Free and open source

Marker-controlled Watershed is an ImageJ/Fiji plugin to segment grayscale images of any type (8, 16 and 32-bit) in 2D and 3D based on the marker-controlled watershed algorithm (Meyer and Beucher, 1990). This algorithm considers the input image as a topographic surface (where higher pixel values mean higher altitude) and simulates its flooding from specific seed points or markers. A common choice for the markers are the local minima of the gradient of the image, but the method works on any specific marker, either selected manually by the user or determined automatically by another algorithm. Marker-controlled Watershed needs at least two images to run: The Input image: a 2D or 3D grayscale image to flood, usually the gradient of an image. The Marker image: an image of the same dimensions as the input containing the seed points or markers as connected regions of voxels, each of them with a different label. They correspond usually to the local minima of the input image, but they can be set arbitrarily. And it can optionally admit a third image: The Mask image: a binary image of the same dimensions as input and marker which can be used to restrict the areas of application of the algorithm. Set to "None" to run the method on the whole input image. Rest of parameters: Calculate dams: select to enable the calculation of watershed lines. Use diagonal connectivity: select to allow the flooding in diagonal directions.

This macro can be used to un-wide a tubular structure and flatten its surface (like peeling of and flattening the skin of a banana). The macro can only process a single channel 3D stack but it is easy to process multiple channels by exporting and importing ROI manager selections. Technically the macro computes the radial average intensity projection inside a ring centred on the radial symmetry axis of the object. The final image is a radial mapping of the intensity (radial angle along X, axial length along Y).

The example image is available in the documentation link. 

This macro implements a filter that is meant to attenuate close to parallel intensity stripes in an image, such as often happening in light sheet microscopy. The results are usually decent even when the stripes show a large angular spread due to light sheet refraction at the sample surface. The filter can process a 3D stack but the processing is performed slice by slice.

Example image is available in the documentation link. 

Morphological Segmentation is an ImageJ/Fiji plugin that combines morphological operations, such as extended minima and morphological gradient, with watershed flooding algorithms to segment grayscale images of any type (8, 16 and 32-bit) in 2D and 3D. Morphological Segmentation runs on any open grayscale image, single 2D image or (3D) stack. If no image is open when calling the plugin, an Open dialog will pop up. The user can pan, zoom in and out, or scroll between slices (if the input image is a stack) in the main canvas as if it were any other ImageJ window. On the left side of the canvas there are three panels of parameters, one for the input image, one with the watershed parameters and one for the output options. All buttons, checkboxes and input panels contain a short explanation of their functionality that is displayed when the cursor lingers over them. Image pre-processing: some pre-processing is included in the plugin to facilitate the segmentation task. However, other pre-preprocessing may be required depending on the input image. It is up to the user to decide what filtering may be most appropriate upstream.

This macro batch processes all the 2D images (tif and jpg files) located in a user defined folder by calling Fiji Weka trainable segmentation to classify each pixel, and reports the areas of each class in a human readable results table. The classifier to be applied to each image should be previously trained on a representative image by an expert and exported to file (Save classifier) into the image folder to be processed.