def segment_images(inpDir, outDir, config_data):
""" Workflow for data with filamentous structures
such as ZO1, Beta Actin, Titin, Troponin 1.
Args:
inpDir : path to the input directory
outDir : path to the output directory
config_data : path to the configuration file
"""
logging.basicConfig(format='%(asctime)s - %(name)-8s - %(levelname)-8s - %(message)s',
datefmt='%d-%b-%y %H:%M:%S')
logger = logging.getLogger("main")
logger.setLevel(logging.INFO)
inpDir_files = os.listdir(inpDir)
for i,f in enumerate(inpDir_files):
logger.info('Segmenting image : {}'.format(f))
# Load image
br = BioReader(os.path.join(inpDir,f))
image = br.read_image()
structure_channel = 0
struct_img0 = image[:,:,:,structure_channel,0]
struct_img0 = struct_img0.transpose(2,0,1).astype(np.float32)
# main algorithm
intensity_scaling_param = config_data['intensity_scaling_param']
struct_img = intensity_normalization(struct_img0, scaling_param=intensity_scaling_param)
gaussian_smoothing_sigma = config_data['gaussian_smoothing_sigma']
if config_data['preprocessing_function'] == 'image_smoothing_gaussian_3d':
structure_img_smooth = image_smoothing_gaussian_3d(struct_img, sigma=gaussian_smoothing_sigma)
elif config_data['preprocessing_function'] == 'edge_preserving_smoothing_3d':
structure_img_smooth = edge_preserving_smoothing_3d(struct_img)
f3_param = config_data['f3_param']
bw = filament_3d_wrapper(structure_img_smooth, f3_param)
minArea = config_data['minArea']
seg = remove_small_objects(bw>0, min_size=minArea, connectivity=1, in_place=False)
seg = seg >0
out_img=seg.astype(np.uint8)
out_img[out_img>0]=255
# create output image
out_img = out_img.transpose(1,2,0)
out_img = out_img.reshape((out_img.shape[0], out_img.shape[1], out_img.shape[2], 1, 1))
# write image using BFIO
bw = BioWriter(os.path.join(outDir,f))
bw.num_x(out_img.shape[1])
bw.num_y(out_img.shape[0])
bw.num_z(out_img.shape[2])
bw.num_c(out_img.shape[3])
bw.num_t(out_img.shape[4])
bw.pixel_type(dtype='uint8')
bw.write_image(out_img)
bw.close_image()
def myRun(path):
"""
scale_x is set based on the estimated thickness of your target filaments.
For example, if visually the thickness of the filaments is usually 3~4 pixels,
then you may want to set scale_x as 1 or something near 1 (like 1.25).
Multiple scales can be used, if you have filaments of very different thickness.
cutoff_x is a threshold applied on the actual filter reponse to get the binary result.
Smaller cutoff_x may yielf more filaments, especially detecting more dim ones and thicker segmentation,
while larger cutoff_x could be less permisive and yield less filaments and slimmer segmentation.
"""
f3_param=[[1, 0.01]]
f3_param=[[5, 0.001], [3, 0.001]]
stackData = tifffile.imread(path)
numSlices = stackData.shape[0]
_printStackParams('stackData', stackData)
stackData = slidingZ(stackData, upDownSlices=1)
stackData = medianFilter(stackData)
# give us a guess for our intensity_scaling_param parameters
low_ratio, high_ratio = my_suggest_normalization_param(stackData)
# try per slice
normData = stackData.astype(np.float64)
normData[:] = 0
for i in range(numSlices):
oneSlice = stackData[i,:,:]
low_ratio, high_ratio = my_suggest_normalization_param(oneSlice)
print(i, low_ratio, high_ratio)
#low_ratio = 0.2
low_ratio -= 0.2
high_ratio -= 1
intensity_scaling_param = [low_ratio, high_ratio]
sliceNormData = intensity_normalization(oneSlice, scaling_param=intensity_scaling_param)
normData[i,:,:] = sliceNormData
#sys.exit()
'''
#intensity_scaling_param = [0.0, 22.5]
intensity_scaling_param = [low_ratio, high_ratio]
print(' === intensity_normalization() intensity_scaling_param:', intensity_scaling_param)
# intensity normalization
print(' === calling intensity_normalization()')
normData = intensity_normalization(stackData, scaling_param=intensity_scaling_param)
_printStackParams('normData', normData)
'''
# smoothing with edge preserving smoothing
print(' === calling edge_preserving_smoothing_3d()')
smoothData = edge_preserving_smoothing_3d(normData)
_printStackParams('smoothData', smoothData)
print(' === calling filament_3d_wrapper() f3_param:', f3_param)
filamentData = filament_3d_wrapper(smoothData, f3_param)
#filamentData = filamentData > 0
_printStackParams('filamentData', filamentData)
#filamentData2 = slidingZ(filamentData, upDownSlices=1)
#
# napari
print('opening in napari')
scale = (1, 0.6, 0.6)
with napari.gui_qt():
viewer = napari.Viewer(title='xxx')
minContrast = 0
maxContrast = 255
myImageLayer = viewer.add_image(stackData, scale=scale, contrast_limits=(minContrast, maxContrast), colormap='green', visible=True, name='stackData')
minContrast = 0
maxContrast = 1
myImageLayer = viewer.add_image(normData, scale=scale, contrast_limits=(minContrast, maxContrast), opacity=0.6, colormap='gray', visible=True, name='normData')
minContrast = 0
maxContrast = 1
myImageLayer = viewer.add_image(filamentData, scale=scale, contrast_limits=(minContrast, maxContrast), opacity=0.6, colormap='blue', visible=True, name='filamentData')
'''
def run(path, f3_param=[[1, 0.01]], minArea=20, saveNumber=0):
"""
use aicssegmentation to pre-process raw data and then make/save a 3D mask
"""
print('=== path:', path)
# load x/y/z voxel size (assumes .tif was saved with Fiji
xVoxel, yVoxel, zVoxel = readVoxelSize(path)
print(' xVoxel:', xVoxel, 'yVoxel:', yVoxel, 'zVoxel:', zVoxel)
# load the data
reader = AICSImage(path)
IMG = reader.data.astype(np.float32)
print(' IMG.shape:', IMG.shape)
structure_channel = 0
struct_img0 = IMG[0, structure_channel, :, :, :].copy()
# give us a guess for our intensity_scaling_param parameters
#from aicssegmentation.core.pre_processing_utils import suggest_normalization_param
#suggest_normalization_param(struct_img0)
low_ratio, high_ratio = my_suggest_normalization_param(struct_img0)
#intensity_scaling_param = [0.0, 22.5]
intensity_scaling_param = [low_ratio, high_ratio]
print('*** intensity_normalization() intensity_scaling_param:',
intensity_scaling_param)
# intensity normalization
print('=== calling intensity_normalization()')
struct_img = intensity_normalization(struct_img0,
scaling_param=intensity_scaling_param)
# smoothing with edge preserving smoothing
print('=== calling edge_preserving_smoothing_3d()')
structure_img_smooth = edge_preserving_smoothing_3d(struct_img)
#
"""
see: notebooks/playground_filament3d.ipynb
scale_x is set based on the estimated thickness of your target filaments.
For example, if visually the thickness of the filaments is usually 3~4 pixels,
then you may want to set scale_x as 1 or something near 1 (like 1.25).
Multiple scales can be used, if you have filaments of very different thickness.
cutoff_x is a threshold applied on the actual filter reponse to get the binary result.
Smaller cutoff_x may yielf more filaments, especially detecting more dim ones and thicker segmentation,
while larger cutoff_x could be less permisive and yield less filaments and slimmer segmentation.
"""
#f3_param = [[1, 0.01]] # [scale_1, cutoff_1]
print('=== calling filament_3d_wrapper() f3_param:', f3_param)
bw = filament_3d_wrapper(structure_img_smooth, f3_param)
#
#minArea = 20 # from recipe
print('=== calling remove_small_objects() minArea:', minArea)
seg = remove_small_objects(bw > 0,
min_size=minArea,
connectivity=1,
in_place=False)
#
# save original file again (with saveNumber
saveNumberStr = ''
if saveNumber > 1:
saveNumberStr = '_' + str(saveNumber)
#
# save mask
seg = seg > 0
out = seg.astype(np.uint8)
out[out > 0] = 255
# save _dvMask
maskPath = os.path.splitext(path)[0] + '_dvMask' + saveNumberStr + '.tif'
print('=== saving 3D mask [WILL FAIL IF FILE EXISTS] as maskPath:',
maskPath)
try:
writer = omeTifWriter.OmeTifWriter(maskPath)
writer.save(out)
except (OSError) as e:
print(' error: file already exists, di dnot resave, maskPath:',
maskPath)
#
# analyze skeleton, take a 3d mask and analyze as a 1-pixel skeleton
retDict0, mySkeleton = myAnalyzeSkeleton(out=out, imagePath=path)
retDict = OrderedDict()
retDict['tifPath'] = path
retDict['maskPath'] = maskPath
retDict['tifFile'] = os.path.basename(path)
retDict['xVoxel'] = xVoxel
retDict['yVoxel'] = yVoxel
retDict['zVoxel'] = zVoxel
#
retDict['params'] = OrderedDict()
retDict['params']['saveNumber'] = saveNumber
retDict['params'][
'intensity_scaling_param'] = intensity_scaling_param # calculated in my_suggest_normalization_param
retDict['params']['f3_param'] = f3_param[
0] # cludge, not sure where to put this. f3_param is a list of list but screws up my .csv output !!!
retDict['params']['minArea'] = minArea
retDict.update(retDict0)
# save 1-pixel skeleton: mySkeleton
# save _dvSkel
skelPath = os.path.splitext(path)[0] + '_dvSkel' + saveNumberStr + '.tif'
print('=== saving 3D skel [WILL FAIL IF FILE EXISTS] as maskPath:',
skelPath)
try:
writer = omeTifWriter.OmeTifWriter(skelPath)
writer.save(mySkeleton)
except (OSError) as e:
print(' error: file already exists, di dnot resave, skelPath:',
skelPath)
return retDict
def myRun(path,
myCellNumber,
genotype,
sex,
saveBase='/Users/cudmore/Desktop/samiVolume2',
f3_param=[1, 0.01],
minArea=20,
verbose=False): #, saveNumber=0):
"""
use aicssegmentation to pre-process raw data and then make/save a 3D mask
path: path to raw tif, the _ch2.tif
myCellNumber: cell number from batch file (per genotype/sex), NOT unique across different (genotype, sex)
saveBase: full path to folder to save to (e.g. /Users/cudmore/Desktop/samiVolume2), must exist
...
saveNumber: not used
"""
print(' === myRun() path:', path, 'saveBase:', saveBase, 'f3_param:',
f3_param, 'minArea:', minArea) #, 'saveNumber:', saveNumber)
#20200608
#saveBase = '/Users/cudmore/Desktop/samiVolume2'
tmpPath, tmpFileName = os.path.split(path)
tmpPath = tmpPath.replace('../data/', '')
tmpFileNameNoExtension, tmpExtension = tmpFileName.split('.')
saveBase = os.path.join(saveBase, tmpPath)
if not os.path.isdir(saveBase):
print(' making output dir:', saveBase)
os.makedirs(saveBase)
#saveBase = os.path.join(saveBase, tmpPath, tmpFileNameNoExtension)
saveBase = os.path.join(saveBase, tmpFileNameNoExtension)
#saveBase = os.path.join(saveBase, os.path.splitext(path)[0].replace('../data/', ''))
if verbose: print(' saveBase:', saveBase)
if not os.path.isfile(path):
print('ERROR: myRun() did not find file:', path)
return None
# load the data
IMG, tifHeader = bimpy.util.bTiffFile.imread(path)
saveDataPath = saveBase + '.tif'
print(' === saving raw data to saveDataPath:', saveDataPath)
bimpy.util.bTiffFile.imsave(saveDataPath,
IMG,
tifHeader=tifHeader,
overwriteExisting=True)
IMG = IMG.astype(np.float32)
# channel 1: load then save (do nothing else with it)
channelOnePath = path.replace('_ch2.tif', '_ch1.tif')
channelOneData, channelOneTiffHeader = bimpy.util.bTiffFile.imread(
channelOnePath)
saveChannelOnePath = saveBase.replace('_ch2', '_ch1.tif')
bimpy.util.bTiffFile.imsave(saveChannelOnePath,
channelOneData,
tifHeader=tifHeader,
overwriteExisting=True)
# load x/y/z voxel size (assumes .tif was saved with Fiji
xVoxel, yVoxel, zVoxel = readVoxelSize(path)
print(' file:', os.path.basename(path), 'has shape:', IMG.shape,
'xVoxel:', xVoxel, 'yVoxel:', yVoxel, 'zVoxel:', zVoxel)
# give us a guess for our intensity_scaling_param parameters
#low_ratio, high_ratio = my_suggest_normalization_param(struct_img0)
low_ratio, high_ratio = my_suggest_normalization_param(IMG)
#intensity_scaling_param = [0.0, 22.5]
intensity_scaling_param = [low_ratio, high_ratio]
if verbose:
print(' === my_intensity_normalization() intensity_scaling_param:',
intensity_scaling_param)
# intensity normalization
if verbose: print(' === calling my_intensity_normalization()')
#struct_img = my_intensity_normalization(struct_img0, scaling_param=intensity_scaling_param)
struct_img = my_intensity_normalization(
IMG, scaling_param=intensity_scaling_param)
# smoothing with edge preserving smoothing
if verbose: print(' === calling edge_preserving_smoothing_3d()')
structure_img_smooth = edge_preserving_smoothing_3d(struct_img)
#
"""
see: notebooks/playground_filament3d.ipynb
scale_x is set based on the estimated thickness of your target filaments.
For example, if visually the thickness of the filaments is usually 3~4 pixels,
then you may want to set scale_x as 1 or something near 1 (like 1.25).
Multiple scales can be used, if you have filaments of very different thickness.
cutoff_x is a threshold applied on the actual filter reponse to get the binary result.
Smaller cutoff_x may yielf more filaments, especially detecting more dim ones and thicker segmentation,
while larger cutoff_x could be less permisive and yield less filaments and slimmer segmentation.
"""
#f3_param = [[1, 0.01]] # [scale_1, cutoff_1]
if verbose:
print(' === calling filament_3d_wrapper() f3_param:', f3_param)
bw = filament_3d_wrapper(structure_img_smooth,
[f3_param]) # f3_param is a list of a list
#
#minArea = 20 # from recipe
if verbose:
print(' === calling remove_small_objects() minArea:', minArea)
seg = remove_small_objects(bw > 0,
min_size=minArea,
connectivity=1,
in_place=False)
#
# save original file again (with saveNumber
saveNumberStr = ''
#if saveNumber>1:
# saveNumberStr = '_' + str(saveNumber)
#
# save _dvMask.tif
seg = seg > 0
out = seg.astype(np.uint8)
out[out > 0] = 255
#
#maskPath = os.path.splitext(path)[0] + '_dvMask' + saveNumberStr + '.tif'
maskPath = saveBase + '_dvMask' + saveNumberStr + '.tif'
print(' === saving 3D mask as maskPath:', maskPath)
#tifffile.imsave(maskPath, out)
bimpy.util.bTiffFile.imsave(maskPath,
out,
tifHeader=tifHeader,
overwriteExisting=True)
'''
try:
writer = omeTifWriter.OmeTifWriter(maskPath)
writer.save(out)
except(OSError) as e:
print(' ******** ERROR: file already exists, did not resave, maskPath:', maskPath)
'''
#
# ################
# analyze skeleton, take a 3d mask and analyze as a 1-pixel skeleton
retDict0, mySkeleton = myAnalyzeSkeleton(out=out,
imagePath=path,
saveBase=saveBase)
# ################
retDict = OrderedDict()
retDict['analysisDate'] = datetime.today().strftime('%Y%m%d')
#
retDict['saveBase'] = saveBase
retDict['myCellNumber'] = myCellNumber
retDict['genotype'] = genotype
retDict['sex'] = sex
#
retDict['path'] = path # 20200713 working on parallel
retDict['tifPath'] = path
retDict['maskPath'] = maskPath
retDict['tifFile'] = os.path.basename(path)
retDict['xVoxel'] = xVoxel
retDict['yVoxel'] = yVoxel
retDict['zVoxel'] = zVoxel
#
retDict['params'] = OrderedDict()
#retDict['params']['saveNumber'] = saveNumber
retDict['params'][
'intensity_scaling_param'] = intensity_scaling_param # calculated in my_suggest_normalization_param
#retDict['params']['f3_param'] = f3_param[0] # cludge, not sure where to put this. f3_param is a list of list but screws up my .csv output !!!
retDict['params'][
'f3_param'] = f3_param # cludge, not sure where to put this. f3_param is a list of list but screws up my .csv output !!!
retDict['params']['minArea'] = minArea
#
retDict.update(
retDict0
) # this has 'keys' that are lists of ('len3d', 'eLen', 'branchType')
# save 1-pixel skeleton: mySkeleton
# save _dvSkel
#skelPath = os.path.splitext(path)[0] + '_dvSkel' + saveNumberStr + '.tif'
skelPath = saveBase + '_dvSkel' + saveNumberStr + '.tif'
print(' === saving 3D skel as maskPath:', skelPath)
#tifffile.imsave(skelPath, mySkeleton)
bimpy.util.bTiffFile.imsave(skelPath,
mySkeleton,
tifHeader=tifHeader,
overwriteExisting=True)
return retDict