that tool needs black objects on a white background or a threshold to be set on the image which contains the desired objects. Next we must run the Analyze Particles tool in Fiji to locate patches of white pixels and count them.Now you have a set of white foreground patches of white pixels, surrounded by black background pixel areas, and we have separated touching objects with a watershed, which put "dams" one pixel wide between the objects.This method only works robustly for roughly circular objects. Notice how the nuclei have been split away from each other.Your watershed image should look like this: Where 2 "Watersheds" meet, it builds a dam to separate them! One could do all these steps manually, but the watershed function automates that for you, which is nice. This method finds the centre of each object (using a morphological erode operation), then calculates a distance map from the object centre points to the edges of the objects, then fills that "topological map" with imaginary water. To run the built in ImageJ watershed method choose menu item: Process - Binary - Watershed.Watershed algorithm - separate touching objects We will use the watershed method built into Fiji for that: Also it is clear that some nuclei are connected to adjacent ones. Black is background, and white is foreground. If you are happy with the automatically calculate threshold, then click "Apply", which will give you a binary image. Indeed, in this case the background is dark! The default method will be previewed automatically when you launch the menu command, and a threshold will be set, something close to 100 intensity. Turn on the check box for "Dark background". Do menu command Image - Adjust - Threshold.You might get a different answer in the end! You can play with different methods if you like. In this case the default method works pretty well, but you can see there is a long list of methods, which give slightly different threshold results for this image. Fiji has a number of built in Automatic Thresholding methods that try to distinguish the background from the foreground. Next we need to separate the objects from the background using pixel intensity thresholding.Pixel Intensity Threshold - find the foreground areas You should get an image that looks like this: You can preview other values to see how they look also. Run menu command: Process - Filters - Gaussian Blur, with a sigma value of 3 pixels. Too high a sigma value, and the objects will be too blurred, making it harder to find their edges precisely and separate them later. A value of sigma too small will mean that the segmentation will be disturbed by the noise and staining pattern. We will use a large sigma value of 3 for this task. Run a Gaussian Blur filter on the image to blur out the "speckle", actually Poisson distributed, statistical "photon shot noise", and also to smooth out the inhomogeneity of the nuclear staining.open the sample image of touching DAPI stained cell nuclei from a confocal laser scanning microscope. ![]() Okay, so how can we denoise, segment, watershed (separate touching objects) and then count / measure the objects in Fiji? Read on.Luckily, there is a method for doing exactly that.So, we are faced with the problem of being able to separate apparently touching, noisy, objects :-(.That gives objects fuzzy edges and adds uncertainty to the intensity values of each pixel, making it harder to segment properly. The PSF is much bigger than the width of a membrane! Just to make matters even worse the image is quite noisy, because it was made with a fast scan on a confocal laser scanning microscope, which inherently has a low signal/noise ratio. There is a membrane or two at least between them, but an optical microscope cannot resolve that. ![]() The nuclei often seem to touch each other, which in reality, of course, they can not. Worse still, in many tissues that are interesting for developmental biology, the cells are tightly packed, and are composed mostly of nucleus with very little cytoplasm separating them. however, the staining is not homogenous, as there are areas of more or less condensation of the chromosomes. The staining delineates the nuclei pretty well, since in a metaphase cell there is DNA all over the nucleus.
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