def membrane_image_segmentation(img, gaussian_sigma=0.75, h_min=None, segmentation_gaussian_sigma=0.5, volume_threshold=None):
voxelsize = np.array(img.voxelsize)
if h_min is None:
h_min = 2 if img.dtype==np.uint8 else 1000
smooth_image = nd.gaussian_filter(img, sigma=gaussian_sigma / voxelsize).astype(img.dtype)
smooth_img = SpatialImage(smooth_image, voxelsize=voxelsize)
ext_img = h_transform(smooth_img, h=h_min, method='min')
seed_img = region_labeling(ext_img, low_threshold=1, high_threshold=h_min, method='connected_components')
seg_smooth_image = nd.gaussian_filter(img, sigma=segmentation_gaussian_sigma / voxelsize).astype(img.dtype)
seg_smooth_img = SpatialImage(seg_smooth_image, voxelsize=voxelsize)
seg_img = segmentation(seg_smooth_img, seed_img, control='first', method='seeded_watershed')
if volume_threshold is not None:
seg_volumes = dict(zip(np.arange(seg_img.max()) + 1,
nd.sum(np.prod(voxelsize) * np.ones_like(seg_img),
seg_img,
index=np.arange(seg_img.max()) + 1)))
labels_to_remove = np.array(list(seg_volumes.keys()))[np.array(list(seg_volumes.values())) > volume_threshold]
print("--> Removing too large labels :", labels_to_remove)
for l in labels_to_remove:
seg_img[seg_img == l] = 1
return seg_img
def test_plugin():
im = imread(data_path('segmentation_src.inr'))
im_ref = imread(data_path('segmentation_seeded_watershed.inr'))
smooth_img = linear_filtering(im, std_dev=2.0, method='gaussian_smoothing')
regext_img = h_transform(smooth_img, h=5, method='h_transform_min')
conn_img = region_labeling(regext_img, low_threshold=1, high_threshold=3, method='connected_components')
wat_img = segmentation(smooth_img, conn_img, control='first', method='seeded_watershed')
np.testing.assert_array_equal(wat_img, im_ref)
def test_plugin():
im = imread(data_path('segmentation_src.inr'))
im_ref = imread(data_path('segmentation_seeded_watershed.inr'))
smooth_img = linear_filtering(im, std_dev=2.0, method='gaussian_smoothing')
regext_img = h_transform(smooth_img, h=5, method='h_transform_min')
conn_img = region_labeling(regext_img,
low_threshold=1,
high_threshold=3,
method='connected_components')
wat_img = segmentation(smooth_img,
conn_img,
control='first',
method='seeded_watershed')
np.testing.assert_array_equal(wat_img, im_ref)
# ext_img = h_transform(asf_img, h=h_min, method='h_transform_min')
smooth_img = linear_filtering(img2seg,
std_dev=std_dev,
method='gaussian_smoothing')
ext_img = h_transform(smooth_img, h=h_min, method='h_transform_min')
seed_img = region_labeling(ext_img,
low_threshold=1,
high_threshold=h_min,
method='connected_components')
print "Detected {} seeds!".format(len(np.unique(seed_img)))
print "\n - Performing seeded watershed segmentation..."
from timagetk.plugins import segmentation
std_dev = 1.0
# smooth_img = linear_filtering(img2seg, std_dev=std_dev, method='gaussian_smoothing')
seg_im = segmentation(smooth_img, seed_img, method='seeded_watershed')
seg_im[seg_im == 0] = back_id
# world.add(seg_im, 'seg', colormap='glasbey', alphamap='constant')
from timagetk.io import imsave
imsave(seg_img_fname, seg_im)
else:
from timagetk.io import imread
seg_im = imread(seg_img_fname)
###############################################################################
# -- MAYAVI VISU:
###############################################################################
# from mayavi import mlab
# from os.path import splitext
# import numpy as np
method='h_transform_min')
print " --> Components labelling..."
con_img = region_labeling(ext_img,
low_threshold=1,
high_threshold=h_min,
method='connected_components')
nb_connectec_comp = len(np.unique(con_img.get_array()))
print nb_connectec_comp, "seeds detected!"
if nb_connectec_comp <= 3:
print "Not enough seeds, aborting!"
continue
# world.add(con_img, 'labelled_seeds', voxelsize=vxs)
del ext_img
print " --> Seeded watershed..."
seg_im = segmentation(img,
con_img,
method='seeded_watershed')
# - Performs topomesh creation from detected seeds:
print " --> Analysing segmented image...",
img_graph = graph_from_image(
seg_im,
background=1,
spatio_temporal_properties=['barycenter', 'L1'],
ignore_cells_at_stack_margins=False)
print img_graph.nb_vertices(
), "seeds extracted by 'graph_from_image'!"
# -- Create a topomesh from 'seed_positions':
print " --> Topomesh creation..."
seed_positions = {
v: img_graph.vertex_property('barycenter')[v] *
microscope_orientation
largest_component = (np.arange(n_components) +
1)[np.argmax(components_area)]
background_img = (
connected_background_components == largest_component).astype(np.uint16)
# ---- Finaly add the background and make a SpatialImage:
seed_img[background_img == 1] = 1
del smooth_img_bck, background_img
seed_img = SpatialImage(seed_img, voxelsize=voxelsize)
# world.add(seed_img,"seed_image", colormap="glasbey", alphamap="constant",voxelsize=microscope_orientation*voxelsize, bg_id=0)
# -- Performs automatic seeded watershed using previously created seed image:
print "\n# - Seeded watershed using seed EXPERT seed positions..."
smooth_img = linear_filtering(img,
std_dev=std_dev,
method='gaussian_smoothing')
seg_im = segmentation(smooth_img, seed_img)
# Use largest bounding box to determine background value:
background = get_background_value(seg_im, microscope_orientation)
print "Detected background value:", background
# world.add(seg_im,"seg_image", colormap="glasbey", alphamap="constant",voxelsize=microscope_orientation*voxelsize, bg_id=background)
# -- Create a vertex_topomesh from detected cell positions:
print "\n# - Extracting 'barycenter' & 'L1' properties from segmented image..."
# --- Compute 'L1' and 'barycenter' properties using 'graph_from_image'
img_graph = graph_from_image(
seg_im,
background=background,
spatio_temporal_properties=['L1', 'barycenter'],
ignore_cells_at_stack_margins=True)
print img_graph.nb_vertices(), " cells detected"
print "\n# - Creating a vertex_topomesh..."
components_area = nd.sum(np.ones_like(connected_background_components),
connected_background_components,
index=np.arange(n_components) + 1)
largest_component = (np.arange(n_components) + 1)[np.argmax(components_area)]
background_img = (connected_background_components == largest_component).astype(
np.uint16)
con_img[background_img == 1] = 1
del smooth_img_bck, background_img
con_img = SpatialImage(con_img, voxelsize=voxelsize)
# world.add(con_img,"seed_image", colormap="glasbey", alphamap="constant",voxelsize=microscope_orientation*voxelsize, bg_id=0)
# -- Performs automatic seeded watershed using previously created seed image:
smooth_img = linear_filtering(img,
std_dev=std_dev,
method='gaussian_smoothing')
seg_im = segmentation(smooth_img, con_img)
# world.add(seg_im,"seg_image", colormap="glasbey", alphamap="constant",voxelsize=microscope_orientation*voxelsize)
# -- Create a vertex_topomesh from detected cell positions:
# --- Get cell barycenters positions:
img_graph = graph_from_image(seg_im,
background=1,
spatio_temporal_properties=['L1', 'barycenter'],
ignore_cells_at_stack_margins=True)
print img_graph.nb_vertices(), " cells detected"
vtx = list(img_graph.vertices())
labels = img_graph.vertex_property('labels')
margin_expert_cells = [
c for c in expert_topomesh.wisps(0) if not c in [labels[v] for v in vtx]
]
for c in margin_expert_cells:
from timagetk.plugins import labels_post_processing
except ImportError:
raise ImportError('Import Error')
out_path = './results/' # to save results
# we consider an input image
# SpatialImage instance
input_img = imread(data_path('input.tif'))
# optional denoising block
smooth_img = linear_filtering(input_img, std_dev=2.0,
method='gaussian_smoothing')
asf_img = morphology(smooth_img, max_radius=3,
method='co_alternate_sequential_filter')
ext_img = h_transform(asf_img, h=150,
method='h_transform_min')
con_img = region_labeling(ext_img, low_threshold=1,
high_threshold=150,
method='connected_components')
seg_img = segmentation(smooth_img, con_img, control='first',
method='seeded_watershed')
# optional post processig block
pp_img = labels_post_processing(seg_img, radius=1,
iterations=1,
method='labels_erosion')
res_name = 'example_segmentation.tif'
imsave(out_path+res_name, pp_img)
def seg_pipe(img2seg,
h_min,
img2sub=None,
iso=True,
equalize=True,
stretch=False,
std_dev=0.8,
min_cell_volume=20.,
back_id=1,
to_8bits=False):
"""Define the sementation pipeline
Parameters
----------
img2seg : str
image to segment.
h_min : int
h-minima used with the h-transform function
img2sub : str, optional
image to subtract to the image to segment.
iso : bool, optional
if True (default), isometric resampling is performed after h-minima
detection and before watershed segmentation
equalize : bool, optional
if True (default), intensity adaptative equalization is performed before
h-minima detection
stretch : bool, optional
if True (default, False), intensity histogram stretching is performed
before h-minima detection
std_dev : float, optional
real unit standard deviation used for Gaussian smoothing of the image to segment
min_cell_volume : float, optional
minimal volume accepted in the segmented image
back_id : int, optional
the background label
to_8bits : bool, optional
transform the image to segment as an unsigned 8 bits image for the h-transform
and seed-labelleing steps
Returns
-------
seg_im : SpatialImage
the labelled image obtained by seeded-watershed
Notes
-----
* Both 'equalize' & 'stretch' can not be True at the same time since they work
on the intensity of the pixels;
* Signal subtraction is performed after intensity rescaling (if any);
* Linear filtering (Gaussian smoothing) is performed before h-minima transform
for local minima detection;
* Gaussian smoothing should be performed on isometric images, if the provided
image is not isometric, we resample it before smoothing, then go back to
original voxelsize;
* In any case H-Transfrom is performed on the image with its native resolution
to speed upd seed detection;
* Same goes for connexe components detection (seed labelling);
* Segmentation will be performed on the isometric images if iso is True, in
such case we resample the image of detected seeds and use the isometric
smoothed intensity image;
"""
t_start = time.time()
# - Check we have only one intensity rescaling method called:
try:
assert equalize + stretch < 2
except AssertionError:
raise ValueError(
"Both 'equalize' & 'stretch' can not be True at once!")
# - Check the standard deviation value for Gaussian smoothing is valid:
try:
assert std_dev <= 1.
except AssertionError:
raise ValueError(
"Standard deviation for Gaussian smoothing should be superior or equal to 1!"
)
ori_vxs = img2seg.voxelsize
ori_shape = img2seg.shape
if img2sub is not None:
print "\n - Performing signal substraction..."
img2seg = signal_subtraction(img2seg, img2sub)
if equalize:
print "\n - Performing z-slices adaptative histogram equalisation on the intensity image to segment..."
img2seg = z_slice_equalize_adapthist(img2seg)
if stretch:
print "\n - Performing z-slices histogram contrast stretching on the intensity image to segment..."
img2seg = z_slice_contrast_stretch(img2seg)
print "\n - Automatic seed detection...".format(h_min)
# morpho_radius = 1.0
# asf_img = morphology(img2seg, max_radius=morpho_radius, method='co_alternate_sequential_filter')
# ext_img = h_transform(asf_img, h=h_min, method='h_transform_min')
min_vxs = min(img2seg.voxelsize)
if std_dev < min_vxs:
print " -- Isometric resampling prior to Gaussian smoothing...".format(
std_dev)
img2seg = isometric_resampling(img2seg)
print " -- Gaussian smoothing with std_dev={}...".format(std_dev)
iso_smooth_img = linear_filtering(img2seg,
std_dev=std_dev,
method='gaussian_smoothing',
real=True)
if std_dev < min_vxs:
print " -- Down-sampling a copy back to original voxelsize (to use with `h-transform`)..."
smooth_img = resample(iso_smooth_img, ori_vxs)
if not np.allclose(ori_shape, smooth_img.shape):
print "WARNING: shape missmatch after down-sampling from isometric image:"
print " -- original image shape: {}".format(ori_shape)
print " -- down-sampled image shape: {}".format(smooth_img.shape)
else:
print " -- Copying original image (to use with `h-transform`)..."
smooth_img = iso_smooth_img
if not iso:
del iso_smooth_img # no need to keep this image after this step!
print " -- H-minima transform with h-min={}...".format(h_min)
if to_8bits:
ext_img = h_transform(smooth_img.to_8bits(),
h=h_min,
method='h_transform_min')
else:
ext_img = h_transform(smooth_img, h=h_min, method='h_transform_min')
if iso:
smooth_img = iso_smooth_img # no need to keep both images after this step!
print " -- Region labelling: connexe components detection..."
seed_img = region_labeling(ext_img,
low_threshold=1,
high_threshold=h_min,
method='connected_components')
print "Detected {} seeds!".format(len(np.unique(seed_img)) -
1) # '0' is in the list!
del ext_img # no need to keep this image after this step!
print "\n - Performing seeded watershed segmentation..."
if iso:
seed_img = isometric_resampling(seed_img, option='label')
if to_8bits:
seg_im = segmentation(smooth_img.to_8bits(),
seed_img,
method='seeded_watershed')
else:
seg_im = segmentation(smooth_img, seed_img, method='seeded_watershed')
# seg_im[seg_im == 0] = back_id
print "Detected {} labels!".format(len(np.unique(seg_im)))
if min_cell_volume > 0.:
from vplants.tissue_analysis.spatial_image_analysis import SpatialImageAnalysis
print "\n - Performing cell volume filtering..."
spia = SpatialImageAnalysis(seg_im, background=None)
vol = spia.volume()
too_small_labels = [
k for k, v in vol.items() if v < min_cell_volume and k != 0
]
if too_small_labels != []:
print "Detected {} labels with a volume < {}µm2".format(
len(too_small_labels), min_cell_volume)
print " -- Removing seeds leading to small cells..."
spia = SpatialImageAnalysis(seed_img, background=None)
seed_img = spia.get_image_without_labels(too_small_labels)
print " -- Performing final seeded watershed segmentation..."
seg_im = segmentation(smooth_img,
seed_img,
method='seeded_watershed')
# seg_im[seg_im == 0] = back_id
print "Detected {} labels!".format(len(np.unique(seg_im)))
print "\nDone in {}s".format(round(time.time() - t_start, 3))
return seg_im
