SarcOptiM (High Frequency Online Sarcomere Length Measurement)

Authors: Côme PASQUALIN - François GANNIER (gannier at univ-tours dot fr) University of Tours (France)
See also: Others productions for ImageJ from the authors
PCCV group main page
History: 2015/07/22: first version
2016/05/26: add MultiCell mode
Source: FFT_OnLine.java
Requires: tested with ImageJ 1.49t.
Limitations:Works on Mac OS, Linux and Windows
Description: SarcOptiM computes the spatial frequency of the striated pattern of a myocyte. It has been successfully tested on rat ventricular and atrial cardiomyocytes. Although not tested, it is expected to work on skeletal muscle fibers.
The analysis can be performed online on video capture inside ImageJ (e.g.: using the webcam plugin or HF_IDS_Cam plugin) or offline on a previously recorded video.
In the online mode, the HF_IDS_Cam plugin with an IDS UI-1220LE camera allows high frequency online recording of sarcomere shortening (up to 1000 Hz) limited only by the frame rate of the camera.
Publication: "SarcOptiM for ImageJ: high frequency online sarcomere length computing on stimulated cardiomyocytes." Côme Pasqualin, François Gannier, Angele Yu, Claire O Malecot, Pierre Bredeloux, Véronique Maupoil
American Journal of Physiology - Cell Physiology 2016,
DOI: 10.1152/ajpcell.00094.2016
Installation: Download and unpack SarcOptiM.zip.
Copy the SarcOptiM folder into the "ImageJ\plugins" folder.
Copy the SarcOptiM.ijm file into the "ImageJ\macros\toolsets" folder.
Start ImageJ or restart it if already opened.
In the "More tools" menu (>>) of the toolbar, select "SarcOptiM". A new set of buttons should now be present on the right side of the toolbar, as shown in the top figure below. SarcOptiM is now ready to use.

N.B.: To have access to the full functionalities of SarcOptiM, USE ONLY the toolbar. SarcOptiM from the "Plugins" menu alone shows some coding elements used in the script.
How to Use: Despite the fact that SarcOptiM can be used on entire image, using a simple line provides the best results.
1 - Setup:
Spatial and temporal calibrations of the video must be done. For spatial calibration, this can easily be done by pressing CTRL+SHIFT+P or the "1µm" button on the toolbar and following the instructions of the dialog box, for example. Spatial calibration is essential for the programme to search the sarcomere length within the limits of lengths given in the analysis parameters. For temporal calibration, one needs to know the camera frequency. On video capture, if the camera module is compatible (HF_IDS_Cam or webcam_Capture plugin v1.2) check "limit to Video Freq" in the different dialog boxes and enter a time out value COMPATIBLE with the video frequency, i.e., greater than 1/frequency. Otherwise, use a time out equivalent to the video frequency (i.e., 1/frequency).

2 - Online/Offline measurements:

A) For FFT on cell axis:
- Trace a line along the cell long axis, this can be moved or resized later. Use the longest line possible: the ideal will be 256+ pixels while 64/128 is possible but give worse results.
- The live display of the "Online FFT Spectrum" module should help you to find the best profile (i.e., a single peak as narrow and high as possible): while the FFT is displayed you can move the line or even change the angle (very useful also if the cell is moving during the experiment).
N.B.: if the size of the line is changed by a factor 2 from a 2n value (i.e.: 32/64/128/256/512...) you must stop and restart the "Online FFT Spectrum" analysis.

- When the best FFT is obtained, press "SPACE" to stop the "Online FFT Spectrum" analysis, be sure the image window is selected then choose the online or offline video analysis tool.

B) For full frame FFT analysis, two modes exist: SingleCell (AVI) or MultiCell (AVImc)
- In "SingleCell Mode": nothing to do, just start the analysis.
- In "MultiCell Mode": this mode needs the number of cell axes to analyse and the angular tolerance. The cell axis must be defined by drawing a line and all angles must be different.
N.B.: During the analysis, the cell must not rotate beyond the angular tolerance.

Tips: Tips to improve sarcomere measurements.
Tips to improve full frame analysis speed:
- This mode is faster in 8-bit grey levels, if your video is in RGB mode convert it using "Image/Type/8-bit" in ImageJ menu.
- A crop of the image can be done with ImageJ by selecting a ROI then use "CTRL-SHIFT-X" or "CTRL-SHIFT-D".
Testing: Maximum rate of the FFT Analysis: open an image of a cell (not a video, here we only test the maximun speed of the computer; however, you can extract just one image of a video). Calibrate and trace a line along the cell. Use the Online_Video_Analysis module, uncheck "limit to VFreq" and enter 0 in "VFreq time out". Check or uncheck "Display SL while acquiring" to test in both conditions. Then check "advanced options" and click "Ok". In the next dialog box, check "Display Framerate" and click "Ok". The analysis begins and after a couple of seconds the graph of framerate is displayed.
In the same way, you can test the speed in different conditions, e.g.: during video acquisition and at different rates of acquisition.
To test SarcOptiM, some videos recorded in different conditions are provided:
- a digital recording of a cell surrounded by dead cells (150 fps, 0.3 µm/pix).

- a analogical recording (Hamamatsu C3077 - CCIR digitized by Pinnacle Studio DC10plus) of a cell with dead cells (25 fps, 0.25 µm/pix).

- a digital recording of a rotating cell surrounded by dead cells (150 fps, 0.3 µm/pix). For a better analysis, use 1.5 and 2.0 µm for respectively min and max sarcomere lengths.

Troubleshootings: - When TTorg and SarcOptiM plugins are both installed, a message ("Duplicate command ...") is displayed when you first start ImageJ. This is due to the presence of the same library (Hanning_Win.jar) in both plugins. This will be automatically corrected by ImageJ, if not, verify that the last versions of SarcOptiM and TTorg are installed.
- During the real time analysis (online analysis), the result window must not be resized with the mouse. If done, the programme will crash. Use the ImageJ menu to resize it if needed (Edit/options/Plots...).
Thanks: We thank the company "STEMMER Imaging" (Suresnes, France) for the loan of an IDS UI-1225 monochrome camera.

[Menu]

Toolbar providing icons for the different modes of analysis.

[Offline]

Offline analysis on high frequency recorded video (500Hz).
Left: FFT spectrum of the current image, right: sarcomere shortening of the cardiomyocyte

[Synthese]

Synthetic cardiomyocyte and Video synthesis module.
Right top: FFT spectrum of the current image, right bottom: sarcomere shortening of the synthetic cardiomyocyte

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