMA:

1. Manual

This allows you to define the timepoint where the start and end cursor will be placed for all the files to be analyzed. This option is particularly handy for eccentric contractions, where the cursors can be placed at the beginning and end of the tetanic plateau region, before lengthening occurs. When enabled, the Automatic Cursor Settings will disable, and the “Start Cursor” and “End Cursor” can be defined under the Manual Cursor Position tab.

2. Automatic

This allows the software to place the cursors around the contraction automatically. When selected, the Manual Cursor Position tab will disable, and the Automatic Cursor Settings can be defined.

Cursor Threshold: allows the user to adjust how the cursors are placed. Higher percentages means the cursors are placed further from the contraction whereas lower percentages place the cursors closer to the contraction.

Slope Threshold: refers to where in the file the cursors can be placed, and if the slope is changing by more than a certain percentage, the software cannot place them properly.

Baseline Deviation Notification Threshold: how much deviation there is between the starting baseline (i.e. pre-contraction) and end baseline (post-contraction) can occur before returning an orange “Baseline Deviation Error

Baseline Slope Notification Threshold: refers to the change in baseline over time. If the slope is rising or falling by more than the set percentage, it will return a yellow Baseline Slope Error.

Data Analysis Colour Legend

When data files are analyzed, they are coloured according to the results. The following table provides an overview of what each colour represents:

Analysis Result	Colour	Definition
Cursor Priority Failure	Blue	The automatic cursors did not place the start and end cursors in the correct order
Analysis Succeeded	Green	The analysis was successful
Baseline Slope Error	Yellow	The baseline changed by more than the set percentage on the Baseline Slope Threshold
Baseline Deviation Error	Orange	The baseline before and after contraction differ by more than the set percentage
Analysis Failure	Red	The software was unable to place the cursors around the contraction correctly
Manual Placement	White	The cursors were manually placed

Loading Files for DMA High Throughput Analysis

There are a couple of different ways to load multiple data files into DMA, depending on the device you are using.

1. Using DMA on the device that the data was collected on:

You can load studies directly from DMC by clicking “Get DMCv6 Study Data” on the main DMA High Throughput Window. This will open up the “Select Study” dialog box from which you can select the study, animals, and the data file that you want to analyze. You can select multiple files at once and analyze them together by selecting one or more animals, or by selecting a specific type of experiment, such as only tetanus experiments. Click “Select Data” to load them into the Results Table.

2. Using DMA on a separate device:

If you’ve transferred the data onto another device, such as a separate PC or laptop, you can go click either “Pick Files” or “Pick Folder” on the main DMA High Throughput window. You can then navigate the different locations on your device and select the data files from the location or folder of your choosing. Once the data files of interest are selected, click “OK” to load them into the Results Table.

Analyzing Multiple Data Files

Once the data files have been loaded into the Results Table, you are ready for the exciting analysis step. To proceed with analysis, click “Analyze” on the main DMA High Throughput window.

The program will automatically colour each of the data file rows according to the results of the analysis. The files will turn green if they were analyzed successfully, meaning that the parameter values were accurate and the contraction is clearly defined within the data file. Refer to the Data Analysis Colour Legend table above to learn about the different analysis results, their associated colours, and their definitions.

For example, if the row of one of your data is red, this often means that the software could not place the cursors around the contraction correctly. If you need to fix this, double-click the data file and move the yellow cursors around the contraction. Then click “Update Cursors to Table”. Upon updating, the data file will turn white, indicating that the cursors were placed manually.

Saving the Data

There are a couple of different ways to save the DMA data, depending on your preferences.

1. ASCII

To export and save the data as an ASCII file, click “Save Table to ASCII”. You can then save the data to the location of your choosing and load this table in Excel or other .txt compatible program on another Windows PC or laptop.

2. Excel

To export and save the data as an Excel file, click “Export Table to Excel”. You can then save the data to the location of your choosing.

Editing DMA Parameters

DMA offers several built-in parameters to provide useful information about the data. The following is an overview on how to edit the parameters, as well as the definition of each of the parameters.

To edit a parameter, open up the program and click “High Throughput”. Select the appropriate option from the drop down menu, for example, “Force-Frequency Analysis”. This will open up the DMA High Throughput window. Note that the default parameters will appear as column headings in the Results Table. To modify these parameters, click “Edit Columns”. This will open up the Column Editor window, which displays the Available Columns on the left, Column Type in the middle, and Selected Columns on the right. The parameters that are selected for analysis will appear under Selected Columns.

To add a parameter, select it from the Available Columns and click “Add This Column”.

To remove a parameter, select it from the Selected Columns, and click “Remove Highlighted”.

To edit the parameter, use the fields in the middle of the Column Editor window (Column Type, Column Heading, Desired %, Desired Start %, Desired End %, Count From, etc.) to adjust the settings.

Once you are satisfied with the modified parameters, click “Save Changes”. The column headings in the Results Table will then update.

Relaxation Parameters

When adding or editing relaxation parameters in particular, “Count From”, found in the middle of the Column Editor window, must be adjusted.

“Count From” should be set to “Peak Force” for data generated from twitch contractions and “End of Stimulation” for tetanic contractions. This is because twitch contractions involve only one stimulation pulse, therefore the relaxation point should be assessed from the peak of the contraction. On the other hand, tetanic contractions involve multiple stimulation pulses, therefore relaxation should be assessed from the final stimulation pulse.

For example, if you are analyzing Time to 50% Relaxation (1/2RT) of a tetanic contraction:

Update the name of the Column Heading to include the type of contraction. For example, “Tetanic Time to % Relaxation” or “1/2 RT Tetanus”
Enter “50” in the “Desired %” field
Ensure “Count From” is set to “End of Stim”
Click “Update Column”

Key Parameter Definitions

Parameter	Definition
Time to % Contraction	The time is takes to go from baseline to a defined percentage of the maximum force. To define this percentage, enter percentage under “Desired %”, then click “Update Column”.
Maximum Rate of Contraction	Equivalent to the “Maximum Derivative’” in Single File analysis. This is the slope of the ascending force trace at an instantaneous point.
Time to % of Maximum Rate of Contraction	The time point that the maximum rate of contraction occurs at.
Avg Rate of Contraction	Takes two points on the ascending curve and generates a slope between the two points. To define these two percentage, enter them under “Desired Start %” and “Desired End %”, then click “Update Column”.
Time to % Relaxation	The time it takes to go from peak force (for a twitch contraction) or force at the end of stimulation (for a tetanus contraction) to a defined percentage of that value. To define this percentage, enter percentage under “Desired %”, then click “Update Column”.
Maximum Rate of Relaxation	Equivalent to the “Minimum Derivative” in Single File analysis. This is the slope of the descending force trace at an instantaneous point. Note that “Count From” should be adjusted depending on the type of contraction.
Time to % of Maximum Rate of Relaxation	The time point the “Maximum Rate of Relaxation” occurs at. Note that “Count From” should be adjusted depending on the type of contraction.
Avg Rate of Relaxation	Takes two points on the descending curve and generates a slope between those two points, similar to average rate of contraction. Note that “Count From” should be adjusted depending on the type of contraction.

Step-by-Step: Analyzing Experiments in DMA

Single File Analysis

High Throughput Analysis

Further Reading