def stk_to_rois(stk, threshold, min_size, max_window=8, downscale_factor=2):
thresholded_stk = stk > threshold
thresholded_stk = remove_small_objects(thresholded_stk, min_size)
distance = ndi.distance_transform_edt(thresholded_stk)
cropped_stk = stk.copy()
cropped_stk[np.logical_not(thresholded_stk)] = 0
combined_stk = cropped_stk + distance/distance.max()
local_max = peak_local_max(combined_stk, indices=False,
footprint=np.ones((max_window, max_window)),
labels=thresholded_stk)
markers = ndi.label(local_max)[0]
labels = watershed(-combined_stk, markers, mask=thresholded_stk)
new_markers = markers.copy()
for i in set(labels.flatten()):
if i == 0: continue
if np.sum(labels==i) < min_size:
new_markers[markers==i] = 0
labels = watershed(-combined_stk, new_markers, mask=thresholded_stk)
labels_set = set(labels.flatten())
rois = []
for label in labels_set:
if label == 0: continue
if np.sum((labels==label).astype(int)) < min_size: continue
nroi = np.zeros((stk.shape[0], stk.shape[1]))
cx,cy = np.where(labels==label)
cx,cy = int(cx.mean()), int(cy.mean())
x,y = np.ogrid[0:nroi.shape[0], 0:nroi.shape[1]]
r = 4
mask = (cx-x)**2 + (cy-y)**2 <= r*r
nroi[mask] = 1
#nroi[labels==label] = 1
rois.append(zoom(nroi, downscale_factor, order=0))
rois = np.array(rois)
return rois, thresholded_stk, labels
def do_watershed(image, markers, tfile, shape, bstruct, algorithm, mg_size, use_ww_wl, wl, ww, q):
mask = np.memmap(tfile, shape=shape, dtype='uint8', mode='r+')
if use_ww_wl:
if algorithm == 'Watershed':
tmp_image = ndimage.morphological_gradient(
get_LUT_value(image, ww, wl).astype('uint16'),
mg_size)
tmp_mask = watershed(tmp_image, markers.astype('int16'), bstruct)
else:
tmp_image = get_LUT_value(image, ww, wl).astype('uint16')
#tmp_image = ndimage.gaussian_filter(tmp_image, self.config.mg_size)
#tmp_image = ndimage.morphological_gradient(
#get_LUT_value(image, ww, wl).astype('uint16'),
#self.config.mg_size)
tmp_mask = watershed_ift(tmp_image, markers.astype('int16'), bstruct)
else:
if algorithm == 'Watershed':
tmp_image = ndimage.morphological_gradient((image - image.min()).astype('uint16'), mg_size)
tmp_mask = watershed(tmp_image, markers.astype('int16'), bstruct)
else:
tmp_image = (image - image.min()).astype('uint16')
#tmp_image = ndimage.gaussian_filter(tmp_image, self.config.mg_size)
#tmp_image = ndimage.morphological_gradient((image - image.min()).astype('uint16'), self.config.mg_size)
tmp_mask = watershed_ift(tmp_image, markers.astype('int8'), bstruct)
mask[:] = tmp_mask
mask.flush()
q.put(1)
def analyse(self, **kwargs):
image_object = kwargs['image']
if image_object is None:
raise RuntimeError()
# Read the image
image = cv2.imread(self.image_utils.getOutputFilename(image_object.id))
if image is None:
print('File not found')
return
# Work on green channel
gray = image[:, :, 1]
# Apply otsu thresholding
thresh = filters.threshold_otsu(gray)
gray[gray < thresh] = 0
# Apply histogram equalization
gray = exposure.equalize_adapthist(gray) * 255
# Create elevation map
elevation_map = filters.sobel(gray)
gray = gray.astype(int)
# Create cell markers
markers = numpy.zeros_like(gray)
markers[gray < 100] = 2 # seen as white in plot
markers[gray > 150] = 1 # seen as black in plot
# Segment with watershed using elevation map
segmentation = morphology.watershed(elevation_map, markers)
segmentation = ndi.binary_fill_holes(segmentation - 1)
# labeled_image, n = ndi.label(segmentation)
# Watershed with distance transform
kernel = numpy.ones((5, 5), numpy.uint8)
distance = ndi.distance_transform_edt(segmentation)
distance2 = cv2.erode(distance, kernel)
distance2 = cv2.dilate(distance2, kernel)
local_max = peak_local_max(distance2, num_peaks=1, indices=False, labels=segmentation)
markers2 = ndi.label(local_max)[0]
labels = morphology.watershed(-distance2, markers2, mask=segmentation)
# Extract regions (caching signifies more memory use)
regions = regionprops(labels, cache=True)
# Filter out big wrong regions
regions = [region for region in regions if region.area < 2000]
# Set result
result = str(len(regions))
return result
def run(self, workspace):
labeled_nuclei = workspace.object_set.get_objects(self.primary_objects.value).get_segmented()
cell_image = workspace.image_set.get_image(self.image_name.value).pixel_data[:,:]
image_collection = []
cell_treshold = otsu(cell_image, min_threshold=0, max_threshold=1)
cell_binary = (cell_image >= cell_treshold)
cell_distance = scipym.distance_transform_edt(cell_binary).astype(np.uint16)
cell_labeled = skm.watershed(-cell_distance, labeled_nuclei, mask=cell_binary)
#
#fil hall and filter on syze the object in cell_labeled
#
cell_labeled = self.filter_on_border(cell_labeled)
cell_labeled = fill_labeled_holes(cell_labeled)
objects = cellprofiler.objects.Objects()
objects.segmented = cell_labeled
objects.parent_image = cell_image
workspace.object_set.add_objects(objects, self.object_name.value)
image_collection.append((cell_image, "Original"))
image_collection.append((cell_labeled, "Labelized image"))
workspace.display_data.image_collection = image_collection
def __call__(self, image, window_size=10, threshold=0, fill_holes=True,
outline_smoothing=2, remove_borderobjects=True, size_min=1,
*args, **kw):
thresh = threshold_adaptive(image, block_size=window_size,
offset=-1*threshold)
if outline_smoothing >= 1:
thresh = outlineSmoothing(thresh, outline_smoothing)
thresh = remove_small_objects(thresh, size_min)
seeds = ndi.label(clear_border(~thresh))[0]
thresh = ndi.binary_fill_holes(thresh)
smask = seeds.astype(bool)
# object don't touch border after outline smoothing
if remove_borderobjects:
thresh = clear_border(thresh)
img = np.zeros(thresh.shape)
img[~smask] = 1
edt = ndi.morphology.distance_transform_edt(img)
edt -= ndi.morphology.distance_transform_edt(seeds)
labels = watershed(edt, seeds)
labels[smask] = 0
labels[~thresh] = 0
return labels
def watershed_with_seeds(image, seed_image, mask_image=None, name='watershed'):
"""Perform watershed segmentation from given seeds. Inputs should be of the
form:
image : grayscale image, with higher values representing more signal
seed_image : grayscale image where each pixel value represents a unique
region"""
if mask_image is None:
mask = None
else:
mask = mask_image.image_array
# We multiply the image by -1 because the algorithm implementation expects
# higher values to be easier for the 'water' to pass
segmented = watershed(-image.image_array,
seed_image.image_array,
mask=mask)
ia = ImageArray(segmented, name)
ia.history = image.history + [name]
return ia
def segment(self, src):
image = src.ndarray[:]
if self.use_adaptive_threshold:
block_size = 25
markers = threshold_adaptive(image, block_size) * 255
markers = invert(markers)
else:
markers = zeros_like(image)
markers[image < self.threshold_low] = 1
markers[image > self.threshold_high] = 255
elmap = sobel(image, mask=image)
wsrc = watershed(elmap, markers, mask=image)
# elmap = ndimage.distance_transform_edt(image)
# local_maxi = is_local_maximum(elmap, image,
# ones((3, 3))
# )
# markers = ndimage.label(local_maxi)[0]
# wsrc = watershed(-elmap, markers, mask=image)
# fwsrc = ndimage.binary_fill_holes(out)
# return wsrc
if self.use_inverted_image:
out = invert(wsrc)
else:
out = wsrc
# time.sleep(1)
# do_later(lambda:self.show_image(image, -elmap, out))
return out
def label_nuclei(binary, min_size):
'''Label, watershed and remove small objects'''
distance = medial_axis(binary, return_distance=True)[1]
distance_blured = gaussian_filter(distance, 5)
local_maxi = peak_local_max(distance_blured, indices=False, labels=binary, min_distance = 30)
markers = measure_label(local_maxi)
# markers[~binary] = -1
# labels_rw = segmentation.random_walker(binary, markers)
# labels_rw[labels_rw == -1] = 0
# labels_rw = segmentation.relabel_sequential(labels_rw)
labels_ws = watershed(-distance, markers, mask=binary)
labels_large = remove_small_objects(labels_ws,min_size)
labels_clean_border = clear_border(labels_large)
labels_from_one = relabel_sequential(labels_clean_border)
# plt.imshow(ndimage.morphology.binary_dilation(markers))
# plt.show()
return labels_from_one[0]
def segment(self, src):
'''
pychron: preprocessing cv.Mat
'''
# image = pychron.ndarray[:]
# image = asarray(pychron)
image = src[:]
if self.use_adaptive_threshold:
# block_size = 25
markers = threshold_adaptive(image, self.block_size)
n = markers[:].astype('uint8')
n[markers == True] = 255
n[markers == False] = 1
markers = n
else:
markers = zeros_like(image)
markers[image < self.threshold_low] = 1
markers[image > self.threshold_high] = 255
elmap = sobel(image, mask=image)
wsrc = watershed(elmap, markers, mask=image)
# wsrc = wsrc.astype('uint8')
return invert(wsrc)
def segment_out_cells(base):
# TODO: try using OTSU for GFP thresholding
sel_elem = disk(2)
gfp_collector = np.sum(base, axis=0)
gfp_clustering_markers = np.zeros(gfp_collector.shape, dtype=np.uint8)
# random walker segment
gfp_clustering_markers[gfp_collector > np.mean(gfp_collector) * 2] = 2
gfp_clustering_markers[gfp_collector < np.mean(gfp_collector) * 0.20] = 1
labels = random_walker(gfp_collector, gfp_clustering_markers, beta=10, mode='bf')
# round up the labels and set the background to 0 from 1
labels = closing(labels, sel_elem)
labels -= 1
# prepare distances for the watershed
distance = ndi.distance_transform_edt(labels)
local_maxi = peak_local_max(distance,
indices=False, # we want the image mask, not peak position
min_distance=10, # about half of a bud with our size
threshold_abs=10, # allows to clear the noise
labels=labels)
# we fuse the labels that are close together that escaped the min distance in local_maxi
local_maxi = ndi.convolve(local_maxi, np.ones((5, 5)), mode='constant', cval=0.0)
# finish the watershed
expanded_maxi_markers = ndi.label(local_maxi, structure=np.ones((3, 3)))[0]
segmented_cells_labels = watershed(-distance, expanded_maxi_markers, mask=labels)
# log debugging data
running_debug_frame.gfp_collector = gfp_collector
running_debug_frame.gfp_clustering_markers = gfp_clustering_markers
running_debug_frame.labels = labels
running_debug_frame.segmented_cells_labels = segmented_cells_labels
return gfp_collector, segmented_cells_labels
def split_object(self, labeled_image):
""" split object when it's necessary
"""
labeled_image = labeled_image.astype(np.uint16)
labeled_mask = np.zeros_like(labeled_image, dtype=np.uint16)
labeled_mask[labeled_image != 0] = 1
#ift structuring element about center point. This only affects eccentric structuring elements (i.e. selem with even num===============================
labeled_image = skr.median(labeled_image, skm.disk(4))
labeled_mask = np.zeros_like(labeled_image, dtype=np.uint16)
labeled_mask[labeled_image != 0] = 1
distance = scipym.distance_transform_edt(labeled_image).astype(np.uint16)
#=======================================================================
# binary = np.zeros(np.shape(labeled_image))
# binary[labeled_image > 0] = 1
#=======================================================================
distance = skr.mean(distance, skm.disk(15))
l_max = skr.maximum(distance, skm.disk(5))
#l_max = skf.peak_local_max(distance, indices=False,labels=labeled_image, footprint=np.ones((3,3)))
l_max = l_max - distance <= 0
l_max = skr.maximum(l_max.astype(np.uint8), skm.disk(6))
marker = ndimage.label(l_max)[0]
split_image = skm.watershed(-distance, marker)
split_image[split_image[0,0] == split_image] = 0
return split_image
def _segment_watershed(image): elevation_map = sobel(image) markers = np.zeros(image.shape) # initialize markers as zero array # determine thresholds for markers sorted_pixels = np.sort(image, axis=None) max_int = np.mean(sorted_pixels[-10:]) min_int = np.mean(sorted_pixels[:10]) #max_int = np.max(orig_image) #min_int = np.min(orig_image) alpha_min = 0.01 alpha_max = 0.4 thresh_background = (1-alpha_min)*min_int + alpha_min*max_int thresh_spots = (1-alpha_max)*min_int + alpha_max*max_int markers[image < thresh_background] = 1 # mark background markers[image > thresh_spots] = 2 # mark background segmentation = watershed(elevation_map, markers) segmentation = segmentation-1 segmentation = ndi.binary_fill_holes(segmentation) # fill holes return segmentation
def testSkimage():
img = Image.open('../img/1.png')
img = np.array(img)
imggray = cv2.cvtColor(img, cv2.COLOR_BGR2GRAY)
# (thresh, imgbw) = cv2.threshold(imggray, 128, 255, cv2.THRESH_BINARY | cv2.THRESH_OTSU)
# canny detector
# from skimage.feature import canny
# edges = canny(imggray/ 255.)
from scipy import ndimage as ndi
# fill_imgbw = ndi.binary_fill_holes(edges)
# label_objects, nb_labels = ndi.label(fill_imgbw)
# sizes = np.bincount(label_objects.ravel())
# mask_sizes = sizes > 20
# mask_sizes[0] = 0
# cleaned_imgbw = mask_sizes[label_objects]
markers = np.zeros_like(imggray)
markers[imggray < 120] = 1
markers[imggray > 150] = 2
from skimage.filters import sobel
elevation_map = sobel(imggray)
from skimage.morphology import watershed
segmentation = watershed(elevation_map, markers)
# from skimage.color import label2rgb
# segmentation = ndi.binary_fill_holes(segmentation - 10)
# labeled_coins, _ = ndi.label(segmentation)
# image_label_overlay = label2rgb(labeled_coins, image=imggray)
plt.imshow(segmentation, cmap='gray')
plt.show()
return
def detectOpticDisc(image):
kernel = octagon(10, 10)
thresh = threshold_otsu(image[:,:,1])
binary = image > thresh
print binary.dtype
luminance = convertToHLS(image)[:,:,2]
t = threshold_otsu(luminance)
t = erosion(luminance, kernel)
labels = segmentation.slic(image[:,:,1], n_segments = 3)
out = color.label2rgb(labels, image[:,:,1], kind='avg')
skio.imshow(out)
x, y = computeCentroid(t)
print x, y
rows, cols, _ = image.shape
p1 = closing(image[:,:,1],kernel)
p2 = opening(p1, kernel)
p3 = reconstruction(p2, p1, 'dilation')
p3 = p3.astype(np.uint8)
#g = dilation(p3, kernel)-erosion(p3, kernel)
#g = rank.gradient(p3, disk(5))
g = cv2.morphologyEx(p3, cv2.MORPH_GRADIENT, kernel)
#markers = rank.gradient(p3, disk(5)) < 10
markers = drawCircle(rows, cols, x, y, 85)
#markers = ndimage.label(markers)[0]
#skio.imshow(markers)
g = g.astype(np.uint8)
#g = cv2.cvtColor(g, cv2.COLOR_GRAY2RGB)
w = watershed(g, markers)
print np.max(w), np.min(w)
w = w.astype(np.uint8)
#skio.imshow(w)
return w
def segment(self, image):
"""
"""
# image = src[:]
if self.use_adaptive_threshold:
bs = self.blocksize
if not bs % 2:
bs += 1
markers = threshold_adaptive(image, bs)
# n = markers[:].astype('uint8')
n = markers.astype('uint8')
# n[markers] = 255
# n[invert(markers)] = 1
# markers = n
return n
else:
markers = zeros_like(image)
# print('image',image.max(), image.min())
# print('le', image<self.threshold_low)
# print('ge', image>self.threshold_high)
markers[image <= self.threshold_low] = 1
markers[image > self.threshold_high] = 255
#elmap = sobel(image, mask=image)
elmap = canny(image, sigma=1)
wsrc = watershed(elmap, markers, mask=image)
return invert(wsrc)
def black_background(image, kernel):
shifted = cv2.pyrMeanShiftFiltering(image, 10, 39)
gray = cv2.cvtColor(shifted, cv2.COLOR_BGR2GRAY)
thresh = cv2.threshold(gray, 0, 255,
cv2.THRESH_BINARY | cv2.THRESH_OTSU)[1]
D = ndimage.distance_transform_edt(thresh)
localMax = peak_local_max(D, indices=False, min_distance=10,
labels=thresh)
# perform a connected component analysis on the local peaks,
# using 8-connectivity, then appy the Watershed algorithm
markers = ndimage.label(localMax, structure=np.ones((3, 3)))[0]
labels = watershed(-D, markers, mask=thresh)
# create a mask
mask2 = np.zeros(gray.shape, dtype="uint8")
# loop over the unique labels returned by the Watershed algorithm for
for label in np.unique(labels):
# if the label is zero, we are examining the 'background' so simply ignore it
if label == 0:
continue
# otherwise, allocate memory for the label region and draw
# it on the mask
mask2[labels == label] = 255
return mask2
def segment(self, data, peaks):
"""Perform a watershed segmentation based on local maxima."""
markers = np.zeros_like(data)
markers[tuple(peaks.T)] = np.arange(len(peaks)) + 1
seg = morphology.watershed(-data, markers, mask=data > 0)
return seg, markers
def Image_ws_tranche(image):
laser = Detect_laser(image)
laser_tranche = tranche_image(laser,60)
image_g = skimage.color.rgb2gray(image)
image_g = image_g * laser_tranche
image_med = rank2.median((image_g*255).astype('uint8'),disk(8))
image_clahe = exposure.equalize_adapthist(image_med, clip_limit=0.03)
image_clahe_stretch = exposure.rescale_intensity(image_clahe, out_range=(0, 256))
image_grad = rank2.gradient(image_clahe_stretch,disk(3))
image_grad_mark = image_grad<20
image_grad_forws = rank2.gradient(image_clahe_stretch,disk(1))
image_grad_mark_closed = closing(image_grad_mark,disk(1))
Labelised = (skimage.measure.label(image_grad_mark_closed,8,0))+1
Watersheded = watershed(image_grad_forws,Labelised)
cooc = coocurence_liste(Watersheded,laser,3)
x,y = compte_occurences(cooc)
return x,y
def segmentation(file_name):
data_x, data_y, data_z = get_data(file_name)
shape_x = len(np.unique(data_x))
shape_y = len(np.unique(data_y))
X = data_x.reshape(shape_x, shape_y)
Y = data_y.reshape(shape_x, shape_y)
Z = data_z.reshape(shape_x, shape_y)
markers = np.zeros_like(Z)
markers[Z < 0.15] = 1
markers[Z > 0.3] = 2
elevation_map = roberts(Z)
fig, (ax1, ax2) = plt.subplots(1, 2, figsize=(6, 3), sharex=True, sharey=True)
# ax.imshow(Z)
# ax.imshow(elevation_map, cmap=plt.cm.jet, interpolation='nearest')
segmentation = watershed(elevation_map, markers)
ax2.imshow(segmentation, interpolation='nearest')
# ax.axis('off')
# ax.set_title('segmentation')
segmentation = ndi.binary_fill_holes(segmentation - 1)
labeled_coins, _ = ndi.label(segmentation)
ax1.imshow(Z, cmap=plt.cm.gray, interpolation='nearest')
ax1.contour(segmentation, [0.5], linewidths=1.2, colors='y')
ax1.axis('off')
ax1.set_adjustable('box-forced')
plt.show()
def watershed(image):
hsv_image = color.rgb2hsv(image)
low_res_image = rescale(hsv_image[:, :, 0], SCALE)
local_mean = mean(low_res_image, disk(50))
local_minimum_flat = np.argmin(local_mean)
local_minimum = np.multiply(np.unravel_index(local_minimum_flat, low_res_image.shape), round(1 / SCALE))
certain_bone_pixels = np.full_like(hsv_image[:, :, 0], False, bool)
certain_bone_pixels[
local_minimum[0] - INITIAL_WINDOW_SIZE/2:local_minimum[0]+INITIAL_WINDOW_SIZE/2,
local_minimum[1] - INITIAL_WINDOW_SIZE/2:local_minimum[1]+INITIAL_WINDOW_SIZE/2
] = True
certain_non_bone_pixels = np.full_like(hsv_image[:, :, 0], False, bool)
certain_non_bone_pixels[0:BORDER_SIZE, :] = True
certain_non_bone_pixels[-BORDER_SIZE:-1, :] = True
certain_non_bone_pixels[:, 0:BORDER_SIZE] = True
certain_non_bone_pixels[:, -BORDER_SIZE:-1] = True
smoothed_hsv = median(hsv_image[:, :, 0], disk(50))
threshold = MU * np.median(smoothed_hsv[certain_bone_pixels])
possible_bones = np.zeros_like(hsv_image[:, :, 0])
possible_bones[smoothed_hsv < threshold] = 1
markers = np.zeros_like(possible_bones)
markers[certain_bone_pixels] = 1
markers[certain_non_bone_pixels] = 2
labels = morphology.watershed(-possible_bones, markers)
return labels
def find_neighbors(self, imbin, max_extension):
background = np.zeros(imbin.shape)
background[imbin==0] = 255
distance = ndi.distance_transform_edt(background)
cell_labels = label(imbin, neighbors=4, background=0)
# this is a hack, as background pixels obtain label -1
# and we do not want to have negative values.
# from version 0.12 this can be removed, probably.
cell_labels = cell_labels - cell_labels.min()
# the mask is an extension of the initial shape by max_extension.
# it can be derived from the distance map (straight forward)
mask = np.zeros(imbin.shape)
mask[distance < max_extension] = 255
# The watershed of the distance transform of the background.
# this corresponds an approximation of the "cells"
labels = watershed(distance, cell_labels, mask=mask)
if self.settings.debug_screen_output:
out_filename = os.path.join(self.settings.img_debug_folder, '%s16_distance.png' % self.prefix)
temp = distance / distance.max()
skimage.io.imsave(out_filename, temp)
out_filename = os.path.join(self.settings.img_debug_folder, '%s16_labels_from_ws.png' % self.prefix)
skimage.io.imsave(out_filename, labels)
return cell_labels, labels
def segment(image, thresh):
#preprocess image
gray = cv2.cvtColor(image, cv2.COLOR_BGR2GRAY)
#perform euclidean distance transform
distances = ndimage.distance_transform_edt(thresh)
localMax = peak_local_max(distances, indices = False, min_distance = 3, labels = thresh)
#perform connected component analysis on local peaks
markers = ndimage.label(localMax, structure = np.ones((3, 3)))[0]
labels = watershed(-distances, markers, mask = thresh)
#loop over labels returned from watershed to mark them
for label in np.unique(labels):
if label == 0:
continue
mask = np.zeros(gray.shape, dtype="uint8")
mask[labels == label] = 255
#find contours in mask and choose biggest contour by area
contours = cv2.findContours(mask.copy(), cv2.RETR_EXTERNAL, cv2.CHAIN_APPROX_SIMPLE)[-2]
contour = max(contours, key = cv2.contourArea)
#draw circle around max size contour
((x, y), r) = cv2.minEnclosingCircle(contour)
cv2.circle(image, (int(x), int(y)), int(r), (0, 255, 0), 2)
#show final image
cv2.imshow("Output", image)
return len(np.unique(labels) - 1)
def waterShed(blob, shape): img = np.zeros(shape, np.uint16) img[zip(*blob)] = 99999 D = ndimage.distance_transform_edt(img) mindist = 7 labels = [1,2,3,4] while len(np.unique(labels)) > 3: mindist += 1 localMax = peak_local_max(D, indices=False, min_distance=mindist, labels=img) markers = ndimage.label(localMax, structure=np.ones((3,3)))[0] labels = watershed(-D, markers, mask=img) subBlobs = [] for label in np.unique(labels): if label == 0: continue ww = np.where(labels==label) bb = zip(ww[0], ww[1]) subBlobs.append(bb) # code.interact(local=locals()) try: return subBlobs, zip(np.where(localMax==True)[0],np.where(localMax==True)[1])[0] except IndexError: return subBlobs, 0
def ImageSegmentation(self):
kernel = np.array(self.coords_cell, np.int32)
circle = np.zeros(self.image.shape[:2], np.uint8)
# link with polylines the coordinates of "left click", thickness could be adjusted,
# Could also fill inside the polyline
cv2.polylines(circle,[kernel],False,(255,0,0), thickness=5)
kernel2 = np.array(self.coords_cell, np.int32)
circle2 = np.zeros(self.image.shape[:2], np.uint8)
cv2.polylines(circle2,[kernel2],False,(255,0,0), thickness=4)
# Segmentation of the protein accumulation using watershed
self.segmentation = morphology.watershed(self.clean_image, self.markers, mask = circle)
self.segmentation[self.segmentation < 1.5] = 0
self.segmentation = self.segmentation.astype('uint8')
# Find contour of the segmented area
contours,hierarchy = cv2.findContours(self.segmentation, 1, 2)
# Find countour of the masked area
contours_circle,hierarchy = cv2.findContours(circle2, 1, 2)
self.area = [cv2.contourArea(cnt) for cnt in contours if (cv2.contourArea(cnt))!=0.0]
self.area = sum(self.area)
self.area_mask = [cv2.contourArea(cnt_cell) for cnt_cell in contours_circle]
self.area_mask = sum(self.area_mask)
if self.area > 0:
self.surface_segmented.append(self.area)
if self.area_mask > 0:
self.surface_masked.append(self.area_mask)
def segmentationize(imageSe):
"""
Divides coherent forms of an image in smaller groups of type integer.
"""
# create an matrix of distances to the next sourrounding area
distance = ndimage.distance_transform_edt(imageSe, sampling=3)
erosed = ndimage.binary_erosion(imageSe, iterations=8).astype(imageSe.dtype)
distanceE = ndimage.distance_transform_edt(erosed, sampling=3)
distance += (2 * distanceE)
labels, num = label(imageSe, background=0, return_num='True')
sizes_image = ndimage.sum(imageSe, labels, range(num))
sizes_image = np.sort(sizes_image, axis=None)
pos = int(0.4 * num)
areal = int(sizes_image[pos] ** 0.5)
if areal <= 10:
areal = 10
elif (areal % 2) != 0:
areal += 1
footer = circarea(areal) # draw circle area
# find the positions of the maxima from the distances
local_maxi = peak_local_max(distance, indices=False, footprint=footer, labels=imageSe)
markers = label(local_maxi)
# watershed algorithm starts at the maxima and returns labels of particles
simplefilter("ignore", FutureWarning) # avoid warning in watershed method
labels_ws = watershed(-distance, markers, mask=imageSe)
simplefilter("default", FutureWarning)
return labels, labels_ws, local_maxi
def expand_watershed(self, pubsub_evt):
markers = self.matrix
image = self.viewer.slice_.matrix
self.viewer.slice_.do_threshold_to_all_slices()
mask = self.viewer.slice_.current_mask.matrix[1:, 1:, 1:]
ww = self.viewer.slice_.window_width
wl = self.viewer.slice_.window_level
if BRUSH_BACKGROUND in markers and BRUSH_FOREGROUND in markers:
tmp_image = ndimage.morphological_gradient(get_LUT_value(image, ww, wl).astype('uint16'), self.mg_size)
tmp_mask = watershed(tmp_image, markers)
if self.viewer.overwrite_mask:
mask[:] = 0
mask[tmp_mask == 1] = 253
else:
mask[(tmp_mask==2) & ((mask == 0) | (mask == 2) | (mask == 253))] = 2
mask[(tmp_mask==1) & ((mask == 0) | (mask == 2) | (mask == 253))] = 253
#mask[:] = tmp_mask
self.viewer.slice_.current_mask.matrix[0] = 1
self.viewer.slice_.current_mask.matrix[:, 0, :] = 1
self.viewer.slice_.current_mask.matrix[:, :, 0] = 1
self.viewer.slice_.discard_all_buffers()
self.viewer.slice_.current_mask.clear_history()
Publisher.sendMessage('Reload actual slice')
def seeded_watershed(boundary, seed_threshold = 0, seed_size = 5, mask=None):
"""Extract seeds from boundary prediction and runs seeded watershed.
Args:
boundary (3D numpy array) = boundary predictions
seed_threshold (int) = Add seeds where boundary prob is <= threshold
seed_size (int) = seeds must be >= seed size
mask (3D numpy array) = true to watershed, false to ignore
Returns:
3d watershed
"""
from skimage import morphology as skmorph
from numpy import bincount
# get seeds
from scipy.ndimage import label as label2
seeds = label2(boundary <= seed_threshold, output=numpy.uint32)[0]
# remove small seeds
if seed_size > 0:
component_sizes = bincount(seeds.ravel())
small_components = component_sizes < seed_size
small_locations = small_components[seeds]
seeds[small_locations] = 0
# mask out background (don't have to 0 out seeds since)
supervoxels = skmorph.watershed(boundary, seeds,
None, None, mask)
return supervoxels
def LargestWatershedRegion(shapes,dims,skipBias):
L=len(shapes)-skipBias
shapes=shapes.reshape((-1,) + dims[1:])
D=len(dims)
num_peaks=4
# structure=np.ones(tuple(3*np.ones((np.ndim(shapes)-1,1))))
for ll in range(L):
temp=shapes[ll]
local_maxi = peak_local_max(gaussian_filter(temp,[1]*(D-1)), exclude_border=False, indices=False, num_peaks=num_peaks)
markers,junk = label(local_maxi)
nonzero_mask=temp>0
if np.sum(nonzero_mask)>(3**3)*num_peaks:
labels = watershed(-temp, markers, mask=nonzero_mask) #watershed regions
ind = 1
temp2 = np.copy(temp)
temp2[labels!=1]=0
total_intensity = sum(temp2.reshape(-1,))
for kk in range(2,labels.max()+1):
temp2 = np.copy(temp)
temp2[labels!=kk]=0
total_intensity2 = sum(temp2.reshape(-1,))
if total_intensity2>total_intensity:
ind = kk
total_intensity=total_intensity2
temp[labels!=ind]=0
shapes[ll]=temp
shapes=shapes.reshape((len(shapes),-1))
return shapes
def watershed(base_image, seed_image=None, threshold_distance=80):
""" execute watershed with chosen seeds
"""
from scipy import ndimage as ndi
from skimage.morphology import watershed
from skimage.feature import peak_local_max
from skimage.morphology import label
import matplotlib.pyplot as plt
distance = ndi.distance_transform_edt(base_image)
# imgplot = plt.imshow(distance)
fig = plt.figure() # a new figure window
ax = fig.add_subplot(1, 1, 1) # specify (nrows, ncols, axnum)
# ax.imshow(distance>threshold_distance, cmap='Greys')
thresh = distance > threshold_distance
ax.imshow(thresh, cmap='Greys')
#local_maxi = peak_local_max(distance, labels=jac, footprint=np.ones((100, 100)), indices=False)
if seed_image is None:
markers = label(thresh)
else:
markers = label(seed_image)
# imgplot = plt.imshow(markers)
watersh = watershed(-distance, markers, mask=base_image)
# plt.imshow(watersh, cmap=plt.cm.viridis, interpolation='nearest')
return watersh
def isolate_single_seed(stack_path, output_path):
"""Load stack then isolate seed."""
sigma = 10
iterations = 1
raw_stack = Image3D.from_path(stack_path)
print raw_stack.shape
smoothed = gaussian_filter(raw_stack, sigma).view(Image3D)
edges = sobel_magnitude_nd(smoothed).view(Image3D)
#edges.save('edges')
labels = np.zeros(raw_stack.shape)
cx, cy, cz = map(lambda x: x/2, raw_stack.shape)
labels[cx, cy, cz] = 1
threshold = threshold_otsu(smoothed)
thresholded = smoothed > threshold
#thresholded.view(Image3D).save('thresh')
segmentation = watershed(edges, markers=labels, mask=thresholded)
#segmentation.view(Image3D).save('seg')
dilated = binary_dilation(segmentation, iterations=iterations)
isolate = np.multiply(raw_stack, dilated)
isolate.view(Image3D).save(output_path)
# plt.figure(5)
# plt.imshow(segmented_ct_scan_Blur[i], cmap=plt.cm.gray)
# plt.figure(6)
# plt.imshow(Nodule_Candidate[i], cmap=plt.cm.gray)
# # plt.figure(7)
# # plt.imshow(Nodule_Candidate_mor[i], cmap=plt.cm.gray)
# plt.show()
start_time = time.time()
Total_Num_Label = 0
nodule_total_array = []
distance1 = ndi.distance_transform_edt(Nodule_Candidate)
local_maxi1 = Dist_Thresholding_3D(distance1)
markers1 = label(local_maxi1)
labels1 = watershed(-distance1, markers1, mask=Nodule_Candidate)
nodule_total_array, Total_Num_Label1 = Nodule_Candidate_Extract(
pix_resampled, segmented_ct_scan, labels1, patients[i], i,
nodule_total_array)
Total_Num_Label = Total_Num_Label + Total_Num_Label1
distance2 = ndi.distance_transform_edt(Nodule_Candidate_Op1)
local_maxi2 = Dist_Thresholding_3D(distance2)
markers = label(local_maxi2)
labels2 = watershed(-distance2, markers, mask=Nodule_Candidate_Op1)
nodule_total_array, Total_Num_Label2 = Nodule_Candidate_Extract(
pix_resampled, segmented_ct_scan, labels2, patients[i], i,
nodule_total_array)
Total_Num_Label = Total_Num_Label + Total_Num_Label2
def segment(self):
# start timing
starttime = time.time()
# DATA IMPORT AND PREPROCESSING
f_directory = os.getcwd()
print('reading ' + self.filename + ' ...')
raw_img = io.imread(self.filename)
default_shape = raw_img.shape
print('raw image imported.')
# next step's gaussian filter
print('performing gaussian filtering...')
gaussian_img = np.zeros(shape=default_shape, dtype='float32')
for i in range(default_shape[0]):
temp_img = np.copy(raw_img[i])
gaussian_img[i] = gaussian_filter(input=raw_img[i], sigma=(2, 2))
print('cytosolic image smoothed.')
print('preprocessing complete.')
# next step's gaussian filter assumes 60x objective
# BINARY THRESHOLDING AND IMAGE CLEANUP
print('thresholding...')
threshold_img = np.copy(gaussian_img)
threshold_img[threshold_img < self.threshold] = 0
threshold_img[threshold_img > 0] = 1
print('filling holes...')
filled_img = np.zeros(shape=default_shape, dtype='float32')
#Goes through each time course separately and creates a filled image.
for i in range(default_shape[0]):
temp_img = np.copy(threshold_img[i])
filled_img[i] = binary_fill_holes(temp_img)
print('2d holes filled.')
print('binary processing complete.')
# DISTANCE AND MAXIMA TRANFORMATIONS TO FIND CELLS
# next two steps assume 60x objective
print('generating distance map...')
dist_map = np.zeros(shape=default_shape, dtype='float32')
#Goes through each time course separately and distance map.
for i in range(default_shape[0]):
temp_img = np.copy(filled_img[i])
dist_map[i] = distance_transform_edt(temp_img, sampling=(1, 1))
print('distance map complete.')
print('smoothing distance map...')
smooth_dist = np.zeros(shape=default_shape, dtype='float32')
#Goes through each timecourse separately and creates a smoothed distance map.
for i in range(default_shape[0]):
temp_img = np.copy(dist_map[i])
smooth_dist[i] = gaussian_filter(temp_img, [4, 4])
print('distance map smoothed.')
print('identifying maxima...')
max_strel_2d = generate_binary_structure(2, 2)
maxima = np.zeros(shape=default_shape, dtype='float32')
for i in range(default_shape[0]):
maxima[i] = maximum_filter(
smooth_dist[i], footprint=max_strel_2d) == smooth_dist[i]
bgrd_2d = smooth_dist[i] == 0
eroded_background_2d = binary_erosion(bgrd_2d,
structure=max_strel_2d,
border_value=1)
maxima[i] = np.logical_xor(maxima[i], eroded_background_2d)
print('maxima identified.')
# WATERSHED SEGMENTATION
labs = np.zeros(shape=default_shape, dtype='float32')
for i in range(default_shape[0]):
labs[i] = self.watershed_labels(maxima[i])
print('watershedding...')
cells = np.zeros(shape=default_shape, dtype='float32')
for i in range(default_shape[0]):
cells[i] = watershed(-smooth_dist[i], labs[i], mask=filled_img[i])
print('raw watershedding complete.')
print('cleaning up cells...')
clean_cells = np.zeros(shape=default_shape, dtype='float32')
for i in range(default_shape[0]):
clean_cells[i] = self.reassign_pixels_2d(cells[i])
print('cell cleanup complete.')
print('SEGMENTATION OPERATION COMPLETE.')
endtime = time.time()
runningtime = endtime - starttime
print('time elapsed: ' + str(runningtime) + ' seconds')
cell_num = [[] for f in range(default_shape[0])]
volume = [[] for f in range(default_shape[0])]
#Assigns a cell number and volume for each segmented cell in the timecourse.
for i in range(default_shape[0]):
cell_num[i], volume[i] = np.unique(clean_cells[i],
return_counts=True)
for j in range(default_shape[0]):
cell_num[j] = cell_num[j].astype('uint16')
volume[j] = volume[j].astype('uint16')
cell_nums = [[] for f in range(default_shape[0])]
volumes = [[] for f in range(default_shape[0])]
#Creates a dictionary to match cell number to its volume as determined by 2D number of pixels.
for k in range(default_shape[0]):
volumes[k] = dict(zip(cell_num[k], volume[k]))
cell_nums[k] = cell_num[k][np.nonzero(cell_num[k])]
#Deletes any artifacts that have a volume of 0.
for vol in range(len(volumes)):
if 0 in volumes[vol]:
del volumes[vol][0]
del volumes[vol][0]
#Determines the distance from center in order to determine which cell is the mother.
print('determining distances...')
distances = self.avg_distance_from_center(clean_cells)
print('distances determined.')
mother = [[] for f in range(default_shape[0])]
#Determines which cell is the mother basedo n the smallest distance and determines if that cell has the largest volume.
for j in range(default_shape[0]):
if volumes[j] == {} or distances[j] == {}:
pass
else:
if self.keywithmaxval(volumes[j]) == self.keywithminval(
distances[j]):
mother[j] = self.keywithminval(distances[j])
print(j)
else:
mother[j] = self.keywithminval(distances[j])
print(
'Maximum size does not agree with minimum distance! Mother may be incorrect, assuming min distance.'
)
print(j)
mother_cell = np.copy(clean_cells)
print('eliminating all but mother cell...')
#Eliminates the masks for all cells that are not the mother and reassigns the mother's number to 1.
for frame in range(default_shape[0]):
mother_cell[frame] = np.copy(clean_cells[frame])
mother_cell[frame][clean_cells[frame] != mother[frame]] = 0
mother_cell[frame][mother_cell[frame] > 0] = 1
print('mothers produced.')
mother_num = [[] for f in range(default_shape[0])]
new_volume = [[] for f in range(default_shape[0])]
#Reassigns mother cells volume calculated previously.
for i in range(default_shape[0]):
mother_num[i], new_volume[i] = np.unique(mother_cell[i],
return_counts=True)
for j in range(default_shape[0]):
mother_cell[j] = mother_cell[j].astype('uint16')
new_volume[j] = new_volume[j].astype('uint16')
mother_nums = [[] for f in range(default_shape[0])]
new_volumes = [[] for f in range(default_shape[0])]
for k in range(default_shape[0]):
new_volumes[k] = dict(zip(mother_num[k], new_volume[k]))
mother_nums[k] = mother_num[k][np.nonzero(mother_num[k])]
for vol in range(len(new_volumes)):
del new_volumes[vol][0]
return SegmentObj(f_directory, self.filename, raw_img, self.threshold,
threshold_img, filled_img, dist_map, smooth_dist,
maxima, labs, cells, clean_cells, cell_nums, volumes,
mother_cell, mother_nums, new_volumes)
def watershed_segmentation(num, img, thresh, gray, manual, outline):
master_data = []
target = []
clean = img.copy()
cleanContours = np.ones((1000, 1000, 3)) * 255
# cv2.imshow('clean', clean)
# cv2.waitKey(0)
# cv2.destroyAllWindows()
D = ndimage.distance_transform_edt(thresh)
localMax = peak_local_max(D, indices=False, min_distance=20, labels=thresh)
# perform a connected component analysis on the local peaks,
# using 8-connectivity, then appy the Watershed algorithm
markers = ndimage.label(localMax, structure=np.ones((3, 3)))[0]
labels = watershed(-D, markers, mask=thresh)
count = 1
for label in np.unique(labels):
# if the label is zero, we are examining the 'background'
# so simply ignore it
if label == 0:
continue
# otherwise, allocate memory for the label region and draw
# it on the mask
mask = np.zeros(gray.shape, dtype="uint8")
mask[labels == label] = 255
# detect contours in the mask and grab the largest one
cnts = cv2.findContours(mask.copy(), cv2.RETR_EXTERNAL,
cv2.CHAIN_APPROX_SIMPLE)[-2]
cleanContours = cv2.drawContours(cleanContours, cnts, -1, (255, 0, 0),
1)
c = max(cnts, key=cv2.contourArea)
if c.shape[0] >= 5:
if manual:
manual_label(c, clean, target)
# features = extractFeatures(c)
# master_data.append(features)
x, y, w, h = cv2.boundingRect(c)
if outline:
roi = cleanContours[y:y + h, x:x + w]
height = np.size(roi, 0)
width = np.size(roi, 1)
# cv2.fillPoly(img, pts =[contours], color=(255,255,255))
print(width, height)
print((128 - w) / 2., (128 - h) / 2.)
top = int(math.ceil((128 - h) / 2.))
bottom = int(math.floor((128 - h) / 2.))
left = int(math.ceil((128 - w) / 2.))
right = int(math.floor((128 - w) / 2.))
roi = cv2.copyMakeBorder(roi, top, bottom, left, right,
cv2.BORDER_CONSTANT, None,
(255, 255, 255))
cv2.imwrite(
rootpath +
"\Images\cells_new\{0}_c{1}.png".format(num, count), roi)
else:
roi = clean[y:y + h, x:x + w]
# cv2.imwrite(rootpath + "\Images\cells_out\{0}_c{1}.png".format(num, count), roi)
cv2.imwrite(
rootpath + "\output\{0}_c{1}.png".format(num, count), roi)
count += 1
# print num, count
return master_data, target
local_maxi = peak_local_max(img2, indices=False, min_distance = 1)
imgm2 = img2.copy();
imgm2[local_maxi] = 3 * imgm2.max();
plt.subplot(2,2,3);
plt.imshow(imgm2, cmap=plt.cm.jet, interpolation='nearest')
local_maxi = peak_local_max(img, indices=False, min_distance = 5)
imgm = img.copy();
imgm[local_maxi] = 3 * imgm.max();
plt.subplot(2,2,4);
plt.imshow(imgm, cmap=plt.cm.jet, interpolation='nearest')
markers = ndi.label(local_maxi)[0]
labelsws = watershed(-img, markers, mask = None);
labels = labelsws.copy();
labels.max()
#if False:
# cls = [[1], range(2,6), range(6, 11), range(11,21), range(21, 26), range(26,31)];
# cls = [range(1,3), range(3, 11), range(11,31)];
# for i, c in enumerate(cls):
# for cc in c:
# labels[labelsws == cc] = i;
fig, axes = plt.subplots(ncols=3, sharex=True, sharey=True, subplot_kw={'adjustable':'box-forced'})
ax0, ax1, ax2 = axes
ax0.imshow(img, cmap=plt.cm.jet, interpolation='nearest')
ax0.set_title('PDF')
# plt.imshow(np.ma.array(green_max,mask=green_max==0),interpolation='none') # plt.show() #------------------------------------------------------------------------------ # SEGMENTATION: EXPANSION BY WATERSHED # Watershedding is a relatively simple but powerful algorithm for expanding # seeds. The image intensity is considered as a topographical map (with high # intensities being "mountains" and low intensities "valleys") and water is # poured into the valleys from each of the seeds. The water first labels the # lowest intensity pixels around the seeds, then continues to fill up. The cell # boundaries are where the waterfronts between different seeds touch. # Get the watershed function and run it from skimage.morphology import watershed green_ws = watershed(green_smooth, green_max) # Show result as transparent overlay # Note: For a better visualization, see "FINDING CELL EDGES" below! # plt.imshow(green_smooth,cmap='gray',interpolation='none') # plt.imshow(green_ws,interpolation='none',alpha=0.7) # plt.show() # Notice that the previously connected cells are now mostly separated and the # membranes are partitioned to their respective cells. # ...however, we now see a few cases of oversegmentation! # This is a typical example of the trade-offs one has to face in any # computational classification task. #------------------------------------------------------------------------------
def segmentByClustering(rgbImage, colorSpace, clusteringMethod,
numberOfClusters):
#import dataset function from main
#Import function check_dataset
from main import check_dataset
#download dataset and unzip it
check_dataset()
#Import libraries
import matplotlib.pyplot as plt
#import os
import os
#import io, color
from skimage import io, color
import numpy as np
from sklearn.cluster import KMeans
from sklearn.mixture import GaussianMixture
from sklearn.cluster import AgglomerativeClustering
from skimage.feature import peak_local_max
from skimage.morphology import watershed
from scipy import ndimage
import cv2
from main import imshow
from scipy import misc
from skimage.transform import resize
#get the current cwd
#cwd = os.getcwd()
#get the image path
#img_file = os.path.join('BSDS_small',rgbImage)
img_file = 'BSR' + '/' + 'BSDS500' + '/' + 'data' + '/' + 'images' + '/' + 'test' + '/' + rgbImage
#show the groundtruth segmentation
#groundtruth(img_file)
#show the groundtruth edges
# from main import groundtruth_edges
# groundtruth_edges(img_file)
#read the image (rgb)
imag = io.imread(img_file)
from skimage.filters import gaussian
rgb = gaussian(imag, sigma=1.5, multichannel=True)
#Check all possible color spaces
#HSV color space
if (colorSpace == 'hsv'):
#convert rgb2hsv
hsv = color.rgb2hsv(rgb)
hsv = cv2.normalize(hsv,
np.zeros((hsv.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
#return hsv
#plot the image in hsv
#plt.imshow(hsv)
#plt.show()
#shape of the image
(fils, cols, channels) = hsv.shape
#Check all clustering Methods
#Kmeans
if (clusteringMethod == 'kmeans'):
#reshape the image for kmeans
X = hsv.reshape(fils * cols, 3)
#Convert data to float
#X.astype(float)
#kmeans with numberofClusters as a parameter
kmeans = KMeans(n_clusters=numberOfClusters, random_state=0).fit(X)
#obtaining the segmented image
segmented_img = kmeans.cluster_centers_[kmeans.labels_]
#reshape the image to the original size
segmented_img = kmeans.labels_
#reshape labels to obtain 2d image
segmented_img = segmented_img.reshape((fils, cols))
#show the segmentation
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'gmm'):
X = hsv.reshape(fils * cols, 3)
gmm = GaussianMixture(n_components=numberOfClusters)
gmm = gmm.fit(X)
cluster = gmm.predict(X)
cluster = cluster.reshape(fils, cols)
#cluster = cluster.astype(np.uint8)
segmented_img = cluster
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'hierarchical'):
hsv2 = resize(hsv, (int(hsv.shape[0] / 4), int(hsv.shape[1] / 4)),
anti_aliasing=True)
hsv2 = cv2.normalize(hsv2,
np.zeros((hsv2.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
(fils, cols, channels) = hsv2.shape
X = hsv2.reshape(fils * cols, 3)
cluster = AgglomerativeClustering(n_clusters=numberOfClusters,
affinity='manhattan',
linkage='complete')
cluster.fit_predict(X)
labels = cluster.labels_
segmented_img = labels.reshape(fils, cols)
#segmented_img = segmented_img.astype(np.uint8)
segmented_img = misc.imresize(segmented_img,
(hsv.shape[0], hsv.shape[1]),
interp='bicubic')
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'watershed'):
hsv = np.mean(hsv, axis=2)
local_maxima = peak_local_max(-1 * hsv,
min_distance=15,
indices=False,
num_peaks=numberOfClusters)
marks = ndimage.label(local_maxima)[0]
segmented_img = watershed(hsv, marks)
segmented_img = segmented_img
#imshow(rgb,segmented_img, title= rgbImage)
#Check lab color space
elif (colorSpace == 'lab'):
#convert rgb image to lab
lab = color.rgb2lab(rgb)
lab = cv2.normalize(lab,
np.zeros((lab.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
#show of the image in lab color space
#shape of the image
(fils, cols, channels) = lab.shape
#Check all possible clustering methods
#Kmeans
if (clusteringMethod == 'kmeans'):
#reshape the image for kmeans
X = lab.reshape(fils * cols, 3)
#Convert data to float
#X.astype(float)
#kmeans with numberofClusters as a parameter
kmeans = KMeans(n_clusters=numberOfClusters, random_state=0).fit(X)
#obtaining the segmented image (labels)
segmented_img = kmeans.labels_
#reshape labels to obtain 2d image
segmented_img = segmented_img.reshape((fils, cols))
#convert it to uint8
#segmented_img = segmented_img.astype(np.uint8)
#show the segmentation
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'gmm'):
X = lab.reshape(fils * cols, 3)
gmm = GaussianMixture(n_components=numberOfClusters)
gmm = gmm.fit(X)
cluster = gmm.predict(X)
cluster = cluster.reshape(fils, cols)
#cluster = cluster.astype(np.uint8)
segmented_img = cluster
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'hierarchical'):
lab2 = resize(lab, (int(lab.shape[0] / 4), int(lab.shape[1] / 4)),
anti_aliasing=True)
lab2 = cv2.normalize(lab2,
np.zeros((lab2.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
(fils, cols, channels) = lab2.shape
X = lab2.reshape(fils * cols, 3)
cluster = AgglomerativeClustering(n_clusters=numberOfClusters,
affinity='euclidean',
linkage='ward')
cluster.fit_predict(X)
labels = cluster.labels_
segmented_img = labels.reshape(fils, cols)
#segmented_img = segmented_img.astype(np.uint8)
segmented_img = misc.imresize(segmented_img,
(rgb.shape[0], rgb.shape[1]),
interp='bicubic')
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'watershed'):
lab = np.mean(lab, axis=2)
local_maxima = peak_local_max(-1 * lab,
min_distance=15,
indices=False,
num_peaks=numberOfClusters)
marks = ndimage.label(local_maxima)[0]
segmented_img = watershed(lab, marks)
segmented_img = segmented_img
#imshow(rgb,segmented_img, title= rgbImage)
#Check rgb+xy color space
elif (colorSpace == 'rgb_xy'):
#size of the image
(fils, cols, channels) = rgb.shape
pos_x = np.ones((fils, cols), dtype='uint8')
pos_y = np.ones((fils, cols), dtype='uint8')
for i in range(0, fils):
for j in range(0, cols):
pos_x[i, :] = i
pos_y[:, j] = j
pos_x = cv2.normalize(pos_x,
np.zeros((pos_x.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
pos_y = cv2.normalize(pos_y,
np.zeros((pos_y.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
rgb_xy = np.dstack((rgb, pos_x, pos_y))
if (clusteringMethod == 'kmeans'):
X = rgb_xy.reshape(fils * cols, 5)
#X.astype(float)
kmeans = KMeans(n_clusters=numberOfClusters, random_state=0).fit(X)
segmented_img = kmeans.labels_
segmented_img = segmented_img.reshape((fils, cols))
#segmented_img = segmented_img.astype(np.uint8)
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'gmm'):
X = rgb_xy.reshape(fils * cols, 5)
gmm = GaussianMixture(n_components=numberOfClusters)
gmm = gmm.fit(X)
cluster = gmm.predict(X)
cluster = cluster.reshape(fils, cols)
#cluster = cluster.astype(np.uint8)
segmented_img = cluster
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'hierarchical'):
#X = rgb_xy.reshape(fils*cols, 5)
rgb2 = resize(rgb, (int(rgb.shape[0] / 4), int(rgb.shape[1] / 4)),
anti_aliasing=True)
rgb2 = cv2.normalize(rgb2,
np.zeros((rgb2.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
(fils, cols, channels) = rgb2.shape
pos_x = np.ones((fils, cols), dtype='uint8')
pos_y = np.ones((fils, cols), dtype='uint8')
for i in range(0, fils):
for j in range(0, cols):
pos_x[i, :] = i
pos_y[:, j] = j
rgb_xy = np.dstack((rgb2, pos_x, pos_y))
X = rgb_xy.reshape(fils * cols, 5)
cluster = AgglomerativeClustering(n_clusters=numberOfClusters,
affinity='euclidean',
linkage='ward')
cluster.fit_predict(X)
labels = cluster.labels_
segmented_img = labels.reshape(fils, cols)
#segmented_img = segmented_img.astype(np.uint8)
segmented_img = misc.imresize(segmented_img,
(rgb.shape[0], rgb.shape[1]),
interp='nearest')
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'watershed'):
rgb_xy = np.mean(rgb, axis=2)
local_maxima = peak_local_max(-1 * rgb_xy,
min_distance=15,
indices=False,
num_peaks=numberOfClusters)
marks = ndimage.label(local_maxima)[0]
segmented_img = watershed(rgb_xy, marks)
segmented_img = segmented_img
#imshow(rgb,segmented_img, title= rgbImage)
elif (colorSpace == 'lab_xy'):
#size of the image
lab = color.rgb2lab(rgb)
lab2 = cv2.normalize(lab,
np.zeros((lab.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
(fils, cols, channels) = lab.shape
pos_x = np.ones((fils, cols)) #dtype='uint8')
pos_y = np.ones((fils, cols)) #dtype='uint8')
for i in range(0, fils):
for j in range(0, cols):
pos_x[i, :] = i
pos_y[:, j] = j
pos_x = cv2.normalize(pos_x,
np.zeros((pos_x.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
pos_y = cv2.normalize(pos_y,
np.zeros((pos_y.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
lab_xy = np.dstack((lab, pos_x, pos_y))
if (clusteringMethod == 'kmeans'):
X = lab_xy.reshape(fils * cols, 5)
#X.astype(float)
kmeans = KMeans(n_clusters=numberOfClusters).fit(X)
segmented_img = kmeans.labels_
segmented_img = segmented_img.reshape((fils, cols))
#segmented_img = segmented_img.astype(np.uint8)
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'gmm'):
X = lab_xy.reshape(fils * cols, 5)
gmm = GaussianMixture(n_components=numberOfClusters)
gmm = gmm.fit(X)
cluster = gmm.predict(X)
cluster = cluster.reshape(fils, cols)
#cluster = cluster.astype(np.uint8)
segmented_img = cluster
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'hierarchical'):
lab2 = resize(lab, (int(lab.shape[0] / 4), int(lab.shape[1] / 4)),
anti_aliasing=True)
lab2 = cv2.normalize(lab2,
np.zeros((lab2.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
(fils, cols, channels) = lab2.shape
pos_x = np.ones((fils, cols), dtype='uint8')
pos_y = np.ones((fils, cols), dtype='uint8')
for i in range(0, fils):
for j in range(0, cols):
pos_x[i, :] = i
pos_y[:, j] = j
lab_xy = np.dstack((lab2, pos_x, pos_y))
X = lab_xy.reshape(fils * cols, 5)
cluster = AgglomerativeClustering(n_clusters=numberOfClusters,
affinity='euclidean',
linkage='ward')
cluster.fit_predict(X)
labels = cluster.labels_
segmented_img = labels.reshape(fils, cols)
#segmented_img = segmented_img.astype(np.uint8)
segmented_img = misc.imresize(segmented_img,
(lab.shape[0], lab.shape[1]),
interp='bicubic')
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'watershed'):
lab_xy = np.mean(lab, axis=2)
local_maxima = peak_local_max(-1 * lab_xy,
min_distance=15,
indices=False,
num_peaks=numberOfClusters)
marks = ndimage.label(local_maxima)[0]
segmented_img = watershed(lab_xy, marks)
segmented_img = segmented_img
#imshow(rgb,segmented_img, title= rgbImage)
#Check if the color space is hsv
elif (colorSpace == 'hsv_xy'):
#size of the image
hsv = color.rgb2hsv(rgb)
hsv = cv2.normalize(hsv,
np.zeros((hsv.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
(fils, cols, channels) = hsv.shape
pos_x = np.ones((fils, cols), dtype='uint8')
pos_y = np.ones((fils, cols), dtype='uint8')
for i in range(0, fils):
for j in range(0, cols):
pos_x[i, :] = i
pos_y[:, j] = j
pos_x = cv2.normalize(pos_x,
np.zeros((pos_x.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
pos_y = cv2.normalize(pos_y,
np.zeros((pos_y.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
hsv_xy = np.dstack((hsv, pos_x, pos_y))
if (clusteringMethod == 'kmeans'):
X = hsv_xy.reshape(fils * cols, 5)
#X.astype(float)
kmeans = KMeans(n_clusters=numberOfClusters, random_state=0).fit(X)
segmented_img = kmeans.labels_
segmented_img = segmented_img.reshape((fils, cols))
#segmented_img = segmented_img.astype(np.uint8)
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'gmm'):
X = hsv_xy.reshape(fils * cols, 5)
gmm = GaussianMixture(n_components=numberOfClusters)
gmm = gmm.fit(X)
cluster = gmm.predict(X)
cluster = cluster.reshape(fils, cols)
#cluster = cluster.astype(np.uint8)
segmented_img = cluster
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'hierarchical'):
hsv2 = resize(hsv, (int(hsv.shape[0] / 4), int(hsv.shape[1] / 4)),
anti_aliasing=True)
hsv2 = cv2.normalize(hsv2,
np.zeros((hsv2.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
(fils, cols, channels) = hsv2.shape
pos_x = np.ones((fils, cols), dtype='uint8')
pos_y = np.ones((fils, cols), dtype='uint8')
for i in range(0, fils):
for j in range(0, cols):
pos_x[i, :] = i
pos_y[:, j] = j
hsv_xy = np.dstack((hsv2, pos_x, pos_y))
X = hsv_xy.reshape(fils * cols, 5)
cluster = AgglomerativeClustering(n_clusters=numberOfClusters,
affinity='euclidean',
linkage='ward')
cluster.fit_predict(X)
labels = cluster.labels_
segmented_img = labels.reshape(fils, cols)
#segmented_img = segmented_img.astype(np.uint8)
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'watershed'):
#hsv_xy=hsv_xy[:,:,2]
hsv_xy = np.mean(hsv, axis=2)
local_maxima = peak_local_max(-1 * hsv_xy,
min_distance=15,
indices=False,
num_peaks=numberOfClusters)
marks = ndimage.label(local_maxima)[0]
segmented_img = watershed(hsv_xy, marks)
segmented_img = segmented_img
#imshow(rgb,segmented_img, title= rgbImage)
elif (colorSpace == 'rgb'):
(fils, cols, channels) = rgb.shape
if (clusteringMethod == 'kmeans'):
X = rgb.reshape(fils * cols, 3)
#X.astype(float)
kmeans = KMeans(n_clusters=numberOfClusters, random_state=0).fit(X)
segmented_img = kmeans.labels_
segmented_img = segmented_img.reshape((fils, cols))
#segmented_img = segmented_img.astype(np.uint8)
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'gmm'):
X = rgb.reshape(fils * cols, 3)
gmm = GaussianMixture(n_components=numberOfClusters)
gmm = gmm.fit(X)
cluster = gmm.predict(X)
cluster = cluster.reshape(fils, cols)
#cluster = cluster.astype(np.uint8)
segmented_img = cluster
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'hierarchical'):
rgb2 = resize(rgb, (int(rgb.shape[0] / 4), int(rgb.shape[1] / 4)),
anti_aliasing=True)
rgb2 = cv2.normalize(rgb2,
np.zeros((rgb2.shape), dtype=np.uint8),
alpha=0,
beta=255,
norm_type=cv2.NORM_MINMAX,
dtype=cv2.CV_8U)
(fils, cols, channels) = rgb2.shape
X = rgb2.reshape(fils * cols, 3)
cluster = AgglomerativeClustering(n_clusters=numberOfClusters,
affinity='euclidean',
linkage='ward')
cluster.fit_predict(X)
labels = cluster.labels_
segmented_img = labels.reshape(fils, cols)
#segmented_img = segmented_img.astype(np.uint8)
segmented_img = misc.imresize(segmented_img,
(rgb.shape[0], rgb.shape[1]),
interp='bicubic')
#imshow(rgb,segmented_img,title = rgbImage)
elif (clusteringMethod == 'watershed'):
rgb = np.mean(rgb, axis=2)
local_maxima = peak_local_max(-1 * rgb,
min_distance=15,
indices=False,
num_peaks=numberOfClusters)
marks = ndimage.label(local_maxima)[0]
segmented_img = watershed(rgb, marks)
segmented_img = segmented_img
#imshow(rgb,segmented_img, title= rgbImage)
return segmented_img
def nucleus_finder_dt(stack_in, name_in, smooth_type, smooth_size, cont_size,
thresh_type, erode, min_radius, max_radius, test, plot,
save):
print('smooth_size', smooth_size)
print('cont_size', cont_size)
print('thresh_type', thresh_type)
print('erode', erode)
print('min_radius', min_radius)
print('max_radius', max_radius)
print('test', test)
print('save', save)
# stack_in = stack_in.max(axis=0)
# z_size=1
z_size, y_size, x_size = int(np.shape(stack_in)[0]), int(
np.shape(stack_in)[1]), int(np.shape(stack_in)[2])
nuclei_count = []
# Need to determine global threshold based on max intensity projetion that has
# undergone same preprocessing as the stacks undergo
max_int_proj = stack_in.max(axis=0)
max_int_proj = preprocessor.smooth(np.copy(max_int_proj), smooth_type,
smooth_size, cont_size)
max_int_proj = preprocessor.contrast(np.copy(max_int_proj), cont_size)
thresh = preprocessor.threshold(np.copy(max_int_proj), thresh_type)
####################################################################
# Currently cluster in x and y then z separately - must fix this
####################################################################
for im in range(0, z_size):
image = stack_in[im]
smoothed = preprocessor.smooth(np.copy(image), smooth_type,
smooth_size, cont_size)
smoothed = preprocessor.contrast(np.copy(smoothed), cont_size)
# thresh = preprocessor.threshold(np.copy(smoothed), thresh_type)
binary = smoothed > thresh
# binary_closed = binary_closing(binary)
binary_closed = binary_fill_holes(binary)
if erode > 0:
print('Performing', str(erode), 'binary erosions')
eroded = binary_closed
# n_erodes = min_radius // 2
for i in range(0, erode):
eroded = binary_erosion(eroded)
elif erode == 0:
eroded = binary_closed
# Distance transform and threshold thereof
distance = ndimage.distance_transform_edt(eroded)
distance = distance > min_radius
distance = ndimage.distance_transform_edt(distance)
# Only look for local maxima if there's anything left after erosion:
if len(np.where(distance != 0)[0]) == 0:
print('Zero distance matrix')
labeled = np.zeros_like(image)
preprocessor.testplot(image, im, [], [], [], [], smoothed,
binary_closed, eroded, distance, labeled)
continue
# local_maxi = peak_local_max(distance, min_radius, exclude_border=False, indices=False, labels=smoothed)
local_maxi = peak_local_max(distance,
exclude_border=False,
indices=False,
footprint=square(3),
labels=smoothed)
# local_maxi = local_maxima(distance)
x, y = np.where(local_maxi != False)[1], np.where(
local_maxi != False)[0]
# List for storing x and y-coords on each slice
x_slice, y_slice = [], []
if len(x) == 0:
print('No peaks detected')
if len(x) == 1:
print('Only one maximum detected')
dist_non_zero = np.where(distance != 0)
print(dist_non_zero)
# Label the 1 remaining region
labeled = distance
labeled[dist_non_zero] = 1
labeled = labeled.astype(int)
print(labeled[np.where(labeled != 0)])
f_prop = regionprops(labeled, intensity_image=image)
for d in f_prop:
print('len(f_prop)', len(f_prop))
radius = (d.area / np.pi)**0.5
nuclei_count.append([
d.weighted_centroid[1], d.weighted_centroid[0], im, d.area,
radius, d.mean_intensity * d.area
])
x_slice.append(d.weighted_centroid[1])
y_slice.append(d.weighted_centroid[0])
# Produce test plot
preprocessor.testplot(image, im, x, y, x_slice, y_slice, smoothed,
binary_closed, eroded, distance, labeled)
if len(x) > 1:
# Use hierarchical clustering algorithm to cluster maxima
# Could alternatively use structure-based labelling
print('Detected', str(len(x)), 'peaks')
positions = np.stack((y, x), axis=1)
# Distance matrix is n-particles x n-particles in size
# This gives the upper triangle of the distance matrix
dist_mat = dist.pdist(positions)
link_mat = hier.linkage(dist_mat)
# fcluster assigns each of the particles in positions a cluster to which it belongs
cluster_idx = hier.fcluster(link_mat,
min_radius,
criterion='distance')
particles = np.unique(cluster_idx)
markers = np.zeros_like(smoothed)
markers[y, x] = cluster_idx
# markers = ndimage.label(local_maxi, structure=square(3))[0]
# Now that maxima have been clustered, label them by watershedding
labeled = watershed(-distance, markers, mask=binary)
f_prop = regionprops(labeled, intensity_image=image)
for d in f_prop:
radius = (d.area / np.pi)**0.5
nuclei_count.append([
d.weighted_centroid[1], d.weighted_centroid[0], im, d.area,
radius, d.mean_intensity * d.area
])
x_slice.append(d.weighted_centroid[1])
y_slice.append(d.weighted_centroid[0])
#Plot binary and watershedded images showing identified peaks
if test == True:
vis.testplot(image, name_in, im, x, y, x_slice, y_slice, smoothed,
binary_closed, eroded, distance, labeled)
# Now we've scanned all the way through the stack and located things
# we think are nuclei in each place. Now we need to cluster them in 3D so
# we link together the slices
values = []
if len(nuclei_count) > 1:
# Cluster nuclei
columns = ('x', 'y', 'z', 'area', 'radius', 'intensity')
nuclei_count = pd.DataFrame(nuclei_count, columns=columns)
# Now cluster nuclei by x, y coordinates
positions = np.stack(
(nuclei_count['x'].values, nuclei_count['y'].values,
nuclei_count['z'].values),
axis=1)
dist_mat = dist.pdist(positions)
link_mat = hier.linkage(dist_mat)
cluster_idx = hier.fcluster(link_mat, min_radius, criterion='distance')
particles = np.unique(cluster_idx)
# Calculate weighted average position of particles
nuclei_count['particle'] = cluster_idx
for j in particles:
current = nuclei_count[nuclei_count['particle'] == j]
# Normalisation constant of weighted average
norm = np.sum(current['intensity'])
x_av = np.sum(current['intensity'] * current['x']) / norm
y_av = np.sum(current['intensity'] * current['y']) / norm
z_av = np.sum(current['intensity'] * current['z']) / norm
a_max = np.amax(current['area'])
# Exclude nuclei on minimum intensity
if norm > 10000:
values.append([x_av, y_av, z_av, a_max, norm])
# Data Frame containing the weighted averages of the locations of the particles
columns = ('x', 'y', 'z', 'a_max', 'total_intensity')
nuclei_averaged = pd.DataFrame(values, columns=columns)
print('Found', len(nuclei_averaged), 'nuclei')
if plot == True:
# Numbers purely for visualisation purposes
plt.clf()
xy_scale, z_scale = 1., 2.
# Plot 3D visualisation of data
fig = plt.figure(figsize=(12, 12))
# xy projection:
ax_xy = fig.add_subplot(111)
ax_xy.imshow(stack_in.max(axis=0), cmap='gray')
ax_xy.scatter(nuclei_averaged['x'],
nuclei_averaged['y'],
facecolors='none',
edgecolors='red',
s=100)
divider = make_axes_locatable(ax_xy)
ax_zx = divider.append_axes("top", 2, pad=0.2, sharex=ax_xy)
ax_zx.imshow(stack_in.max(axis=1),
aspect=z_scale / xy_scale,
cmap='gray')
ax_zx.scatter(nuclei_averaged['x'],
nuclei_averaged['z'],
facecolors='none',
edgecolors='red',
s=100)
ax_yz = divider.append_axes("right", 2, pad=0.2, sharey=ax_xy)
ax_yz.imshow(stack_in.max(axis=2).T,
aspect=xy_scale / z_scale,
cmap='gray')
ax_yz.scatter(nuclei_averaged['z'],
nuclei_averaged['y'],
facecolors='none',
edgecolors='red',
s=100)
plt.draw()
if save == True:
outname = name_in + '_nuclei_py.tif'
plt.savefig(outname, bbox_inches='tight')
print(outname, 'saved')
plt.close()
return nuclei_averaged
def nps_finder_2d_stack(image_in,
name_in,
z_in=0,
thresh_type='yen',
smooth_type='mean',
smooth_size=1,
gauss_size=0,
cont_size=0,
sep_method='watershed',
mass_cutoff=200,
area_cutoff=0,
max_radius=5,
min_radius=1,
test=True):
# np.copy() used because peak_local_max does weird things to the image histogram if we use the actual image
print('mass_cutoff is', mass_cutoff)
y_size, x_size = np.shape(image_in)
smoothed = np.copy(image_in)
# Calculate threshold
thresh = preprocessor.threshold(np.copy(smoothed), thresh_type)
print('thresh is', thresh)
im_max = smoothed.max()
print('im_max is', im_max)
binary = smoothed > thresh
# Two approaches
# 1. Identify local maxima in real-space image - separate by watershedding
if sep_method == 'old_watershed':
print('old_watershed')
local_maxi = peak_local_max(np.copy(smoothed),
min_distance=min_radius,
threshold_abs=thresh,
indices=False,
labels=np.copy(smoothed))
labeled_image = ndimage.label(local_maxi, structure=disk(1))[0]
# markers = ndimage.label(local_maxi)[0]
labeled_image = watershed(-labeled_image, labeled_image, mask=binary)
# print(labeled_image)
if sep_method == 'binary_label':
print('binary_label')
labeled_image = ndimage.label(binary, structure=disk(1))[0]
# print(labeled_image)
if sep_method == 'peak_label':
print('peak_label')
local_maxi = peak_local_max(np.copy(smoothed),
min_distance=min_radius,
threshold_abs=thresh,
indices=False,
labels=np.copy(smoothed))
labeled_image = ndimage.label(local_maxi, structure=disk(1))[0]
# print(labeled_image)
columns = ('x', 'y', 'area', 'intensity')
if len(np.where(labeled_image != 0)[0]) == 0:
properties = pd.DataFrame([], columns=columns)
if len(np.where(labeled_image != 0)[0]) > 0:
f_prop = regionprops(labeled_image, intensity_image=smoothed)
properties = []
for d in f_prop:
properties.append([
d.weighted_centroid[1], d.weighted_centroid[0], d.area,
d.mean_intensity * d.area
])
properties = pd.DataFrame(properties, columns=columns)
if test == True:
preprocessor.testplot_parts(image_in, name_in, z_in, properties['x'],
properties['y'], smoothed, binary,
labeled_image)
return properties
def watershed_segmentation(rgb_img, mask, distance=10):
"""Uses the watershed algorithm to detect boundary of objects. Needs a marker file which specifies area which is
object (white), background (grey), unknown area (black).
Inputs:
rgb_img = image to perform watershed on needs to be 3D (i.e. np.shape = x,y,z not np.shape = x,y)
mask = binary image, single channel, object in white and background black
distance = min_distance of local maximum
Returns:
watershed_header = shape data table headers
watershed_data = shape data table values
analysis_images = list of output images
:param rgb_img: numpy.ndarray
:param mask: numpy.ndarray
:param distance: int
:return watershed_header: list
:return watershed_data: list
:return analysis_images: list
"""
# # Will be depricating opencv version 2
# if cv2.__version__[0] == '2':
# dist_transform = cv2.distanceTransform(mask, cv2.cv.CV_DIST_L2, maskSize=0)
# else:
dist_transform = cv2.distanceTransformWithLabels(mask, cv2.DIST_L2, maskSize=0)[0]
localMax = peak_local_max(dist_transform, indices=False, min_distance=distance, labels=mask)
markers = ndi.label(localMax, structure=np.ones((3, 3)))[0]
dist_transform1 = -dist_transform
labels = watershed(dist_transform1, markers, mask=mask)
img1 = np.copy(rgb_img)
for x in np.unique(labels):
rand_color = color_palette(len(np.unique(labels)))
img1[labels == x] = rand_color[x]
img2 = apply_mask(img1, mask, 'black')
joined = np.concatenate((img2, rgb_img), axis=1)
estimated_object_count = len(np.unique(markers)) - 1
analysis_image = []
analysis_image.append(joined)
watershed_header = (
'HEADER_WATERSHED',
'estimated_object_count'
)
watershed_data = (
'WATERSHED_DATA',
estimated_object_count
)
if params.debug == 'print':
print_image(dist_transform, os.path.join(params.debug_outdir, str(params.device) + '_watershed_dist_img.png'))
print_image(joined, os.path.join(params.debug_outdir, str(params.device) + '_watershed_img.png'))
elif params.debug == 'plot':
plot_image(dist_transform, cmap='gray')
plot_image(joined)
# Store into global measurements
if not 'watershed' in outputs.measurements:
outputs.measurements['watershed'] = {}
outputs.measurements['watershed']['estimated_object_count'] = estimated_object_count
# Store images
outputs.images.append(analysis_image)
return watershed_header, watershed_data, analysis_image
markers = np.zeros_like(coins)
markers[coins < 30] = 1
markers[coins > 150] = 2
fig, ax = plt.subplots(figsize=(4, 3))
ax.imshow(markers, cmap=plt.cm.spectral, interpolation='nearest')
ax.axis('off')
ax.set_title('markers')
"""
.. image:: PLOT2RST.current_figure
Finally, we use the watershed transform to fill regions of the elevation map
starting from the markers determined above:
"""
segmentation = morphology.watershed(elevation_map, markers)
fig, ax = plt.subplots(figsize=(4, 3))
ax.imshow(segmentation, cmap=plt.cm.gray, interpolation='nearest')
ax.axis('off')
ax.set_title('segmentation')
"""
.. image:: PLOT2RST.current_figure
This last method works even better, and the coins can be segmented and labeled
individually.
"""
from skimage.color import label2rgb
# Segment to connected objects
auto_segmented_smoothed_threshold_mask = measure.label(smoothed_threshold_mask)
# Watershed
from scipy import ndimage as ndi
from skimage.morphology import watershed
from skimage.feature import peak_local_max
distance = ndi.distance_transform_edt(smoothed_threshold_mask)
local_maxi = peak_local_max(distance,
indices=False,
footprint=np.ones((4, 4)),
labels=smoothed_threshold_mask)
markers = ndi.label(local_maxi)[0]
labels = watershed(-distance, markers, mask=smoothed_threshold_mask)
plt.imshow(smoothed_threshold_mask)
plt.show()
plt.imshow(labels)
plt.show()
len(np.unique(labels))
plt.imshow(gray_image, cmap='gray')
#
#from scipy import ndimage
#from skimage import morphology
## Black tophat transformation (see https://en.wikipedia.org/wiki/Top-hat_transform)
#hat = ndimage.black_tophat(gray_image, 7)
## Combine with denoised image
#hat -= 0.3 * gray_image
#load image
image = cv2.imread(infile)
#convert to greyscale
grey = cv2.cvtColor(image, cv2.COLOR_BGR2GRAY)
#thresholding
ret, thresh = cv2.threshold(grey, 49, 255, cv2.THRESH_BINARY + cv2.THRESH_OTSU)
#watershed algorithm
D = ndimage.distance_transform_edt(thresh)
localMax = peak_local_max(D, indices=False, min_distance=20, labels=thresh)
markers = ndimage.label(localMax, structure=np.ones((3, 3)))[0]
labels = watershed(-D, markers, mask=thresh)
for label in np.unique(labels):
if label == 0:
continue
mask = np.zeros(grey.shape, dtype="uint8")
mask[labels == label] = 255
contours = cv2.findContours(mask.copy(), cv2.RETR_EXTERNAL,
cv2.CHAIN_APPROX_SIMPLE)
contours = imutils.grab_contours(contours)
# draw contours
drawn = cv2.drawContours(grey, contours, -1, (255, 255, 255), 1)
#drawing a rectangle in which text will live
x, y, w, h = 0, 0, 375, 75
def watershedContours(image):
# =============================================================================
# # load the image and perform pyramid mean shift filtering
# # to aid the thresholding step
# shifted = cv2.pyrMeanShiftFiltering(image, 21, 51)
# =============================================================================
bw = cv2.cvtColor(image, cv2.COLOR_BGR2GRAY)
_, binarized = cv2.threshold(bw, 20, 255.0, cv2.THRESH_BINARY)
opened = morphOperation(binarized,
operation='opening',
times=1,
kernel_size=5)
# =============================================================================
# showImage(opened, name="Output")
# =============================================================================
closed = morphOperation(opened,
operation='closing',
times=1,
kernel_size=5)
eroded = eroded = morphOperation(closed,
operation='erosion',
times=1,
kernel_size=35)
# =============================================================================
# showImage(closed, name="Output")
# =============================================================================
# compute the exact Euclidean distance from every binary
# pixel to the nearest zero pixel, then find peaks in this
# distance map
D = ndimage.distance_transform_edt(eroded)
localMax = peak_local_max(D,
indices=False,
min_distance=30,
exclude_border=False)
# perform a connected component analysis on the local peaks,
# using 8-connectivity, then appy the Watershed algorithm
markers = ndimage.label(localMax, structure=np.ones((3, 3)))[0]
labels = watershed(-D, markers, mask=closed)
# loop over the unique labels returned by the Watershed
# algorithm
contours = []
for label in np.unique(labels):
# if the label is zero, we are examining the 'background'
# so simply ignore it
if label == 0:
continue
# otherwise, allocate memory for the label region and draw
# it on the mask
mask = np.zeros(bw.shape, dtype="uint8")
mask[labels == label] = 255
# detect contours in the mask and grab the largest one
cnts, hierarchy = cv2.findContours(mask.copy(), cv2.RETR_TREE,
cv2.CHAIN_APPROX_SIMPLE)
c = max(cnts, key=cv2.contourArea)
contours.append(c)
contours = np.array(contours)
return contours
image = io.imread(
'/Users/mahdi/Desktop/1005479.svs (1, 63517, 19793, 927, 1727)1.png', 1)
# denoise image
denoised = rank.median(image, disk(2))
# find continuous region (low gradient -
# where less than 10 for this image) --> markers
# disk(5) is used here to get a more smooth image
markers = rank.gradient(denoised, disk(1)) < 10
markers = ndi.label(markers)[0]
# local gradient (disk(2) is used to keep edges thin)
gradient = rank.gradient(denoised, disk(2))
# process the watershed
labels = watershed(gradient, markers)
# display results
fig, axes = plt.subplots(nrows=2,
ncols=2,
figsize=(8, 8),
sharex=True,
sharey=True)
ax = axes.ravel()
ax[0].imshow(image, cmap=plt.cm.gray, interpolation='nearest')
ax[0].set_title("Original")
ax[1].imshow(gradient, cmap=plt.cm.nipy_spectral, interpolation='nearest')
ax[1].set_title("Local Gradient")
def nuclear_expansion_watershed(label, membrane):
new_labels = morph.watershed(membrane, markers=label, watershed_line=False)
return new_labels
# read pixel size information
orig_file = os.path.basename(im_file_list[0]).replace('im_seed_nan_', '')
im = PIL.Image.open(os.path.join(training_dir, orig_file))
xres = 0.0254 / im.info['dpi'][0] * 1e6 # um
yres = 0.0254 / im.info['dpi'][1] * 1e6 # um
# load data
full_dataset, full_file_list, full_shuffle_idx = \
cytometer.data.load_datasets(im_file_list, prefix_from='im', prefix_to=['im', 'lab'], nblocks=1)
# remove borders between cells in the lab_train data. For this experiment, we want labels touching each other
for i in range(full_dataset['lab'].shape[0]):
full_dataset['lab'][i, :, :, 0] = watershed(
image=np.zeros(shape=full_dataset['lab'].shape[1:3],
dtype=full_dataset['lab'].dtype),
markers=full_dataset['lab'][i, :, :, 0],
watershed_line=False)
# relabel background as "0" instead of "1"
full_dataset['lab'][full_dataset['lab'] == 1] = 0
# plot example of data
if DEBUG:
i = 0
plt.clf()
plt.subplot(121)
plt.imshow(full_dataset['im'][i, :, :, :])
plt.subplot(122)
plt.imshow(full_dataset['lab'][i, :, :, 0])
def _skimage_ws(self, NEED_WL):
return morphology.watershed(self.dist, self.markers,
connectivity=ndimage.generate_binary_structure(3, 3),
watershed_line=NEED_WL)
'im', 'lab', 'seg', 'mask', 'predlab_kfold_' + str(i_fold).zfill(2)
],
nblocks=1)
train_im = datasets['im']
train_seg = datasets['seg']
train_mask = datasets['mask']
train_reflab = datasets['lab']
train_predlab = datasets['predlab_kfold_' + str(i_fold).zfill(2)]
del datasets
# remove borders between labels
for i in range(train_reflab.shape[0]):
train_reflab[i, :, :,
0] = watershed(image=np.zeros(shape=train_reflab[i, :, :,
0].shape,
dtype=np.uint8),
markers=train_reflab[i, :, :, 0],
watershed_line=False)
# change the background label from 1 to 0
train_reflab[train_reflab == 1] = 0
if DEBUG:
i = 250
plt.clf()
plt.subplot(221)
plt.imshow(train_im[i, :, :, :])
plt.subplot(222)
plt.imshow(train_seg[i, :, :, 0])
plt.subplot(223)
plt.imshow(train_reflab[i, :, :, 0])
plt.subplot(224)
def watershed_segmentation(rgb_img, mask, distance=10):
"""Uses the watershed algorithm to detect boundary of objects. Needs a marker file which specifies area which is
object (white), background (grey), unknown area (black).
Inputs:
rgb_img = image to perform watershed on needs to be 3D (i.e. np.shape = x,y,z not np.shape = x,y)
mask = binary image, single channel, object in white and background black
distance = min_distance of local maximum
Returns:
analysis_images = list of output images
:param rgb_img: numpy.ndarray
:param mask: numpy.ndarray
:param distance: int
:return analysis_images: list
"""
params.device += 1
# Store debug mode
debug = params.debug
params.debug = None
# # Will be depricating opencv version 2
# if cv2.__version__[0] == '2':
# dist_transform = cv2.distanceTransform(mask, cv2.cv.CV_DIST_L2, maskSize=0)
# else:
dist_transform = cv2.distanceTransformWithLabels(mask,
cv2.DIST_L2,
maskSize=0)[0]
localMax = peak_local_max(dist_transform,
indices=False,
min_distance=distance,
labels=mask)
markers = ndi.label(localMax, structure=np.ones((3, 3)))[0]
dist_transform1 = -dist_transform
labels = watershed(dist_transform1, markers, mask=mask)
img1 = np.copy(rgb_img)
for x in np.unique(labels):
rand_color = color_palette(len(np.unique(labels)))
img1[labels == x] = rand_color[x]
img2 = apply_mask(img1, mask, 'black')
joined = np.concatenate((img2, rgb_img), axis=1)
estimated_object_count = len(np.unique(markers)) - 1
# Reset debug mode
params.debug = debug
if params.debug == 'print':
print_image(
dist_transform,
os.path.join(params.debug_outdir,
str(params.device) + '_watershed_dist_img.png'))
print_image(
joined,
os.path.join(params.debug_outdir,
str(params.device) + '_watershed_img.png'))
elif params.debug == 'plot':
plot_image(dist_transform, cmap='gray')
plot_image(joined)
outputs.add_observation(variable='estimated_object_count',
trait='estimated object count',
method='plantcv.plantcv.watershed',
scale='none',
datatype=int,
value=estimated_object_count,
label='none')
# Store images
outputs.images.append([dist_transform, joined])
return joined
def extract_binary_masks_blob(A,
neuron_radius,
dims,
num_std_threshold=1,
minCircularity=0.5,
minInertiaRatio=0.2,
minConvexity=.8):
"""
Function to extract masks from data. It will also perform a preliminary selectino of good masks based on criteria like shape and size
Parameters:
----------
A: scipy.sparse matris
contains the components as outputed from the CNMF algorithm
neuron_radius: float
neuronal radius employed in the CNMF settings (gSiz)
num_std_threshold: int
number of times above iqr/1.349 (std estimator) the median to be considered as threshold for the component
minCircularity: float
parameter from cv2.SimpleBlobDetector
minInertiaRatio: float
parameter from cv2.SimpleBlobDetector
minConvexity: float
parameter from cv2.SimpleBlobDetector
Returns:
--------
masks: np.array
pos_examples:
neg_examples:
"""
params = cv2.SimpleBlobDetector_Params()
params.minCircularity = minCircularity
params.minInertiaRatio = minInertiaRatio
params.minConvexity = minConvexity
# Change thresholds
params.blobColor = 255
params.minThreshold = 0
params.maxThreshold = 255
params.thresholdStep = 3
params.minArea = np.pi * ((neuron_radius * .75)**2)
params.filterByColor = True
params.filterByArea = True
params.filterByCircularity = True
params.filterByConvexity = True
params.filterByInertia = True
detector = cv2.SimpleBlobDetector_create(params)
masks_ws = []
pos_examples = []
neg_examples = []
for count, comp in enumerate(A.tocsc()[:].T):
print(count)
comp_d = np.array(comp.todense())
gray_image = np.reshape(comp_d, dims, order='F')
gray_image = (gray_image - np.min(gray_image)) / \
(np.max(gray_image) - np.min(gray_image)) * 255
gray_image = gray_image.astype(np.uint8)
# segment using watershed
markers = np.zeros_like(gray_image)
elevation_map = sobel(gray_image)
thr_1 = np.percentile(gray_image[gray_image > 0], 50)
iqr = np.diff(np.percentile(gray_image[gray_image > 0], (25, 75)))
thr_2 = thr_1 + num_std_threshold * iqr / 1.35
markers[gray_image < thr_1] = 1
markers[gray_image > thr_2] = 2
edges = watershed(elevation_map, markers) - 1
# only keep largest object
label_objects, _ = ndi.label(edges)
sizes = np.bincount(label_objects.ravel())
if len(sizes) > 1:
idx_largest = np.argmax(sizes[1:])
edges = (label_objects == (1 + idx_largest))
edges = ndi.binary_fill_holes(edges)
else:
print('empty component')
edges = np.zeros_like(edges)
masks_ws.append(edges)
keypoints = detector.detect((edges * 200.).astype(np.uint8))
if len(keypoints) > 0:
pos_examples.append(count)
else:
neg_examples.append(count)
return np.array(masks_ws), np.array(pos_examples), np.array(neg_examples)
def get_ws():
energy = np.array(h.File(heatmap_20_path, 'r')['main'])[0]
#energy = energy[np.newaxis, :, :]
##CC
seg = get_seg(energy, None, 16)
nlabels, count = np.unique(seg,
return_counts=True) #count return the times
indices = np.argsort(count)
nlabels = nlabels[indices]
count = count[indices]
least_index = np.where(count >= 1000)[0][0]
count = count[least_index:]
nlabels = nlabels[least_index:]
rl = np.arange(seg.max() + 1).astype(seg.dtype)
for i in range(seg.max() + 1):
if i not in nlabels:
rl[i] = 0
seg = rl[seg]
# segcc_path = folder + 'WholeSlice/SegCC/whole_segcc_' + str(z) + '.h5'
# writeh5(segcc_path,'main',seg)
## Watershed
energy = np.array(h.File(heatmap_20_path,
'r')['main'])[0].astype(np.float32)
threshold = 150
energy_thres = energy - threshold
markers_unlabelled = (energy_thres > 0).astype(int)
markers, ncomponents = label_scipy(markers_unlabelled)
labels_d, count_d = np.unique(markers, return_counts=True)
rl = np.arange(markers.max() + 1).astype(markers.dtype)
pixel_threshold = 100
for i in range(len(labels_d)):
if count_d[i] < pixel_threshold:
rl[labels_d[i]] = 0
markers = rl[markers]
mask = (seg > 0).astype(int) # uses cc
labels = watershed(-energy, mask=mask, markers=markers)
segws_path = folder + 'seg_' + '_' + str(threshold) + '.h5'
writeh5(segws_path, 'main', labels)
seg_eval = (labels > 0).astype(int)
gt = np.array(
h.File('/n/pfister_lab2/Lab/xingyu/Human/Dataset/test_gt60.h5',
'r')['main'])
gt_eval = (gt > 0).astype(int)
print(np.shape(seg_eval))
print(np.shape(gt_eval))
tp = np.sum(gt_eval & seg_eval)
fp = np.sum((~gt_eval) & seg_eval)
tn = np.sum(gt_eval & (~seg_eval))
print(tp, fp, tn)
precision = float(tp) / (tp + fp)
recall = tp / (tp + tn)
print(precision, recall)
def hierarchical_segmentation(grayscale, pickle_me=False):
'''
Segments a grayscale image by first applying adaptive histogram
equalization to enhance contrast followed by an Otsu threshold to
isolate cell colonies. An adaptive thresholding method is then used to
isolate clusters of close-packed cells. Estimated regions of interest for
individual cells are finally generated using the Watershed algorithm, and
the cell regions are given unique labels and various measurements are
calculated for the regions from the original grayscale image.
Inputs:
-------
grayscale: A grayscale image loaded into a NumPy array
pickle_me: Boolean, dumps NumPy arrays of intermediate images to pickle
files in the current working directory if True
Outputs:
--------
labels: The labels associated with the thresholded regions
props: The properties of the regions-of-interest measured from the original
grayscale image
'''
# Apply CLAHE
equalized = equalize_adapthist(grayscale,
ntiles_x=16,
ntiles_y=16,
clip_limit=0.01,
nbins=256)
# Otsu threshold of CLAHE equalized "grayscale"
otsu1 = threshold_otsu(equalized)
print "Otsu threshold: {0}".format(otsu1)
thresh1 = remove_small_objects(equalized > otsu1)
colonies = thresh1 * equalized
thresh2 = threshold_adaptive(colonies, 21)
# Use morphological opening to help separate clusters and remove noise
opened = binary_opening(thresh2, selem=disk(3))
clusters = opened * equalized
# Generate labels for Watershed using local maxima of the distance
# transform as markers
distance = distance_transform_edt(opened)
local_maxi = peak_local_max(distance,
min_distance=6,
indices=False,
labels=opened)
markers = ndimage.label(local_maxi)[0]
# plt.imshow(markers)
# Apply Watershed
labels = watershed(-distance, markers, mask=opened)
# plt.imshow(label2rgb(labels))
# Measure labeled region properties in the illumination-corrected image
# (not the contrast stretched image)
props = regionprops(labels, intensity_image=grayscale)
# fig, axs = plt.subplots(2, 4)
# axs[0, 0].imshow(equalized, cmap=plt.cm.gray)
# axs[0, 0].set_title('CLAHE Equalized')
# axs[0, 1].imshow(thresh1, cmap=plt.cm.gray)
# axs[0, 1].set_title('Threshold 1, Otsu')
# axs[0, 2].imshow(colonies, cmap=plt.cm.gray)
# axs[0, 2].set_title('Colonies')
# axs[0, 3].imshow(thresh2, cmap=plt.cm.gray)
# axs[0, 3].set_title('Threshold 2, Adaptive')
# axs[1, 0].imshow(opened, cmap=plt.cm.gray)
# axs[1, 0].set_title('Threshold 2, Opened')
# axs[1, 1].imshow(clusters, cmap=plt.cm.gray)
# axs[1, 1].set_title('Clusters')
# axs[1, 2].imshow(distance, cmap=plt.cm.gray)
# axs[1, 2].set_title('Distance Transform')
# axs[1, 3].imshow(label2rgb(labels))
# axs[1, 3].set_title('Labelled Segmentation')
if pickle_me:
equalized.dump('CLAHE_equalized.p')
thresh1.dump('thresh1_otsu.p')
colonies.dump('colonies.p')
thresh2.dump('thresh2_adaptive.p')
opened.dump('opened_thresh2.p')
clusters.dump('clusters.p')
distance.dump('distance_transform.p')
markers.dump('max_dist_markers.p')
return (labels, props)
pixel_threshold = COUNT_THRESH2
for i in range(len(labels_d)):
if count_d[i] < pixel_threshold:
rl[labels_d[i]] = 0
markers = rl[markers]
# Mask for watershed from CC output
mask = (seg > 0).astype(int)
# Watershed with markers and mask
labels = watershed(-energy, mask=mask, markers=markers)
# # show contrast results
# plt.subplot(1,4,1)
# plt.imshow(markers_unlabelled[0,:,:])
# plt.title('original seed')
# plt.subplot(1,4,2)
# plt.imshow(labels[0])
# plt.title('original seg')
# --------------------------------------------------------------------------------------
# iterative watershed
# --------------------------------------------------------------------------------------
flags = needIteration(labels, VESICLE_AREA)
erosion_map = np.zeros(labels.shape)
# Identifiy markers in distance map
localMax = peak_local_max(distance_withred,
indices=False,
min_distance=minimum_distance_map_distance,
labels=thresh)
# perform a connected component analysis on the local peaks,
# using 8-connectivity, then apply the Watershed algorithm
markers_withred = ndimage.label(localMax, structure=np.ones((3, 3)))[0]
markersim = np.array(markers * 1000000, dtype="uint8")
# In[]: Identify each rsegmented region
labels = watershed(-distance_withred, markers_withred, mask=thresh)
print("{} unique segments found".format(len(np.unique(labels)) - 1))
# loop over the unique labels returned by the Watershed algorithm
maskim = np.zeros(np.shape(image), dtype="uint8")
val = 0
radius = 6
r = np.zeros(len(np.unique(labels)) + 1, dtype="uint8")
mean_coloursBRG = np.zeros((len(np.unique(labels)) + 1, 4), dtype="uint8")
colour_threshold = 160 #120 #COLOUR THRESHOLD
for label in np.unique(labels):
# if the label is zero, we are examining the 'background' so simply ignore it
val = val + 1
cv2.KMEANS_RANDOM_CENTERS)
center = np.uint8(center)
res = center[label.flatten()]
res2 = res.reshape((img.shape))
h, s, v = cv2.split(res2)
# Now we want to separate the two objects in image
# Generate the markers as local maxima of the distance to the background
distance = ndi.distance_transform_edt(v)
local_maxi = peak_local_max(distance,
indices=False,
footprint=np.ones((3, 3)),
labels=v)
markers = ndi.label(local_maxi)[0]
labels = watershed(-distance, markers, mask=v)
fig, axes = plt.subplots(ncols=3,
figsize=(9, 3),
sharex=True,
sharey=True,
subplot_kw={'adjustable': 'box-forced'})
ax = axes.ravel()
ax[0].imshow(v, cmap=plt.cm.gray, interpolation='nearest')
ax[0].set_title('Overlapping objects')
ax[1].imshow(-distance, cmap=plt.cm.gray, interpolation='nearest')
ax[1].set_title('Distances')
ax[2].imshow(labels, cmap=plt.cm.spectral, interpolation='nearest')
ax[2].set_title('Separated objects')
for a in ax:
def cell_boundary(tubulin, hoechst, threshold=80, markers=None):
def build_gabor_filters():
filters = []
ksize = 9
for theta in np.arange(0, np.pi, np.pi / 8):
kern = cv2.getGaborKernel((ksize, ksize),
4.0,
theta,
6.0,
0.5,
0,
ktype=cv2.CV_32F)
kern /= kern.sum()
filters.append(kern)
return filters
def process_gabor(img, filters):
accum = np.zeros_like(img)
for kern in filters:
fimg = cv2.filter2D(img, cv2.CV_16UC1, kern)
np.maximum(accum, fimg, accum)
return accum
p2 = np.percentile(tubulin, 2)
p98 = np.percentile(tubulin, 98)
tubulin = exposure.rescale_intensity(tubulin, in_range=(p2, p98))
p2 = np.percentile(hoechst, 2)
p98 = np.percentile(hoechst, 98)
hoechst = exposure.rescale_intensity(hoechst, in_range=(p2, p98))
# img = np.maximum(tubulin, hoechst)
img = tubulin
img = morphology.erosion(img, morphology.square(3))
filters = build_gabor_filters()
gabor = process_gabor(img, filters)
gabor = cv2.convertScaleAbs(gabor, alpha=(255.0 / 65535.0))
ret, bin1 = cv2.threshold(gabor, threshold, 255, cv2.THRESH_BINARY)
# gaussian blur on gabor filter result
ksize = 31
blur = cv2.GaussianBlur(bin1, (ksize, ksize), 0)
ret, cells_mask = cv2.threshold(blur, threshold, 255, cv2.THRESH_OTSU)
# ret, bin2 = cv2.threshold(blur, 70, 255, cv2.THRESH_BINARY)
if markers is None:
# get markers for watershed from hoescht channel
hoechst_8 = cv2.convertScaleAbs(hoechst, alpha=(255.0 / 65535.0))
blur_nuc = cv2.GaussianBlur(hoechst_8, (ksize, ksize), 0)
ret, bin_nuc = cv2.threshold(blur_nuc, 0, 255, cv2.THRESH_OTSU)
markers = ndi.label(bin_nuc)[0]
labels = morphology.watershed(-gabor, markers, mask=cells_mask)
boundaries_list = list()
# loop over the labels
for (i, l) in enumerate([l for l in np.unique(labels) if l > 0]):
# find contour of mask
cell_boundary = np.zeros(shape=labels.shape, dtype=np.uint8)
cell_boundary[labels == l] = 255
cnts = cv2.findContours(cell_boundary.copy(), cv2.RETR_EXTERNAL,
cv2.CHAIN_APPROX_SIMPLE)[-2]
contour = cnts[0]
boundary = np.array([[x, y] for x, y in [i[0] for i in contour]],
dtype=np.float32)
if len(boundary) >= 3:
boundaries_list.append({'id': l, 'boundary': Polygon(boundary)})
return boundaries_list, cells_mask > 255
# Compute compacity of granular material
compacity = mask[100:500, 200:600].mean()
# Separate the different coins
# ------------------------------------------------
erosion = morphology.binary_erosion(mask, morphology.disk(9))
erosion = morphology.binary_erosion(erosion, morphology.disk(5))
labs = morphology.label(erosion, background=0)
labs += 1
from scipy import ndimage
elevation_map = -ndimage.distance_transform_edt(mask)
regions = morphology.watershed(elevation_map, markers=labs, mask=mask)
plt.figure(figsize=(12, 3))
plt.subplot(131)
plt.imshow(labs, cmap='spectral')
plt.axis('off')
plt.subplot(132)
plt.imshow(elevation_map, cmap='spectral')
plt.axis('off')
plt.subplot(133)
plt.imshow(regions, cmap='spectral')
plt.axis('off')
plt.tight_layout()
# remove borders and relabel regions
l0, l1 = img.shape
C_binary_cut = np.zeros([DIM_LAT, DIM_LON])
# r = 500 #the bounding box side = 2r
# C_binary_cut[I_idx[0]-r:I_idx[0]+r,I_idx[1]-r:I_idx[1]+r] = C_binary[I_idx[0]-r:I_idx[0]+r,I_idx[1]-r:I_idx[1]+r]
C_binary8 = C_binary.astype(np.uint8)
kernel = np.ones((3, 3), np.uint8)
opening = cv2.morphologyEx(C_binary8,
cv2.MORPH_OPEN,
kernel,
iterations=2)
dist_transform = cv2.distanceTransform(opening, cv2.DIST_L2, 0)
ret, sure_fg = cv2.threshold(dist_transform,
0.04 * dist_transform.max(), 255, 0)
dist_transform = cv2.distanceTransform(opening, cv2.DIST_L2, 0)
labels_ws = watershed(-dist_transform, C_flag_core, mask=C_binary8)
C_binary8_second = np.where(labels_ws > 0, C_binary8, 0)
C_flag_overflow = np.where(labels_ws == 0, 0, labels_ws)
C_flag_overflow = C_flag_overflow.astype(np.uint8)
#%de')
#% Compare the mask obtained from previous mask and the current overflow mask
C_flag_compared = C_flag_overflow - C_flag_core
blobs_labels_compared = measure.label(
C_flag_compared, neighbors=4,
background=0) # identify separate blobs
volume_core = np.count_nonzero(C_flag_core)
C_flag = C_flag_core[:]
def nps_finder_2d(stack_in,
name_in,
thresh_type,
smooth_type,
smooth_size=1,
gauss_size=0,
cont_size=0,
sep_method='watershed',
mass_cutoff=200,
area_cutoff=0,
max_radius=5,
min_radius=1,
test=True,
plot=True,
save=True):
# np.copy() used because peak_local_max does weird things to the image histogram if we use the actual image
print('mass_cutoff is', mass_cutoff)
z_size, y_size, x_size = np.shape(stack_in)
# Generate flatted image from stack
max_int_proj = stack_in.max(axis=0)
# Image to store labels
labeled_image = np.zeros_like(max_int_proj)
# Smooth data
smoothed = preprocessor.smooth(np.copy(max_int_proj), smooth_type,
smooth_size)
smoothed = preprocessor.bandpass(np.copy(smoothed), gauss_size)
smoothed = preprocessor.contrast(np.copy(smoothed), cont_size)
# Calculate threshold
thresh = preprocessor.threshold(np.copy(smoothed), thresh_type)
# thresh = threshold_local(smoothed, block_size = 31, offset=40)
print('thresh is', thresh)
im_max = smoothed.max()
print('im_max is', im_max)
binary = smoothed > thresh
# Two approaches
# 1. Identify local maxima in real-space image - separate by watershedding
if sep_method == 'watershed':
print('watershedding')
local_maxi = peak_local_max(np.copy(smoothed),
min_distance=min_radius,
threshold_abs=thresh,
indices=False,
labels=np.copy(smoothed))
labeled_image = ndimage.label(local_maxi, structure=square(3))[0]
markers = ndimage.label(local_maxi)[0]
labeled_image = watershed(-labeled_image, markers, mask=binary)
# Calculate properties of particles
# Properties - area, coords, label, radius
properties = []
columns = ('x', 'y', 'area', 'radius', 'intensity')
indices = []
# f_prop = regionprops(labeled_image, intensity_image = max_int_proj)
f_prop = regionprops(labeled_image, intensity_image=max_int_proj)
for d in f_prop:
radius = (d.area / np.pi)**0.5
properties.append([
d.weighted_centroid[1], d.weighted_centroid[0], d.area, radius,
d.mean_intensity * d.area
])
indices.append(d.label)
# if not len(indices):
# all_props = pd.DataFrame([], index=[])
indices = pd.Index(indices, name='label')
properties = pd.DataFrame(properties, index=indices, columns=columns)
properties = properties[properties['intensity'] < 10000]
properties = properties[properties['intensity'] > mass_cutoff]
properties = properties[properties['area'] > area_cutoff]
properties['np_smooth_type'] = smooth_type
properties['np_smooth_size'] = smooth_size
properties['np_thresh_method'] = thresh_type
properties['separation_method'] = sep_method
particles_averaged = properties
# ################################
# # Create data array in which clustered particles are averaged
# # Store computed values
# values = []
# if len(properties) == 0:
# values.append(['NaN', 'NaN', 'NaN', 'NaN', 'NaN'])
# elif len(properties) > 0:
# if len(properties) == 1:
# properties['particle'] = 1
# elif len(properties) > 1:
# # Clustering to eliminate maxima that are too close together
# positions = properties[['x', 'y']].values
# # Distance matrix is n-particles x n-particles in size - reckon it gives the interparticle separation
# # This gives the upper triangle of the distance matrix
# dist_mat = dist.pdist(positions)
# link_mat = hier.linkage(dist_mat)
# # fcluster assigns each of the particles in positions a cluster to which it belongs
# cluster_idx = hier.fcluster(link_mat, max_radius, criterion='distance')
# properties['particle'] = cluster_idx
# particles = np.unique(properties['particle'].values)
# for i in particles:
# # Calculate weighted average position of particles
# current = properties[properties['particle'] == i]
# # Normalisation constant of weighted average
# norm = np.sum(current['intensity'])
# x_av = np.mean(current['x'])
# y_av = np.mean(current['y'])
# a_total = np.sum(current['area'])
# # Geometric cut-off to particle size
# if a_total > 2 and a_total < 400:
# values.append([x_av, y_av, a_total, norm, i])
# # Data Frame containing the weighted averages of the locations of the particles
# columns = ('x', 'y', 'a_total', 'intensity', 'particle')
# particles_averaged = pd.DataFrame(values, columns = columns)
# print 'Found', len(particles_averaged), 'particles'
if test == True:
vis.testplot_parts(max_int_proj, name_in, particles_averaged['x'],
particles_averaged['y'], smoothed, binary,
labeled_image)
if plot == True:
# Plot data
plt.clf()
fig = plt.figure(figsize=(12, 12))
ax = fig.add_subplot(111)
ax.imshow(max_int_proj, cmap='gray', vmin=0, vmax=255)
ax.scatter(particles_averaged['x'],
particles_averaged['y'],
facecolors='none',
edgecolors='red',
s=100)
if save == True:
outname = name_in + '_particles.tif'
plt.savefig(outname, bbox_inches='tight')
print(outname, 'saved')
plt.close()
return particles_averaged
def water(image, image_rgb, image_labels, index, type_trainortest):
# 滤波 过滤噪声
#中值滤波器（median): 返回图像局部区域内的中值，用此中值取代该区域内所有像素值。
denoised = filters.rank.median(image, morphology.disk(5))
# 梯度计算
#返回图像的局部梯度值（最大值 - 最小值），用此梯度值代替区域内所有像素值。
markers_t = filters.rank.gradient(denoised,
morphology.disk(5)) ##半径为5的圆形滤波器
ax_img = plt.subplot(2, 2, 1)
ax_img.set_title("gradient")
ax_img.imshow(markers_t, 'gray')
print("markers_t")
print(markers_t)
# 显示直方图
ax_hist = plt.subplot(2, 2, 2)
ax_hist.set_title('hist')
ax_hist.hist(markers_t.ravel(), bins=256) #ravel将多维数组降为一维
ax_hist.ticklabel_format(axis='y', style='scientific', scilimits=(0, 0))
ax_hist.set_xlabel('Pixel intensity')
# 梯度计算并选取梯度小的区域作为初始区域 将梯度(中值)值低于20的作为开始标记点
markers = filters.rank.gradient(denoised, morphology.disk(3)) < 10
print("markers111")
print(markers)
# 根据连通性对局部区域标记
markers = ndi.label(markers)[0]
print("matkers222")
print(markers)
# 梯度计算
gradient = filters.rank.gradient(denoised, morphology.disk(3))
# 根据标记的局部区域进行分水岭分割
labels = morphology.watershed(gradient, markers, mask=image)
print("labels:")
#print(labels)
print(labels.shape[0])
print(labels.shape[1])
# print(labels.shape, labels.max())
# 区域分割效果图转换为边缘提取效果图
oriimg = image_rgb.copy()
bwimg = image.copy()
for i in range(1, labels.shape[0] - 1):
for j in range(1, labels.shape[1] - 1):
rect = labels[i - 1:i + 2, j - 1:j + 2]
#print(np.mean(rect))
if labels[i, j] != np.mean(rect): #算数平均值。i,j点的像素值与走位区域的平均值不相等，则是边缘
oriimg[i, j] = [255, 0, 0]
bwimg[i, j] = 255
else:
bwimg[i, j] = 0
#用于灰度扩张
bwimg_wide = ndi.grey_dilation(bwimg, size=(5, 5))
# bwimg_wide = ndi.grey_dilation(bwimg, footprint=ndi.generate_binary_structure(2,1))
# bwimg_wide = ndi.grey_dilation(bwimg_wide, footprint=ndi.generate_binary_structure(2, 1))
# 输出
cv2.imwrite(
'data/' + type_trainortest + '/water_gradient10_kuo3/' + str(index) +
'.png', oriimg)
cv2.imwrite(
'data/' + type_trainortest + '/wateredge_gradient10_kuo3/' +
str(index) + '.png', bwimg)
cv2.imwrite(
'data/' + type_trainortest + '/wateredgewide_gradient10_kuo3/' +
str(index) + '.png', bwimg_wide)
ax_oriimg = plt.subplot(2, 2, 3)
ax_oriimg.set_title('original')
ax_oriimg.imshow(image_rgb)
ax_segimg = plt.subplot(2, 2, 4)
ax_segimg.set_title('edge')
ax_segimg.imshow(oriimg)
#plt.show()
fig, axes = plt.subplots(nrows=2, ncols=2, figsize=(6, 6))
axes = axes.ravel()
ax0, ax1, ax2, ax3 = axes
ax0.imshow(image, cmap=plt.cm.gray, interpolation='nearest')
ax0.set_title("Original")
ax1.imshow(image_labels, cmap=plt.cm.gray, interpolation='nearest')
ax1.set_title("Labels")
ax2.imshow(gradient, cmap=plt.cm.gray, interpolation='nearest')
ax2.set_title("Gradient")
ax3.imshow(bwimg_wide, cmap=plt.cm.gray, interpolation='nearest')
ax3.set_title("WideEdge")
for ax in axes:
ax.axis('off')
fig.tight_layout()
