![]() Where it is black (zero pixel value), the pixels will be ignored: not included in the analysis”. “where the mask image is white (255 pixel value for an 8 bit greyscale image) colocalization will be analyzed for those pixels only. Could this be caused by having a sepatare ROI for each Hmm interesting… I had read that page, but I didn’t notice: I was able to do this using the old “Colocalization Threshold” but with pretty inconsistent results. The problem I am currently trying to solve is that it works right up until the colocalization, at which point it seems to be running a separate colocalization for every slice in the image rather than one for the whole stack. SaveAs("Text",dir+imageTitle+"_coloc2_log.txt") Run("Coloc 2", "channel_1=Ch2 channel_2=Ch3 roi_or_mask= threshold_regression=Costes spearman's_rank_correlation manders'_correlation psf=3 costes_randomisations=10") Rename to be called in Coloc2 (using run("Coloc 2", "channel_1=C2-"+imageTitle". Batch mode not working? Nothing seems to be added to ROI manager when in batch mode Automatically assumes Channel 1 is DAPIĭir=getDirectory("Choose a Directory - MUST CONTAIN IMAGES ONLY") Processes a folder of images as long as that folder only contains image files Here’s what I have so far: //Z-stack Colocalisation with Costes method using Coloc 2 The macro I am trying to produce will select a folder, and for each the image stack in the folder run Coloc2 with Costes thresholding, primary to produce Mander’s coefficients for channel 2 and 3 (1 is DAPI). Thank you in advance for your help and your time. Should I somehow automatically generate an ROI for each slice in the stack (maybe easier because I already have an intensity of 0 outside of the cell) then apply Coloc 2 to the ROI’d stack? I believe the Costes method used was somehow inaccurate, and fixed in Coloc 2? However, Coloc 2 freezes (or takes many days, I haven’t left it running because it wouldn’t be feasible anyway) when trying to calculate Costes thresholds.Īnyway, how can I calculate a Costes threshold on a z-stack, ignoring zero-zero pixels, then with that threshold generate Mander’s coefficients etc.? 7/8 images I tried to analyse today were thresholded wrong and had a tM1/tM2 of 1.0. However, I am now getting very inconsistent thresholding with this plugin. This seemed to work on most of my images. ![]() There appears to be no option to ignore zero-zero pixels in any other colocalization plugin. ![]() I then open that file in Fiji and running the old ‘colocalization threshold’ plugin set to ignore zero-zero pixels. The only method I have gotten to work so far is using Imaris to create a 3D surface around the cell body, then masking the cell so that all pixels outside are 0 and saving the file. I’ve done quite a bit of reading on all the methods available, including using rolling ball or local median background subtraction rather than Costes thresholding. These stacks are images of cells and I want to only colocalize signal within them, ignoring all signal outside. Looking for some advice about getting any of the ImageJ plugins to play ball for colocalization of largeish z-stacks (~130 slices).
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