Spots colocalization (ComDet)
|File||ComDet.jar + for ImageJ Jama lib|
|Initial release||23 November 2012|
|Latest version||12 April 2018|
Plugin for finding and/or analyzing colocalization of bright intensity spots (cells, particles, vesicles, comets, dots, etc) in images with heterogeneous background (microscopy, astronomy, engineering, etc).
For help with plugin installation, see instructions.
How to use plugin
Open the image to be analyzed in ImageJ. In general plugin works in two modes:
- particles detection (whole image or ROI)
- particles detection and colocalization analysis (whole image or ROI).
For colocalization you need an image containing two color channels. Plugin auto-detects the number of channels and if it is equal to two then the plugin will automatically switch to second mode (see below).
Plugin works with time/z-stacks.
Launch the detection by choosing Plugins->ComDet->Detect Particles.
Simple detection (1 channel)
If the image is not two channels image, the following dialog window will appear:
Specify estimated particles size and intensity threshold (particle brightness) and press OK. To have good detection usually you need to play with parameters and see how it goes.
By default, plugin looks only for particles of specified size. If you check "Include larger particles?" box, it will also try to quantify bigger spots.
If you choose Add to ROI Manager option ("All particles"), plugin will add detected rectangular ROIs around particles to ROI Manager. ROIs will have names in the format of d(detection# in Results table)_ch(#channel)_sl(#slice)_fr(#frame)_c(0 if particle is not colocalized and 1 if it is, see below).
After pressing OK plugin runs and add rectangles in overlay on top of detected particles.
Also it will provide you Results table containing particles' coordinates (see below) and Summary table. I recommend to play with parameters to get a nice detection result.
Detection in ROI
If you want detection to be performed in some specific region instead of whole image, select some ROI using any ImageJ ROI selection tools before launching plugin:
In this case only particles in that ROI will be detected:
Detection and colocalization in 2 channels
If your image contains two channels then after pressing Detect Particles the dialog will look a bit more complex:
In this case you can specify the same parameters of detection for each channel separately. To get colocalization analysis you need to choose "Two channels detection: Detect in both channels independently" option and check "Calculate colocalization?" box. If you uncheck it, then only detection will be performed.
There is an additional parameter in case of colocalization: maximum distance between spots' centers. It defines at what distance (in pixels) two spots in different channels are considered to be localized.
So colocalization is based on the distance between spots' centers!
After detection is finished ComDet marks detected particles with rectangles of their own channel colour and colocalized particles in yellow:
Also Add to ROI Manager option is available. ROIs will have names in the format of d(detection# in Results table)_ch(#channel)_sl(#slice)_fr(#frame)_c(0 if particle is not colocalized and 1 if it is). You can add all detected particles or only those that colocalize.
For two channels detection, there are three options: "Detect in both channels independently" or "Detect in channel 1(2), quantify in channel 2(1)". In last two cases, detection of spots will be done only in the first specified channel. For the other channel, same spots intensities would be quantified over the same area as in the "detection" channel (whether there is a spot there or not).
Also only ROI detection mode (as described above) works in this case.
Here is example of Results table (Summary table is kind of self-explanatory).
- Abs_frame = absolute frame number in stack (unique frame number)
- X_(px) = x coordinate of spot (centroid fitting)
- Y_(px) = y coordinate of spot (centroid fitting)
- Channel = corresponding channel number
- Slice = corresponding slice number (if z-stack)
- Frame = corresponding frame number (if timelapse)
- xMin, yMin, xMax, yMax = coordinate of rectangle around spot (in pixels)
- NArea = thresholded area of the spot in pixels
- IntegratedInt = sum of all pixels intensity inside the thresholded area minus average spot-specific backgrounds. Background value is calculates as average intensity of pixels on perimeter of the rectangle.
- Colocalized = 0 if spot does not colocalize with another and 1 if it does.
- IntensityRatio = Ratio of spot's IntegratedInt to IntegratedInt of corresponding colocalized spot.
- ColocIndex = corresponds to the detection index in Results table, corresponding to the particle in anothe channels (works only for two channel independent detection).