I am trying to overlap multiple images I have of a single neuron taken from different brains, and see the overlap of these neurons. I am trying to visualize variability in this neuron between brains. I am using the ‘Name Landmarks and Register’ plugin to achieve image registration, and the manual registration of these images works well. However, whenever I want to add another neuron to this image registration, FIJI automatically resets the color of my previous image and combines them all into one color. I have included an image below to help demonstrate what I mean.
In the image on the left, I have registered 2 images together, one neuron is in magenta, the neuron from a separate image is in green, and their overlap is in white. I save this image, and perform image registration again, using the image on the left as my template. When I add in a third neuron (image on the right), the template image is all blended into one color (magenta), and the neuron from the third brain I have added is in green. This prevents me from seeing where all of the individual neurons are, and it prevents me from seeing where there are areas of high overlap, and areas of little overlap.
Is FIJI able to overcome this issue? Ideally, I would like to threshold each image, register the images, and have ‘sum’ the overlap, so that if I have 5 images registered with one another, I would see all areas with a pixel value of 5 in magenta, areas with a pixel value of 4 in another color, and so on. I was able to accomplish this with zstack projections in MATLAB, but I cannot register these 3D images in MATLAB manually. Any help would be greatly appreciated , thank you!