title graphic AIF graphic

dias.gif (758 bytes)
(Dynamic Image Analysis System) instructions

On 10 August 2004 we got a new G4 running OSX and installed new DIAS 3.4.1.  Thus, new instructions.
First Draft, of course.  Email changes here or scribble on a printout & drop off at Michael's office.

IMAGEJ-TRACING.gif (1383 bytes)


If you are tracing in ImageJ to prepare for DIAS or for other measurements, such as using the tracings as masks for measuring fluorescence levels, BEFORE TRACING run this macro prepare-for-tracing02.txt or do an equivalent yourself using the Process-->Math command in ImageJ.

The point of this is to make sure that the traced areas are a unique value not used anywhere else in the image.  For instance, the image will be displayed using the grayscale range 2 to 253 with all your traced areas being 0 or 255.


The simplest way to trace is to

  1. Open the time lapse into ImageJ.
  2. Run the prepare for tracing macro.
    This macro must be run once before doing any tracings.
    You may instead use a math function of your choosing.
  3. Make sure the background colors are set to some combination of white and black (0 and 255).
  4. At each timepoint, trace the cell.  It may be selected as a traced object by simply cutting <ctrl>x or filling <ctrl>f.   (The first time, drag the mouse over the traced are and check that the value is 0 or 255).
  5. Save repeatedly while tracing so that if you make a mistake, you don't lose all the tracings.
  6. When you are done with all the tracings, threshold and apply the threshold in ImageJ so that your image consists of traced areas in white and cells in black (or vice versa).
  7. Save as a TIF or uncompressed AVI.
  8. Move ther file to a Macintosh.
  9. On the Macintosh convert the files to a folder of tifs with the DIAS numbering convention which must be 1, 2, 3... not 01, 02, 03...
    Use this macro to-DIAS-ver201macver.txt to do this.   The instructions are at the beginning of the macro which is a simple text file.   You may read them.

file-formats.gif (1708 bytes)

Files must be one of a few very specific formats.

Here are the two simplest:

  1. Quicktime.  Files must be saved with NO COMPRESSION if you want tracings from the AIF's IimageJ macros to be compatible.
  2. A unique folder for each sequence containing uncompressed 8 bit grayscale tif files numbered beginning with "1" to n.
    The numbers must not have leading zeros.  The sequence must begin with "1" and not "0".
    The files may not have any extension.  In DOS this may be rectified by:
    command-prompt.gif (2023 bytes)
    Any mistakes in a file's name will result in failure of opening the entire sequence.

More detailed instructions:

  1. Make a series of new folders.
    Each folder will contain one and only one time lapse sequence for DIAS.
    No folder may contain a space or plus sign "+" in the name.   "HDKrun3" is an okay name and "HDK run3" is not ok.
    This is a good structure for saving files:
    arrangement-files.gif (33005 bytes)
  2. Run ImageJ.
    On a Macintosh:
    a01.gif (5713 bytes)
    On a PC:
    a01pc.gif (2492 bytes)
  3. Install the macros.  Plugins-->Macros-->Install
    On a Macintosh OSX: to-DIAS-ver201macver.txt
  4. With the stack open, run the macro
    a07.gif (21869 bytes)
  5. In the macro, choose the directory to save the files in and click "Select".
    Do not go into the directory where the files are supposed to go.
    a08.gif (16096 bytes)

dias-protocol.gif (1361 bytes)

  1. Open the files.  (Dragging the first file onto DIAS icon also works.)
    a11.gif (8236 bytes)
  2. Click on the first in the sequence.
    Picture-2.gif (15860 bytes)
  3. Yes!
    a13.gif (2156 bytes)
  4. Give it a save name.
    a16.gif (1756 bytes)
  5. We're using DIAS, so save as DIAS.
    a17.gif (3991 bytes)
  6. I'd click No.
    a18.gif (2418 bytes)
  7. Trace.
    a19.gif (10750 bytes)
  8. Ho many frames do you want to process?
    a20.gif (2849 bytes)
  9. No selects the entire field which is usually the correct choice.
    Yes asks you to draw a region on the image.
    a21.gif (2154 bytes)
  10. This is the window you get if you clicked No.  Just click OK.
    a22.gif (4756 bytes)
  11. Look at the numbers used here.  Use the same ones.
    a23.gif (12650 bytes)
    You may save the settings for use on other files.
    a24.gif (1994 bytes)
  12. You need to make a Path File before you can do any quantification.
    a25.gif (27724 bytes)
  13. This makes the paths.
    a26.gif (9494 bytes)
  14. Interior pixel data are not relevent for these types of tracings so click No.
    a27.gif (2706 bytes)
  15. Luminance is not relevent for these types of tracings so click No.
    a28.gif (2395 bytes)
  16. Example result.
    Picture-22.gif (35584 bytes)
  17. The paths must be saved with the correct information.
    For instance, 15 second intervals = 4 fr/min.
    2X2 binning with 60X on CCD#1,2,4,5 0.11 um/pixel.  (More click here.)
    And use Perimeter Based Centroids.
    The entry shown here is for one image every five minutes with our Diaphot time lapse setup with a 10X objective and the video card in the SVGA collection setting.
    a31-editfileheader.gif (10938 bytes)
  18. Now you may do measurements.
    a33.gif (26805 bytes)
  19. Open the path file.
    Picture-28openpathfile.gif (22157 bytes)
  20. Check what you want to know.
    The Database File may be very useful for more detailed analysis later.
    a34computerp.gif (23778 bytes)
  21. Ok.
    a35.gif (3036 bytes)
  22. Yes!
    Picture-31.gif (3284 bytes)
  23. Example results:

    Immediately rename this results table to prevent loss when creating a new table!

Picture-32results.gif (14082 bytes)


Other Features:

a41-drawpaths.gif (19333 bytes)

a42.gif (18288 bytes)

a43-stack-image.gif (22625 bytes)



Here's a quick-and-dirty non-DIAS way to see where the cell dwells.

non-dias.gif (12592 bytes)

The white shows the locations where the cell dwelled at every timepoint.  Black shows where the cell didn't go at all.  From violet (brief dwell) to green to red shows the cell staying more in a given location.

This image was generated from the tracings simply by an average pixel projection with LUT applied.

Another similar method here.

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