Morphological filters

Prerequisites

Before starting this lesson, you should be familiar with:

• Segmentation

• Median filter

• Connected component labeling

• Neighbourhood filters

Learning Objectives

After completing this lesson, learners should be able to:

• Understand how to design morphological filters using rank filters

• Execute morphological filters on binary or label images and understand the output

Motivation

Morphological filters (MFs) are used to clean up segmentation masks and achieve a change in morphology and/or size of the objects. For example, MFs are used to remove wrongly assigned foreground pixels, separate touching objects, or identify objects boundaries.

Concept map

Figure

Activities

Explore various morphological filters

Perform some or all of the activities below

• Dilation and erosion of binary
  • Open xy_8bit_binary__two_spots_different_size.tif
  • Explore erosion and dilation
  • Explore how structures grow and shrink depending on the size of the structuring element
• Opening and closing of binary
  • Open xy_8bit_binary__for_open_and_close.tif
  • Perform erosion followed by dilation - opening. Explains it effects in removing thin structures, smoothing borders. If applicable show that opening runs as single command.
  • Perform dilation followed by erosion - closing. Explains it effects on filling small holes, connecting gaps. If applicable show that opening runs as single command.
• Morphological internal gradient of binary
  • Open xy_8bit_binary__h2b.tif
  • Perform an erosion
  • Subtract eroded image from binary image and discuss the results (Internal Gradient)
  • If applicable show where the morphological gradient runs as a single command

ImageJ Macro & GUI: Dilation and erosion

run("Close All");
//Make sure black background in Process > Binary > Options is set to true
setOption("black background", true);

open("https://github.com/NEUBIAS/training-resources/raw/master/image_data/xy_8bit_binary__two_spots_different_size.tif");
// Image > Rename
rename("binary");
run("Set... ", "zoom=400 x=29 y=25");
// Erosion, use default binary IJ binary operations
// It is a radius 1 squared SE, i.e. 3x3 SE

// Image > Duplicate, name it eroded
run("Duplicate...", "title=eroded");
//Process > Binary > Erode
run("Erode");
run("Set... ", "zoom=400 x=29 y=25");
// Apply second erosion will remove small dot
// Image > Duplicate, name it eroded_twice
run("Duplicate...", "title=eroded_twice");
// Process > Binary > Erode
run("Erode");
run("Set... ", "zoom=400 x=29 y=25");
// Use MorpholibJ and a radius 2, i.e. 5x5 squared structuring element
// This corresponds to 2 sequntial 3x3 erosions
selectWindow("binary");
// Plugins > MorpholibJ > Filtering > Morphological Filters 
// Select Erosion, Element Square 
run("Morphological Filters", "operation=Erosion element=Square radius=2");
rename("erosion_radius2");
run("Set... ", "zoom=400 x=29 y=25");
// Dilation, use MorpholibJ
selectWindow("binary");
// Plugins > MorpholibJ > Filtering > Morphological Filters
// Use MorpholibJ and a radius 2, i.e. 5x5 squared structuring element
run("Morphological Filters", "operation=Dilation element=Square radius=1");
rename("dilation_radius1");
run("Set... ", "zoom=400 x=29 y=25");
// Dilation, use MorpholibJ
selectWindow("binary");
// Plugins > MorpholibJ > Filtering > Morphological Filters
// Use MorpholibJ and a radius 2, i.e. 5x5 squared structuring element
run("Morphological Filters", "operation=Dilation element=Square radius=2");
rename("dilation_radius2");
run("Set... ", "zoom=400 x=29 y=25");
run("Tile")

ImageJ Macro & GUI: Closing and opening

run("Close All");
//Make sure black background in Process > Binary > Options is set to true
setOption("black background", true);
open("https://github.com/NEUBIAS/training-resources/raw/master/image_data/xy_8bit_binary__for_open_and_close.tif")
rename("binary");
run("Set... ", "zoom=400 x=29 y=25");
// Opening, use default binary IJ binary operations in sequence
run("Duplicate...", "title=opening");
//Process > Binary > Erode
run("Erode");
//Process > Binary > Dilate
run("Dilate");
run("Set... ", "zoom=400 x=29 y=25");
// See how the thin structure disappear

// Opening, use default binary IJ binary operations
selectWindow("binary");
run("Duplicate...", "title=opening2");
//Process > Binary > Open
run("Open");
run("Set... ", "zoom=400 x=29 y=25");

// Opening, use MorpholibJ, try different radii
selectWindow("binary");
// Plugins > MorpholibJ > Filtering > Morphological Filters
run("Morphological Filters", "operation=Opening element=Square radius=1");
rename("binary-Opening_radius=1");
run("Set... ", "zoom=400 x=29 y=25");
selectWindow("binary");
// Plugins > MorpholibJ > Filtering > Morphological Filters
run("Morphological Filters", "operation=Opening element=Square radius=3");
rename("binary-Opening_radius=3");
run("Set... ", "zoom=400 x=29 y=25");
// see how also the small blob disappear, side of large blob are deformed


// Closing, use MorpholibJ
selectWindow("binary");
// Plugins > MorpholibJ > Filtering > Morphological Filters
run("Morphological Filters", "operation=Closing element=Square radius=1");
rename("binary-Closing_radius=1");
run("Set... ", "zoom=400 x=29 y=25");
// Fill the hole in big blob
// Closes the gap
run("Tile")

ImageJ Macro & GUI: Internal Gradient

run("Close All");
//Make sure black background in Process > Binary > Options is set to true
setOption("black background", true);

open("https://raw.githubusercontent.com/NEUBIAS/training-resources/master/image_data/xy_8bit_binary__h2b.tif");
rename("binary");

// Internal gradient is the original - eroded image
// Shown as step by step process
// Erode image
// Plugins > MorpholibJ > Filtering >  Morphological Filters
//   Operation = Erosion
//   Element = Square
//   Radius (in pixels) = 1
run("Morphological Filters", "operation=Erosion element=Square radius=1");
rename("eroded")
// Process > Image Calculator ... 
//    image1 = binary
//    operation = subtract
//    image2 = binary-Erosion
//    [x] Create enew window
imageCalculator("Subtract create", "binary","eroded");
rename("internal_gradient");

// Add internal_gradient as overlay
selectImage("binary");
// Image > Overlay > Add..
run("Add Image...", "image=internal_gradient x=0 y=0 opacity=50");

//Internal gradient with MorpholibJ

selectWindow("binary");
// Plugins > MorpholibJ > Filtering >  Morphological Filters
//   Operation = Erosion
//   Element = Square
//   Radius (in pixels) = 1
run("Morphological Filters", "operation=[Internal Gradient]  element=Square radius=1");

run("Tile")

skimage napari

#######################################################
## To follow along you need to complete the tool
## installation activity for skimage napari.
#######################################################

#%%
# Import python packages.
from OpenIJTIFF import open_ij_tiff
from napari.viewer import Viewer
from skimage.morphology import square, disk
from skimage.morphology import erosion, dilation
from skimage.morphology import opening, closing

#%% 
# download and read tif file
fpath = "https://github.com/NEUBIAS/training-resources/raw/master/image_data/xy_8bit_binary__two_spots_different_size.tif"
image_2spots, _, _, _ = open_ij_tiff(fpath)

# Create a napari_viewer and visualize image and labels.
napari_viewer = Viewer()
napari_viewer.add_image(image_2spots, name='image')

#%%
# Perform erosion and dilation with default structuring element (cross, connectivity=1).
eroded = erosion(image_2spots)
dilated = dilation(image_2spots)

#%%
# Now try with connectivity 2 (square).
square3 = square(3)
print(square3)
eroded_square3 = erosion(image_2spots, square3)
dilated_square3 = dilation(image_2spots, square3)

#%%
# add images to the viewer and inspect the results
napari_viewer.add_image(eroded, name='eroded1')
napari_viewer.add_image(dilated, name='dilated1')
napari_viewer.add_image(eroded_square3, name='eroded3')
napari_viewer.add_image(dilated_square3, name='dilated3')

# Now try with bigger square (e.g. square(5))
# or a different structuring element (e.g. disk(1))
# Also refer to:
# https://scikit-image.org/docs/stable/api/skimage.morphology.html

#%%
# Explore opening and closing combining erosion and dilation.
# Verify that they give the same results.
fpath = "https://github.com/NEUBIAS/training-resources/raw/master/image_data/xy_8bit_binary__for_open_and_close.tif"
image_structures, _, _, _ = open_ij_tiff(fpath)

#%%
# Opening: remove thin structures, smoothen objects.
opening_manual = dilation(erosion(image_structures, square(3)), square(3))
opening_1step = opening(image_structures, square(3))
print((opening_manual==opening_1step).all())

#%%
# Closing: fill holes, connect gaps.
closing_manual = erosion(dilation(image_structures, square(3)), square(3))
closing_1step = closing(image_structures, square(3))
print((closing_manual==closing_1step).all())

#%%
# Try using the default structuring element (disk(1)).
# Explain what has changed and why.
closing_disk = closing(image_structures, disk(1))

#%%
# Create a napari_viewer and visualize images.
napari_viewer = Viewer()
napari_viewer.add_image(image_structures, name='image')
napari_viewer.add_image(opening_1step, name='opening')
napari_viewer.add_image(closing_1step, name='closing')
napari_viewer.add_image(closing_disk, name='closing_disk')

#%%
# Explore internal gradient.
fpath = "https://github.com/NEUBIAS/training-resources/raw/master/image_data/xy_8bit_binary__h2b.tif"
image, _, _, _ = open_ij_tiff(fpath)

# Internal gradient is the difference between the image and the eroded version of it.
eroded = erosion(image)
internal_gradient = image-eroded

#%%
# Create a napari_viewer and visualize images.
napari_viewer = Viewer()
napari_viewer.add_image(image, name='image')
napari_viewer.add_image(internal_gradient, name='internal_gradient')

# The internal gradient represents the edge of the object.
# Discuss when and how this can be useful.

Exercises

Measure intensity on the nuclear membrane

In image xyc_16bit__nup__nuclei.tif we would like to measure the intensity along the nuclear membrane (channel 1) using the information from the DNA (channel 2). We designed two exercises that provide a workflow using morphological filters.

Clean up segmentation

• Use a combination of opening and closing operations to improve the segmentation of the DNA channel xy_8bit_binary__nuclei_noisy.tif.
• The goal is to achieve something like xy_8bit_binary__nuclei.tif that can be used for further processing and identification of membrane regions.

Define nuclear rim

• Use morphological filtering to define an inner rim of width 3 pixels using the label mask: xy_8bit_labels__nuclei.tif
• (Optional) Measure the mean and total intensity in the first channel of xyc_16bit__nup__nuclei.tif using the modified labels masks.

ImageJ GUI: Clean up segmentation

• Open the image xy_8bit_binary__nuclei_noisy.tif
• [Plugins > MorpholibJ > Filtering > Morphological Filters]
  • operation : Opening
  • element : Square
  • radius : 1
• [Plugins > MorpholibJ > Filtering > Morphological Filters]
  • operation : Closing
  • element : Square
  • radius : 16
• An alternative to the closing operation is also [Plugins > MorpholibJ > Filtering > Fill Holes (Binary/Gray)]

ImageJ GUI: Define nuclear rim

• Open xy_8bit_labels__nuclei.tif
• [Plugins > MorpholibJ > Filtering > Morphological Filters]
  • Operation : Internal Gradient
  • Element : Square
  • Radius : 3
• [ Image › Rename… ]
  • “rim”
• Open xyc_16bit__nup_nuclei
• [Image > Duplicate…]
  • Title : Ch1
  • Duplicate hyperstack
  • Channels (c): 1
• [Plugins > MorpholibJ > Analyze > Intensity Measurements 2D/3D]
  • Input : Ch1
  • Labels : rim

ImageJ Macro: Clean up segmentation

run("Close All");
open("https://github.com/NEUBIAS/training-resources/raw/master/image_data/xyc_16bit__nup_nuclei/xy_8bit_binary__nuclei_noisy.tif");
run("Morphological Filters", "operation=Opening element=Square radius=1");
run("Morphological Filters", "operation=Closing element=Square radius=16");
run("Connected Components Labeling", "connectivity=4 type=[8 bits]");
run("glasbey_on_dark");

ImageJ Macro: Define nuclear rim

run("Close All");
open("https://github.com/NEUBIAS/training-resources/raw/master/image_data/xyc_16bit__nup_nuclei/xy_8bit_labels__nuclei.tif");
run("Morphological Filters", "operation=[Internal Gradient] element=Square radius=3");
rename("rim");
open("https://github.com/NEUBIAS/training-resources/raw/master/image_data/xyc_16bit__nup_nuclei.tif");
run("Duplicate...", "title=Ch1 duplicate channels=1");
run("Intensity Measurements 2D/3D", "input=Ch1 labels=rim mean stddev max min median  numberofvoxels");

Assessment

Fill in the blanks

Using those words fill in the blanks: closing, opening, min, shrinks, decreases, enlarges, max.

1. An erosion _____ objects in a binary image.
2. An erosion in a binary image _____ the number of foreground pixels.
3. A dilation _____ objects in a binary image.
4. An erosion of a binary image correspods to a ___ rank operation.
5. An dilation of a binary image correspods to a ___ rank operation.
6. A dilation followed by an erosion is called ___.
7. An erosion followed by a dilation is called ___ .

Solution

1. shrinks
2. decreases
3. enlarges
4. min
5. max
6. closing
7. opening

True of false?

Discuss with your neighbour!

1. Morphological openings on binary images never decrease the number of foreground pixels.
2. Morphological closings on binary images never decreases the number of foreground pixels.
3. Performing a morphological closing twice in a row does not make sense, because the second closing does not further change the image.
4. Performing a morphological closing with radius 2 element is equivalent to two subsequent closing operation with radius 1.

Solution

1. False
2. True
3. True
4. False

Explanations

Follow-up material

Recommended follow-up modules:

Learn more:

• Morphological gradient (Wikimedia)

• Lecture on filters and segmentation - Refining masks (R. Haase)

• Morphological filters on grayscale images (MorphoLibJ)