The program performs colocalization analysis on pairs of images in Matlab and calculates
- the Pearson correlation coefficient
- the confidence interval of the Pearson correlation coefficient assuming the absence of correlation (r=0) using Costes randomization (as described in his paper in Biophysical Journal)
- the Manders coefficients (for a review see "A guided tour into subcellular colocalization analysis in light microscopy" in Journal of Microscopy)
The program semi-automatically analyses all images in a user-selected folder. After starting the program by typing "processAllInDirForRConf" at the command prompt the following dialog box is displayed:
Default method of segmentation: manual, intermeans, Otsu or maxentropy
Boolean operation: "or" or "and". This selection determines whether a Boolean "or" or "and" operation is performed for the masks calculated for the two images. A mask includes all the pixel above the threshold.
Size of units to reshuffle: the width of units in pixel which are reshuffled in Costes randomization
Number of scramblings: how many times Costes randomization is performed
Confidence interval: the confidence limits calculated for the correlation coefficient assuming the absence of correlation after Costes randomization
Sigma of Gaussian: the SD of the Gaussian filter used for smoothing the images
- single images: pairs of images are stored separately. The two images must have names which differ at the end (e.g. control_1.tif and control_2.tif).
- color image: the pairs of images are stored as color layers in an RGB image. The user will have to select which channels to analyze.
- gray-scale stack: the pairs of images are stored as layers in a 3D stack.
After pressing "OK" the user will have to select the images to be analyzed.
The user either selects one single image in the folder and all the other files in the folder will be analyzed, or every image to be evaluated must be selected.
If a single image file contains multiple images (color image or gray-scale stack selected as image type) the program automatically reads the images if the color image or the gray-scale stack contains two images. Otherwise, the user will have to specify which channels correspond to the images to be analyzed.
After pressing OK in the window above the colocalization analysis is performed. A message is displayed in the Matlab command window and a TXT file is saved into the image folder. An exemplary text file is shown below.
Syntax: type processAllInDirForRConf at the Matlab command prompt. Optionally, an output argument can be provided which will contained a Matlab structure whose field names describe variables stored in them.
Requirement: The program requires DipImage, freely available for download at the following URL: http://www.diplib.org/download
Updating: The program checks for updates after each startup. If an update is available, the user will be asked to press the "Update" button.