Analysis performed for the Belyaeva et.al PNAS 2017 Paper
Executed in FIJI (ImageJ v 1.51n). Requires Bio-Formats and R(v3.3)
DESCRIPTION: Fiji script takes a 3D image stack(x,y,z) with four channels: Nucleus, chromosomes (A and B) and Protein, and identifies the overlapping regions between chromosome A and B in 3D. It also measures the amount of protein levels in these overlapping regions. Then, the R script reads in these values and calculates the intermingling degree and protein enrichment.
IMAGE PROPERTIES: The input image is a confocal z-stack (slice height=0.5microns) with 4 channels, the channel One contains the nucleus and then there are two channels for the chromosomes and one channel for RNA polymerase2. The channel numbers for the chromosomes and Pol2 are specified.
IMAGE ANALYSIS ALGORITHM: Two dialog box opens where the user inputs the source (where the images are stored) the results (where results need to be saved) directory. The program opens nucleus channel, uses the Gaussian filter (sigma=0.15, scaled) to the stack and the thresholds the image and asks the user to check the thresholded image (optional). The user can then adjust the threshold for precision. The program then converts the thresholded image to a binary image and these images are stored in the results folder. For the chromosome channels, it identifies nuclear region and then it performs the aforementioned processes. The threshold values are stored in the datafiles directory. The binary chromosome images are opened in combination (2-3) and passed through the AND filter to identify the intermingling regions (IMR) and the images saved in the results directory. Next, the volumes of the binary nucleus, chromosomes and IMRs are measured and saved in the datafiles directory. Then, the amount of pol2 in the total image, nucleus, and the IMR are calculated and stored in the datafiles directory.
DATA ANALYSIS: The R script uses the features measured by the image analysis script to calculate the intermingling degree and the Protein enrichment at the IMR using the following equations.
Intermingling degree (IMD) = (Volume of IMR between chromosomes A and B)/Volume of chromosome A + Volume of chromosome B)
Protein enrichment (PR) = (Mean Intensity of protein in the IMR) / (Mean intensity of the protein in the nucleus)
The difference between the IMD and PR between samples is visualized as a boxplot and the significance of this difference is tested using the Welsh two sample t-test.
For more details visit our paper.