def test_cwatershed():
S = np.array([
[0,0,0,0],
[0,1,2,1],
[1,1,1,1],
[0,0,1,0],
[1,1,1,1],
[1,2,2,1],
[1,1,2,2]
])
M = np.array([
[0,0,0,0],
[0,0,1,0],
[0,0,0,0],
[0,0,0,0],
[0,0,0,0],
[0,2,0,0],
[0,0,0,0],
])
W = pymorph.cwatershed(2-S,M)
assert np.all(W ==
np.array([[1, 1, 1, 1],
[1, 1, 1, 1],
[1, 1, 1, 1],
[2, 2, 1, 1],
[2, 2, 2, 2],
[2, 2, 2, 2],
[2, 2, 2, 2]]))
def colorscheme(url):
im=image_read(url)
pylab.imshow(im)
dnaf = ndimage.gaussian_filter(im, 1)
rmax = pymorph.regmax(dnaf)
seeds,nr_nuclei = ndimage.label(rmax)
T = mahotas.thresholding.otsu(dnaf)
dist = ndimage.distance_transform_edt(dnaf > T)
dist = dist.max() - dist
dist -= dist.min()
dist = dist/float(dist.ptp()) * 255
dist = dist.astype(np.uint8)
nuclei = pymorph.cwatershed(dist, seeds)
whole = mahotas.segmentation.gvoronoi(nuclei)
im0=pylab.imshow(whole)
pylab.show()
def watershed(img):
# Diminui ruidos
imgf = ndimage.gaussian_filter(img, 16)
mahotas.imsave("dnaf.jpeg", imgf)
rmax = pymorph.regmax(imgf)
T = mahotas.thresholding.otsu(imgf)
dist = ndimage.distance_transform_edt(imgf > T)
dist = dist.max() - dist
dist -= dist.min()
dist = dist / float(dist.ptp()) * 255
dist = dist.astype(np.uint8)
mahotas.imsave("dist.jpeg", dist)
seeds, nr_nuclei = ndimage.label(rmax)
nuclei = pymorph.cwatershed(dist, seeds)
mahotas.imsave("watershed.jpeg", nuclei)
def start(self):
"""Segment frame.
The returned value is a labeled uint16 image.
"""
# Preprocessing: subtract minimum pixel value.
I = self._image - self._image.min()
# 'Bandpass' filtering.
I_s = ndimage.filters.gaussian_filter(I, self._sigma_s) # Foreground.
I_b = ndimage.filters.gaussian_filter(I, self._sigma_b) # Background.
I_bp = I_s - self._alpha * I_b
# Thresholding: create binary image.
I_bin = (I_bp > self._tau)
# Hole filling.
I_bin = ndimage.binary_fill_holes(I_bin > 0)
I_cells = ndimage.label(I_bin)[0]
# Avoid merging nearby cells using watershed.
if self._watershed:
# Distance transfrom on which to apply the watershed algorithm.
I_dist = ndimage.distance_transform_edt(I_bin)
I_dist = I_dist/float(I_dist.max()) * 255
I_dist = I_dist.astype(np.uint8)
# Find markers for the watershed algorithm.
# Reduce false positive using Gaussian smoothing.
I_mask = ndimage.filters.gaussian_filter(I_dist, 8)*I_bin
rmax = pymorph.regmax(I_mask)
I_markers, num_markers = ndimage.label(rmax)
I_dist = I_dist.max() - I_dist # Cells are now the basins.
I_cells = pymorph.cwatershed(I_dist, I_markers)
# Remove cells with area less than threshold.
if self._a_min:
for label in np.unique(I_cells)[1:]:
if (I_cells == label).sum() < self._a_min:
I_cells[I_cells == label] = 0
# Remove cells with summed intensity less than threshold.
if self._s_min:
for label in np.nditer(np.unique(I_cells)[1:]):
if I_bp[I_cells == label].sum() < self._s_min:
I_cells[I_cells == label] = 0
return I_cells.astype('uint16') # This data type is used by ISBI.
def effect(url):
im=image_read(url)
pylab.imshow(im)
dnaf = ndimage.gaussian_filter(im, 1)
rmax = pymorph.regmax(dnaf)
T = mahotas.thresholding.otsu(dnaf)
seeds,nr_nuclei = ndimage.label(rmax)
T = mahotas.thresholding.otsu(dnaf)
T = mahotas.thresholding.otsu(dnaf)
dist = ndimage.distance_transform_edt(dnaf > T)
dist = dist.max() - dist
dist -= dist.min()
dist = dist/float(dist.ptp()) * 255
dist = dist.astype(np.uint8)
nuclei = pymorph.cwatershed(dist, seeds)
pylab.imshow(nuclei)
# pylab.save("out.png")
pylab.show()
def MNUC(datatype, maxrange, outputfile, outputfiletype):
h = open(outputfile, outputfiletype)
TC = 0
for i in range(0, maxrange + 1):
A = datatype[i][0]
rmax = pymorph.regmax(A)
seeds, nr_nuclei = ndimage.label(rmax)
T = mahotas.thresholding.otsu(A)
C = A.copy()
if T < 1:
C[ C <= T ] = 0
C[ C > T ] = 1
else:
C[ C < T ] = 0
C[ C >= T ] = 1
filled = scipy.ndimage.morphology.binary_fill_holes(C)
filled = filled.astype(np.uint8)
dist = ndimage.distance_transform_edt(filled > T)
dist = dist.max() - dist
dist -= dist.min()
dist = dist/float(dist.ptp()) * 255
dist = dist.astype(np.uint8)
nuclei = pymorph.cwatershed(dist, seeds)
find = mahotas.label(nuclei)
for n in range(0, find[1] + 1):
CD = np.where(find[0] == n)
XY1 = np.vstack((CD[0], CD[1]))
edges = filter.canny(XY1, sigma=1)
edges = edges.astype(np.uint8)
edges = np.where(edges == 1)
TC += len(CD[0])
XY1 = np.vstack((CD[0], CD[1], [i*5]*len(CD[0]), [n]*len(CD[0])))
for p in range(0, len(XY1[0])):
for yel in range(0, len(XY1)):
h.write(str(XY1[yel][p]) + '\t')
h.write('\n')
h.write(str(TC) + '\n')
h.write('.' + '\n')
h.close()
def watershedimage(self):
"""Watershed and extract region centers of mass"""
# Generate distribution transform first.
self.distimage()
if not hasattr(self, "labels"):
self.labelimage()
# Load regions if available (takes a bit).
if not self.load_archive("nps"):
self.nps = pymorph.cwatershed(self.dist, self.labels, Bc=numpy.ones((3,3), dtype=bool))
if not self.load_archive("coms"):
self.coms = numpy.array(ndimage.center_of_mass(self.filtered, self.nps, range(1,self.nlabels+1)))
self.regcom = numpy.zeros(self.img.shape, dtype="uint8")
for icom,com in enumerate(self.coms):
self.regcom[round(com[0]),round(com[1])] = 1
self.npcount = len(self.coms)
self.npconc = 10.0**6*self.npcount/self.area
def runWatershed(arr,seeds):
return pymorph.cwatershed(arr,seeds)
pylab.show() writeimg(pymorph.overlay(dna, rmax), 'dnaf-16-rmax-overlay.jpeg') seeds, nr_nuclei = ndimage.label(rmax) print nr_nuclei T = pit.thresholding.otsu(dnaf) dist = ndimage.distance_transform_edt(dnaf > T) dist = dist.max() - dist dist = ((dist - dist.min()) / float(dist.ptp()) * 255).astype(np.uint8) pylab.imshow(dist) pylab.show() savejet(dist, 'dnaf-16-dist.jpeg') nuclei = pymorph.cwatershed(dist, seeds) pylab.imshow(nuclei) pylab.show() savejet(nuclei, 'nuclei-segmented.png') whole = pit.segmentation.gvoronoi(nuclei) pylab.imshow(whole) pylab.show() savejet(whole, 'whole-segmented.png') borders = np.zeros(nuclei.shape, np.bool) borders[0, :] = 1 borders[-1, :] = 1 borders[:, 0] = 1 borders[:, -1] = 1 at_border = np.unique(nuclei[borders])
def _segment(self, I, first):
"""Return the segmented frame 'I'.
If 'first is True, then this is the first segmentation iteration,
otherwise the second.
The returned value is a labeled image of type uint16, in order to be
compatible with ISBI's tool.
"""
# Compute global threshold.
otsu_thresh = mh.thresholding.otsu(I.astype('uint16'))
# Threshold using global and local thresholds.
fnc = fnc_class(I.shape)
I_bin = ndimage.filters.generic_filter(I, fnc.filter, size=self._r,
extra_arguments=(I, self._min_var,
otsu_thresh))
I_med = ndimage.filters.median_filter(I_bin, size=self._r_med)
# Remove cells which are too small (leftovers).
labeled = mh.label(I_med)[0]
sizes = mh.labeled.labeled_size(labeled)
too_small = np.where(sizes < self._a_min)
I_cleanup = mh.labeled.remove_regions(labeled, too_small)
I_cleanup = mh.labeled.relabel(I_cleanup)[0]
# Fill holes.
I_holes = ndimage.morphology.binary_fill_holes(I_cleanup > 0)
# Binary closing.
if first and self._r1:
# First iteration.
I_morph = morph.binary_closing(I_holes, morph.disk(self._r1))
elif not first and self._r2:
# Second iteration.
I_morph = morph.binary_closing(I_holes, morph.disk(self._r2))
else:
# No binary closing.
I_morph = I_holes
# Fill yet to be filled holes.
labels = measure.label(I_morph)
labelCount = np.bincount(labels.ravel())
background = np.argmax(labelCount)
I_morph[labels != background] = True
# Separate touching cells using watershed.
# Distance transfrom on which to apply the watershed algorithm.
I_dist = ndimage.distance_transform_edt(I_morph)
I_dist = I_dist/float(I_dist.max()) * 255
I_dist = I_dist.astype(np.uint8)
# Find markers for the watershed algorithm.
# Reduce false positive using Gaussian smoothing.
I_mask = ndimage.filters.gaussian_filter(I_dist, 8)*I_morph
rmax = pymorph.regmax(I_mask)
I_markers, _ = ndimage.label(rmax)
I_dist = I_dist.max() - I_dist # Cells are now the basins.
I_label = pymorph.cwatershed(I_dist, I_markers)
if self._debug:
plt.subplot(2, 4, 1)
plt.imshow(I)
plt.title('Original Image')
plt.subplot(2, 4, 2)
plt.imshow(I_bin)
plt.title('After Thresholding')
plt.subplot(2, 4, 3)
plt.imshow(I_med)
plt.title('After Median Filter')
plt.subplot(2, 4, 4)
plt.imshow(I_cleanup)
plt.title('After Cleanup')
plt.subplot(2, 4, 5)
plt.imshow(I_holes)
plt.title('After Hole Filling')
plt.subplot(2, 4, 6)
plt.imshow(I_morph)
plt.title('After Closing')
plt.subplot(2, 4, 7)
plt.imshow(I_label)
plt.title('Labeled Image')
plt.show()
return I_label.astype('uint16')
def main():
# Load and show original images
pylab.gray() # set gray scale mode
print
print "0. Reading and formatting images..."
images = {f: loadAndFormat(f) for f in IMAGE_FILES}
for f in IMAGE_FILES:
mShow(images[f])
###########################
# -----> Thresholding
print
print "1. Thresholding images..."
thresholdedImages = {f: getThresholdedImage(images[f]) for f in IMAGE_FILES}
for name in IMAGE_FILES:
mShow(thresholdedImages[name])
###########################
# -----> Count objects
# 1st attempt: label the thresholded image from task 1
print
print "2. Object counting"
pylab.jet() # back to color mode
print "\t1st approach: Label thresholded images"
for name in IMAGE_FILES:
labeled, nrRegions = ndimage.label(thresholdedImages[name])
print "\t" + name + ": " + str(nrRegions)
mShow(labeled)
# 2nd attempt: Changing threshold level
print
print "\t2nd approach: Tuned thresholds"
# For 'objects.png' some objects are very small (e.g.: screw) or
# have many shades (e.g.: spoon) which makes them disappear or appear
# fragmented after thresholding.
# The advantage of this image is that the background is very dark,
# so we can try using a lower threshold to make all shapes more definite
objImage = images['objects.png']
T = mahotas.thresholding.otsu(objImage)
thresholdedImage = objImage > T * 0.7
# Looks better, but...
labeled, nrRegions = ndimage.label(thresholdedImage)
print '\tobjects.png' + ": " + str(nrRegions)
# it returns 18!
# 3rd attempt: Smoothing before thresholding
print
print "\t3rd approach: Smoothing + Tuned threshold"
# Let's apply some Gaussian smoothing AND a lower threshold
smoothImage = ndimage.gaussian_filter(objImage, 3)
T = mahotas.thresholding.otsu(smoothImage)
thresholdedImage = smoothImage > T * 0.7
labeled, nrRegions = ndimage.label(thresholdedImage)
print '\tobjects.png' + ": " + str(nrRegions)
# it worked! Let's save the labeled images for later
# (we will use them for center calculation)
labeledImages = {}
labeledImages['objects.png'] = (labeled, nrRegions)
mShow(labeled)
# Let's see if this approach works on the other images
for name in ['circles.png', 'peppers.png']:
img = images[name]
smoothImage = ndimage.gaussian_filter(img, 3)
T = mahotas.thresholding.otsu(smoothImage)
thresholdedImage = smoothImage > T * 0.7
labeled, nrRegions = ndimage.label(thresholdedImage)
print '\t' + name + ": " + str(nrRegions)
# Again no luck with the circles!
# (We will take a closer look at the peppers later)
# 4th attempt:
# 'circles.png': The problem is that some circles appear "glued together".
# Let's try another technique:
# - smoothing the picture with a Gaussian filter
# - then searching for local maxima and counting regions
# (smoothing avoids having many scatter maxima and a higher level
# must be used than in the previous attempt)
# - use watershed with the maxima as seeds over the thresholded image
# to complete the labelling of circles
print
print "\t4th approach: Smoothing + Local maxima + Watershed"
smoothImage = ndimage.gaussian_filter(images['circles.png'], 10)
localmaxImage = pymorph.regmax(smoothImage)
# A distance transform must be applied before doing the watershed
dist = ndimage.distance_transform_edt(thresholdedImages['circles.png'])
dist = dist.max() - dist
dist -= dist.min()
dist = dist / float(dist.ptp()) * 255
dist = dist.astype(np.uint8)
seeds, nrRegions = ndimage.label(localmaxImage)
labeled = pymorph.cwatershed(dist, seeds)
print "\t" + 'circles.png' + ": " + str(nrRegions)
# worked right only for 'circles.png' !
labeledImages['circles.png'] = (labeled, nrRegions)
mShow(labeled)
print
print "\t5th approach: Smoothing + Multi-threshold +" +\
" Morphology labeling + Size filtering"
# 5th approach (only peppers left!)
imagePeppers = images['peppers.png']
# Problems with peppers are:
# - very different colours, they cause thresholding to work poorly
# - each pepper has some brighter parts which are detected as local maxima
# We propose to address those issues as follows:
# - gaussian filter to smooth regions of light or shadow within each pepper
smoothImage = ndimage.gaussian_filter(imagePeppers, 2)
# - instead of thresholding to create a binary image,
# create multiple thresholds to separate the most frequent colors.
# In this case, 3 thresholds will be enough
mthrImagePeppers = multiThreshold(smoothImage, 3)
# - ndimage.label didn't give good results, we try another
# labelling algorithm
from skimage import morphology
labeled = morphology.label(mthrImagePeppers)
nrRegions = np.max(labeled) + 1
print "\t\tTotal number of regions"
print "\t\t\t" + 'peppers.png' + ": " + str(nrRegions)
# - after counting regions, filter to keep only the sufficiently big ones
filtered, nrRegions = filterRegions(labeled, 0.05)
print "\t\tBig enough regions"
print "\t\t\t" + 'peppers.png' + ": " + str(nrRegions)
labeledImages['peppers.png'] = (filtered, nrRegions)
mShow(filtered)
###########################
# -----> Find center points
print
print "3. Centers for objects"
for img in IMAGE_FILES:
labeledImage, nr_objects = labeledImages[img]
CenterOfMass = ndimage.measurements.center_of_mass
labels = range(1, nr_objects + 1)
centers = CenterOfMass(labeledImage, labeledImage, labels)
centers = [(int(round(x)), int(round(y))) for (x, y) in centers]
print '\t' + img + ": " + str(centers)
pylab.imshow(pymorph.overlay(dna, rmax)) seeds,nr_nuclei = ndimage.label(rmax) print nr_nuclei # prints 22 # Watershed to distance transform of threshold T = mahotas.thresholding.otsu(dnaf) dist = ndimage.distance_transform_edt(dnaf > T) dist = dist.max() - dist dist -= dist.min() dist = dist/float(dist.ptp()) * 255 dist = dist.astype(np.uint8) pylab.imshow(dist) pylab.show() nuclei = pymorph.cwatershed(dist, seeds) pylab.imshow(nuclei) pylab.show() # Extend segmentation to whole plane using generalized voronoi - each px -> nearest nucleus whole = mahotas.segmentation.gvoronoi(nuclei) pylab.imshow(whole) pylab.show() # Quality control - remove cells whose nucleus touches border borders = np.zeros(nuclei.shape, np.bool) # builds an array of zeroes with nuclei shape and np.bool borders[ 0,:] = 1 borders[-1,:] = 1 borders[:, 0] = 1 borders[:,-1] = 1 # Sets borders of array to 1/True at_border = np.unique(nuclei[borders]) # nuclei[borders] gets True borders, unique returns only unique values
import ImageEnhance
I_contrast = pow(I_show.astype("uint16"), 3)
#find regional maxima
regionalMax = pymorph.regmax((I_contrast).astype("uint16"))
#find seeds and cell number
seeds,numCells = ndimage.label(regionalMax)
print numCells
#edge detection
T = mahotas.thresholding.otsu(I_contrast.astype("uint16"))
dist = ndimage.distance_transform_edt(I_contrast.astype("uint16") > T)
dist = dist.max() - dist
dist -= dist.min()
dist = dist/float(dist.ptp()) * 255
dist = dist.astype(np.uint8)
#watershed
I_mask = pymorph.cwatershed(dist, seeds)
frames, x, y = Is.shape
label_mask, num_cells = ndimage.label(I_mask.astype(bool))
print frames
print num_cells
traces = np.zeros((frames, num_cells))
baselined_traces = np.zeros_like(traces)
normed_traces = np.zeros_like(traces)
for cell in range(1,num_cells):
traces[:,cell] = Is[:,label_mask==cell].mean(axis=1)
print "Found %d unique cells" % traces.shape[1]
print traces
