I have a Z-stack of 2D confocal microscopy images (2D slices) and I want to segment cells. The Z-stack of 2D images is actually a 3D data. In different slices along the Z-axis, I see same cells do appear in multiple slices. I am interested in cell shape in the XY so I want to preserve the largest cell area from different Z-axis slices. I thought to combine the consecutive 2D slices after converting them to labelled binary images but I am having few issues and I need some help to proceed further.
I have two images img_a and img_b. I first converted them to binary images using OTSU, then applied some morphological operations and then used cv2.connectedComponentsWithStats() to obtain labelled objects. After labeling images, I combined them using cv2.bitwise_or() but it messes up with the labels. You can see this in the attached processed image (cell higlighted by red circles). I see multiple labels for overlapping cell. However, I want to assign one unique label for every combined overlapping object.
What I want at the end is that when I combine two labelled images, I want to assign one single label (a unique value) to the combined overlapping objects and keep the largest cell area by combining both images. Does anyone know how to do it?
Here is the code:
from matplotlib import pyplot as plt
from skimage import io, color, measure
from skimage.util import img_as_ubyte
from skimage.segmentation import clear_border
import cv2
import numpy as np
cells_a=img_a[:,:,1] # get the green channel
#Threshold image to binary using OTSU.
ret_a, thresh_a = cv2.threshold(cells_a, 0, 255, cv2.THRESH_BINARY+cv2.THRESH_OTSU)
# Morphological operations to remove small noise - opening
kernel = np.ones((3,3),np.uint8)
opening_a = cv2.morphologyEx(thresh_a,cv2.MORPH_OPEN,kernel, iterations = 2)
opening_a = clear_border(opening_a) #Remove edge touchingpixels
numlabels_a, labels_a, stats_a, centroids_a = cv2.connectedComponentsWithStats(opening_a)
img_a1 = color.label2rgb(labels_a, bg_label=0)
## now do the same with image_b
cells_b=img_b[:,:,1] # get the green channel
#Threshold image to binary using OTSU.
ret_b, thresh_b = cv2.threshold(cells_b, 0, 255, cv2.THRESH_BINARY+cv2.THRESH_OTSU)
# Morphological operations to remove small noise - opening
opening_b = cv2.morphologyEx(thresh_b,cv2.MORPH_OPEN,kernel, iterations = 2)
opening_b = clear_border(opening_b) #Remove edge touchingpixels
numlabels_b, labels_b, stats_b, centroids_b = cv2.connectedComponentsWithStats(opening_b)
img_b1 = color.label2rgb(labels_b, bg_label=0)
## Now combined two images
combined = cv2.bitwise_or(labels_a, labels_b) ## combined both labelled images to get maximum area per cell
combined_img = color.label2rgb(combined, bg_label=0)
plt.imshow(combined_img)
Images can be found here:
Based on the comments from Christoph Rackwitz and beaker, I started to look around for 3D connected components labeling. I found one python library that can handle such things and I installed it and give it a try. It seems to be doing pretty good. It does assign labels in each slice and keeps the labels same for the same cells in different slices. This is exactly what I wanted.
Here is the link to the library that I used to label objects in 3D. https://pypi.org/project/connected-components-3d/