Object Separation with Watershed¶

Tools for separating touching or overlapping objects stored in the Selection, Mask, or Model layer.

Overview¶

The Object separation tool uses watershed transformation to divide connected objects into smaller, distinct units. Objects can originate from the Selection, Mask, or Model layer, making the tool useful for refining segmentation results where objects are fused or touching.

Launch via Ribbon → Tools → Object separation.

Mode panel¶

The Mode panel controls the segmentation scope.

• 2D, current slice only — applies watershed only to the currently displayed slice in the Image View panel
• 2D, slice-by-slice — applies 2D watershed to each slice individually
• 3D, volume — performs 3D watershed across the entire dataset or a selected subvolume (see Subarea panel below)
• Aspect ratio — voxel aspect ratio used for 3D distance-transform watershed, expressed as three space-separated values (e.g., 1.00 1.00 3.50). Computed automatically from the pixel sizes in Dataset → Parameters.

Subarea panel¶

The Subarea panel limits processing to a portion of the dataset.

• X — pixel width range, e.g. 1:512
• Y — pixel height range
• Z — slice range
• from Selection — fills X, Y, and Z from the bounding box of the Selection layer
• Current View — limits X and Y to the visible area in the Image View panel
• Bin, xtimes: — binning factor XY; Z to downsample before processing (e.g., 2; 1 halves XY resolution)
• Reset — restores all fields to the full dataset dimensions

Objects to separate panel¶

Defines the source layer and separation options.

Source layer (radio buttons):

• Selection — use the Selection layer as the object source
• Mask — use the Mask layer
• Model, select material — use a specific material from the Model layer; choose the material from the dropdown that appears below the radio buttons

Options:

• Use seeds — enables seeded watershed; when checked, the Layer with seeds and Watershed source panels appear and Reduce oversegmentation is hidden
• Reduce oversegmentation — (standard watershed only, hidden when Use seeds is checked) applies extended minima suppression to reduce excessive fragmentation
• Update materials — refreshes the material dropdown lists from the current model

Layer with seeds panel¶

(Visible only when Use seeds is checked)

Specifies the layer that contains the seed markers used to guide the seeded watershed.

• Selection — use the Selection layer as seeds
• Mask — use the Mask layer as seeds
• Model, select material — use a specific material from the Model layer; choose the material from the dropdown below

Watershed source panel¶

(Visible only when Use seeds is checked)

Controls which data the watershed algorithm uses to define catchment basins.

• Distance map — watershed runs on the Euclidean distance transform of the object mask (default; shape-based separation)
• Image intensity — watershed runs on the raw image grey values (intensity-based separation; requires seeds). See Steve Eddins's blog on cell segmentation. When Image intensity is selected, two additional controls appear:
  • Color channel — color channel used for intensity-based watershed
  • Signal — polarity of the image signal: white-on-black, signal is bright (fluorescence, light microscopy) or black-on-white, signal is dark (transmitted light, electron microscopy)

Object separation example¶

The following example separates fused mitochondria using unseeded watershed:

1. Open Ribbon → Tools → Object separation
2. Select Mask in the Objects to separate panel
3. Click Separate objects

Unseeded watershed can over-segment long objects. To address this, switch to seeded watershed:

1. Check Use seeds
2. In the Layer with seeds panel, select Model, select material and choose the Seeds material
3. Click Separate objects

Each object with a single seed label is extracted individually (highlighted in green).

Algorithm¶

The diagram above illustrates the step-by-step process of the watershed-based object separation algorithm.

Batch scripting¶

This tool supports batch scripting for automation.

BatchOpt.Mode          = {'mode3dRadio', {'mode2dCurrentRadio','mode2dRadio','mode3dRadio'}};
BatchOpt.ObjectSource  = {'Mask', {'Selection','Mask','Model'}};
BatchOpt.UseSeeds      = false;
BatchOpt.ReduceOversegmentation = false;
BatchOpt.XSubarea      = '1:512';
BatchOpt.YSubarea      = '1:512';
BatchOpt.ZSubarea      = '1:100';
BatchOpt.Binning       = '1; 1';

obj.mibController.startController('controllers.ObjectSeparator', [], BatchOpt);

References¶

• Watershed transform Q&A from tech support by Steve Eddins
• Cell segmentation with seeded watershed by Steve Eddins

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