def analyze_single_image(self,
Rawimage,
axis=None,
show_result=True,
show_each_cell=False):
MLresults = self.DetectionOnImage(Rawimage,
axis=axis,
show_result=show_result)
cell_Data, cell_counted_inRound, total_cells_counted_in_coord = \
ProcessImage.retrieveDataFromML(Rawimage, MLresults, show_each_cell = show_each_cell)
print(
"Number of cells counted so far: {}".format(cell_counted_inRound))
print("Number of cells counted in image: {}".format(
total_cells_counted_in_coord))
return cell_Data
def analyze_images_in_folder(self,
folder,
generate_zmax=False,
show_result=True,
save_mask=True,
save_excel=True):
"""
Given the folder, perform general analysis over the images in it.
Parameters
----------
folder : str
Path to the folder.
generate_zmax : bool, optional
Whether to calcaulate the z-max projection first. The default is False.
show_result : bool, optional
If show the machine learning segmentation results. The default is True.
save_mask : bool, optional
DESCRIPTION. The default is True.
save_excel : bool, optional
DESCRIPTION. The default is True.
Returns
-------
cell_Data : pd.dataframe
DESCRIPTION.
"""
flat_cell_counted_in_folder = 0
total_cells_counted_in_folder = 0
# If need to do zmax projection first
if generate_zmax == True:
ProcessImage.cam_screening_post_processing(folder)
# Here a new folder for maxProjection is generated inside, change the path
folder = os.path.join(folder, 'maxProjection')
# If background images are taken
if os.path.exists(os.path.join(folder, 'background')):
# If the background image is taken to substract out
background_substraction = True
# Get all the background files names
background_fileNameList = []
for file in os.listdir(os.path.join(folder, 'background')):
if "tif" in file:
background_fileNameList.append(
os.path.join(folder, 'background', file))
background_image = ProcessImage.image_stack_calculation(
background_fileNameList, operation="mean")
# Get a list of file names
fileNameList = []
for file in os.listdir(folder):
if "tif" in file and "LED" not in file:
fileNameList.append(file)
print(fileNameList)
# Analyse each image
for image_file_name in fileNameList:
print(image_file_name)
Rawimage = imread(os.path.join(folder, image_file_name))
if background_substraction == True:
Rawimage = np.abs(Rawimage - background_image)
# Analyze each image
# Run the detection on input image.
MLresults = self.DetectionOnImage(Rawimage,
axis=None,
show_result=show_result)
if save_mask == True:
if not os.path.exists(os.path.join(folder, 'ML_masks')):
# If the folder is not there, create the folder
os.mkdir(os.path.join(folder, 'ML_masks'))
fig, ax = plt.subplots()
# Set class_names = [None,None,None,None] to mute class name display.
visualize.display_instances(
Rawimage,
MLresults['rois'],
MLresults['masks'],
MLresults['class_ids'],
class_names=[None, None, None, None],
ax=ax,
centre_coors=MLresults['Centre_coor'],
Centre_coor_radius=2,
WhiteSpace=(
0, 0)) #MLresults['class_ids'],MLresults['scores'],
# ax.imshow(fig)
fig.tight_layout()
# Save the detection Rawimage
fig_name = os.path.join(
folder, 'ML_masks', 'ML_mask_{}.png'.format(
image_file_name[0:len(image_file_name) - 4]))
plt.savefig(fname=fig_name,
dpi=200,
pad_inches=0.0,
bbox_inches='tight')
if flat_cell_counted_in_folder == 0:
cell_Data, flat_cell_counted_in_folder, total_cells_counted_in_coord = \
ProcessImage.retrieveDataFromML(Rawimage, MLresults, image_file_name, flat_cell_counted_in_folder)
else:
Cell_Data_new, flat_cell_counted_in_folder, total_cells_counted_in_coord = \
ProcessImage.retrieveDataFromML(Rawimage, MLresults, image_file_name, flat_cell_counted_in_folder)
if len(Cell_Data_new) > 0:
cell_Data = cell_Data.append(Cell_Data_new)
total_cells_counted_in_folder += total_cells_counted_in_coord
if save_excel == True:
# Save to excel
cell_Data.to_excel(
os.path.join(
folder, 'CellsProperties_{}flat_outof_{}cells.xlsx'.format(
flat_cell_counted_in_folder,
total_cells_counted_in_folder)))
return cell_Data
def analyze_images_in_folder(
self,
folder,
generate_zmax=False,
show_result=True,
save_mask=True,
save_excel=True,
):
"""
Given the folder, perform general analysis over the images in it.
Parameters
----------
folder : str
Path to the folder.
generate_zmax : bool, optional
Whether to calcaulate the z-max projection first. The default is False.
show_result : bool, optional
If show the machine learning segmentation results. The default is True.
save_mask : bool, optional
DESCRIPTION. The default is True.
save_excel : bool, optional
DESCRIPTION. The default is True.
Returns
-------
cell_Data : pd.dataframe
DESCRIPTION.
"""
flat_cell_counted_in_folder = 0
total_cells_counted_in_folder = 0
background_substraction = False
root_folder = folder
# If need to do zmax projection first
if generate_zmax == True:
ProcessImage.cam_screening_post_processing(root_folder)
# Here a new folder for maxProjection is generated inside, change the path
folder = os.path.join(root_folder, "maxProjection")
# If background images are taken
if os.path.exists(os.path.join(root_folder, "background")):
# If the background image is taken to substract out
background_substraction = True
print("Run background substraction.")
# Get all the background files names
background_fileNameList = []
for file in os.listdir(os.path.join(root_folder, "background")):
if "calculated background" not in file:
if "tif" in file or "TIF" in file:
background_fileNameList.append(
os.path.join(root_folder, "background", file))
# Average over multiple images
background_image = ProcessImage.image_stack_calculation(
background_fileNameList, operation="mean")
# # Smooth the image
# background_image = ProcessImage.average_filtering(
# background_image, filter_side_length = 25)
# Save the individual file.
with skimtiff.TiffWriter(
os.path.join(root_folder, "background",
"calculated background.tif"),
imagej=True,
) as tif:
tif.save(background_image.astype(np.uint16), compress=0)
# Get a list of file names
fileNameList = []
for file in os.listdir(folder):
if "tif" in file and "LED" not in file:
fileNameList.append(file)
print(fileNameList)
# Analyse each image
for image_file_name in fileNameList:
print(image_file_name)
Rawimage = imread(os.path.join(folder, image_file_name))
if background_substraction == True:
Rawimage = np.abs(Rawimage - background_image).astype(
np.uint16)
camera_dark_level = 100
# # Normalize to the illumination intensity
# Rawimage = np.uint16(Rawimage \
# / ((background_image - camera_dark_level)\
# /(np.amin(background_image) - camera_dark_level)))
# Analyze each image
# Run the detection on input image.
MLresults = self.DetectionOnImage(Rawimage,
axis=None,
show_result=show_result)
if save_mask == True:
if not os.path.exists(os.path.join(folder, "ML_masks")):
# If the folder is not there, create the folder
os.mkdir(os.path.join(folder, "ML_masks"))
fig, ax = plt.subplots()
# Set class_names = [None,None,None,None] to mute class name display.
visualize.display_instances(
Rawimage,
MLresults["rois"],
MLresults["masks"],
MLresults["class_ids"],
class_names=[None, None, None, None],
ax=ax,
centre_coors=MLresults["Centre_coor"],
Centre_coor_radius=2,
WhiteSpace=(0, 0),
) # MLresults['class_ids'],MLresults['scores'],
# ax.imshow(fig)
fig.tight_layout()
# Save the detection Rawimage
fig_name = os.path.join(
folder,
"ML_masks",
"ML_mask_{}.png".format(
image_file_name[0:len(image_file_name) - 4]),
)
plt.savefig(fname=fig_name,
dpi=200,
pad_inches=0.0,
bbox_inches="tight")
if flat_cell_counted_in_folder == 0:
(
cell_Data,
flat_cell_counted_in_folder,
total_cells_counted_in_coord,
) = ProcessImage.retrieveDataFromML(
Rawimage, MLresults, image_file_name,
flat_cell_counted_in_folder)
else:
(
Cell_Data_new,
flat_cell_counted_in_folder,
total_cells_counted_in_coord,
) = ProcessImage.retrieveDataFromML(
Rawimage, MLresults, image_file_name,
flat_cell_counted_in_folder)
if len(Cell_Data_new) > 0:
cell_Data = cell_Data.append(Cell_Data_new)
total_cells_counted_in_folder += total_cells_counted_in_coord
if save_excel == True:
# Save to excel
cell_Data.to_excel(
os.path.join(
folder,
"CellsProperties_{}flat_outof_{}cells.xlsx".format(
flat_cell_counted_in_folder,
total_cells_counted_in_folder),
))
return cell_Data
def FluorescenceAnalysis(self, folder, round_num, save_mask=True):
"""
# =============================================================================
# Given the folder and round number, return a dictionary for the round
# that contains each scanning position as key and structured array of detailed
# information about each identified cell as content.
#
# Returned structured array fields:
# - BoundingBox of cell ROI
# - Mean intensity of whole cell area
# - Mean intensity of cell membrane part
# - Contour soma ratio
# =============================================================================
Parameters
----------
folder : string.
The directory to folder where the screening data is stored.
round_num : string.
The target round number of analysis.
save_mask: bool.
Whether to save segmentation masks.
Returns
-------
cell_Data : pd.DataFrame.
Sum of return from func: retrieveDataFromML, for whole round.
"""
RoundNumberList, CoordinatesList, fileNameList = self.retrive_scanning_scheme(
folder, file_keyword='Zmax')
# RoundNumberList, CoordinatesList, fileNameList = self.retrive_scanning_scheme(folder, file_keyword = 'Zfocus')
if not os.path.exists(
os.path.join(folder, 'MLimages_{}'.format(round_num))):
# If the folder is not there, create the folder
os.mkdir(os.path.join(folder, 'MLimages_{}'.format(round_num)))
for EachRound in RoundNumberList:
cells_counted_in_round = 0
if EachRound == round_num:
# Start numbering cells at each round
self.cell_counted_inRound = 0
for EachCoord in CoordinatesList:
# =============================================================================
# For tag fluorescence:
# =============================================================================
print(EachCoord)
#-------------- readin image---------------
for Eachfilename in enumerate(fileNameList):
if EachCoord in Eachfilename[
1] and EachRound in Eachfilename[1]:
if '0Zmax' in Eachfilename[1]:
ImgNameInfor = Eachfilename[1][
0:len(Eachfilename[1]) -
14] # get rid of '_PMT_0Zmax.tif' in the name.
elif '0Zfocus' in Eachfilename[1]:
ImgNameInfor = Eachfilename[1][
0:len(Eachfilename[1]) -
16] # get rid of '_PMT_0Zfocus.tif' in the name.
_imagefilename = os.path.join(
folder, Eachfilename[1])
#------------------------------------------
# =========================================================================
# USING MASKRCNN...
# =========================================================================
# Imagepath = self.Detector._fixPathName(_imagefilename)
Rawimage = imread(_imagefilename)
# if ClearImgBef == True:
# # Clear out junk parts to make it esaier for ML detection.
# RawimageCleared = self.preProcessMLimg(Rawimage, smallest_size=300, lowest_region_intensity=0.16)
# else:
# RawimageCleared = Rawimage.copy()
image = ProcessImage.convert_for_MaskRCNN(Rawimage)
# Run the detection on input image.
results = self.Detector.detect([image])
MLresults = results[0]
if save_mask == True:
fig, ax = plt.subplots()
# Set class_names = [None,None,None,None] to mute class name display.
visualize.display_instances(
image,
MLresults['rois'],
MLresults['masks'],
MLresults['class_ids'],
class_names=[None, None, None, None],
ax=ax,
centre_coors=MLresults['Centre_coor'],
Centre_coor_radius=2,
WhiteSpace=(0, 0)
) #MLresults['class_ids'],MLresults['scores'],
# ax.imshow(fig)
fig.tight_layout()
# Save the detection image
fig_name = os.path.join(
folder, 'MLimages_{}\{}.tif'.format(
round_num, ImgNameInfor))
plt.savefig(fname=fig_name,
dpi=200,
pad_inches=0.0,
bbox_inches='tight')
# segmentationImg = Image.fromarray(fig) #generate an image object
# segmentationImg.save(os.path.join(folder, 'MLimages_{}\{}.tif'.format(round_num, ImgNameInfor)))#save as tif
if self.cell_counted_inRound == 0:
cell_Data, self.cell_counted_inRound, total_cells_counted_in_coord = \
ProcessImage.retrieveDataFromML(Rawimage, MLresults, str(ImgNameInfor), self.cell_counted_inRound)
else:
Cell_Data_new, self.cell_counted_inRound, total_cells_counted_in_coord = \
ProcessImage.retrieveDataFromML(Rawimage, MLresults, str(ImgNameInfor), self.cell_counted_inRound)
if len(Cell_Data_new) > 0:
cell_Data = cell_Data.append(Cell_Data_new)
# Count in total how many flat and round cells are identified.
cells_counted_in_round += total_cells_counted_in_coord
print("Number of round/flat cells in this round: {}".format(
cells_counted_in_round))
# Save to excel
cell_Data.to_excel(
os.path.join(
os.path.join(
folder, round_num + '_' +
datetime.now().strftime('%Y-%m-%d_%H-%M-%S') +
'_CellsProperties.xlsx')))
return cell_Data
def FluorescenceAnalysis(self, folder, round_num, save_mask=True):
"""
# =============================================================================
# Given the folder and round number, return a dictionary for the round
# that contains each scanning position as key and structured array of detailed
# information about each identified cell as content.
#
# Returned structured array fields:
# - BoundingBox of cell ROI
# - Mean intensity of whole cell area
# - Mean intensity of cell membrane part
# - Contour soma ratio
# =============================================================================
Parameters
----------
folder : string.
The directory to folder where the screening data is stored.
round_num : string.
The target round number of analysis.
save_mask: bool.
Whether to save segmentation masks.
Returns
-------
cell_Data : pd.DataFrame.
Sum of return from func: retrieveDataFromML, for whole round.
"""
RoundNumberList, CoordinatesList, fileNameList = self.retrive_scanning_scheme(
folder, file_keyword="Zmax")
# RoundNumberList, CoordinatesList, fileNameList = self.retrive_scanning_scheme(folder, file_keyword = 'Zfocus')
if not os.path.exists(
os.path.join(folder, "MLimages_{}".format(round_num))):
# If the folder is not there, create the folder to store ML segmentations
os.mkdir(os.path.join(folder, "MLimages_{}".format(round_num)))
for EachRound in RoundNumberList:
cells_counted_in_round = 0
background_substraction = False
# =============================================================================
# For background_substraction
# =============================================================================
# If background images are taken
background_images_folder = os.path.join(
folder, "background {}".format(EachRound))
# print(background_images_folder)
if os.path.exists(background_images_folder):
# If the background image is taken to substract out
background_substraction = True
print("Run background substraction.")
# Get all the background files names
background_fileNameList = []
for file in os.listdir(background_images_folder):
if "calculated background" not in file:
if "tif" in file or "TIF" in file:
background_fileNameList.append(
os.path.join(background_images_folder, file))
background_image = ProcessImage.image_stack_calculation(
background_fileNameList, operation="mean")
# # Smooth the background image
# background_image = ProcessImage.average_filtering(
# background_image, filter_side_length = 25)
# Save the individual file.
with skimtiff.TiffWriter(
os.path.join(background_images_folder,
"calculated background.tif"),
imagej=True,
) as tif:
tif.save(background_image.astype(np.uint16), compress=0)
if EachRound == round_num:
# Start numbering cells at each round
self.cell_counted_inRound = 0
for EachCoord in CoordinatesList:
# =============================================================================
# For fluorescence:
# =============================================================================
print(EachCoord)
# -------------- readin image---------------
for Eachfilename in enumerate(fileNameList):
if (EachCoord in Eachfilename[1]
and EachRound in Eachfilename[1]):
if "Zmax" in Eachfilename[1]:
try:
ImgNameInfor = Eachfilename[1][
0:Eachfilename[1].index(
"_PMT"
)] # get rid of '_PMT_0Zmax.tif' in the name.
except:
ImgNameInfor = Eachfilename[1][
0:Eachfilename[1].index(
"_Cam"
)] # get rid of '_Cam_Zmax.tif' in the name.
elif "Zfocus" in Eachfilename[1]:
ImgNameInfor = Eachfilename[1][
0:len(Eachfilename[1]) -
16] # get rid of '_PMT_0Zfocus.tif' in the name.
elif "Zpos1" in Eachfilename[1]:
ImgNameInfor = Eachfilename[1][0:len(
Eachfilename[1]
)] # get rid of '_PMT_0Zfocus.tif' in the name.
_imagefilename = os.path.join(
folder, Eachfilename[1])
# ------------------------------------------
# =========================================================================
# USING MASKRCNN...
# =========================================================================
# Imagepath = self.Detector._fixPathName(_imagefilename)
Rawimage = imread(_imagefilename)
# Background substraction
if background_substraction == True:
Rawimage = np.abs(Rawimage - background_image)
camera_dark_level = 100
# # Normalize to the illumination intensity
# Rawimage = np.uint16(Rawimage \
# / ((background_image - camera_dark_level)\
# /(np.amin(background_image) - camera_dark_level)))
# if ClearImgBef == True:
# # Clear out junk parts to make it esaier for ML detection.
# RawimageCleared = self.preProcessMLimg(Rawimage, smallest_size=300, lowest_region_intensity=0.16)
# else:
# RawimageCleared = Rawimage.copy()
image = ProcessImage.convert_for_MaskRCNN(Rawimage)
# Run the detection on input image.
results = self.Detector.detect([image])
MLresults = results[0]
if save_mask == True:
fig, ax = plt.subplots()
# Set class_names = [None,None,None,None] to mute class name display.
visualize.display_instances(
image,
MLresults["rois"],
MLresults["masks"],
MLresults["class_ids"],
class_names=[None, None, None, None],
ax=ax,
centre_coors=MLresults["Centre_coor"],
Centre_coor_radius=2,
WhiteSpace=(0, 0),
) # MLresults['class_ids'],MLresults['scores'],
# ax.imshow(fig)
fig.tight_layout()
# Save the detection image
fig_name = os.path.join(
folder, "MLimages_{}\{}.tif".format(
round_num, ImgNameInfor))
plt.savefig(fname=fig_name,
dpi=200,
pad_inches=0.0,
bbox_inches="tight")
# segmentationImg = Image.fromarray(fig) #generate an image object
# segmentationImg.save(os.path.join(folder, 'MLimages_{}\{}.tif'.format(round_num, ImgNameInfor)))#save as tif
# Use retrieveDataFromML from ImageProcessing.py to extract numbers.
if self.cell_counted_inRound == 0:
(
cell_Data,
self.cell_counted_inRound,
total_cells_counted_in_coord,
) = ProcessImage.retrieveDataFromML(
Rawimage,
MLresults,
str(ImgNameInfor),
self.cell_counted_inRound,
show_each_cell=False)
else:
(
Cell_Data_new,
self.cell_counted_inRound,
total_cells_counted_in_coord,
) = ProcessImage.retrieveDataFromML(
Rawimage,
MLresults,
str(ImgNameInfor),
self.cell_counted_inRound,
show_each_cell=False)
if len(Cell_Data_new) > 0:
cell_Data = cell_Data.append(Cell_Data_new)
# Count in total how many flat and round cells are identified.
cells_counted_in_round += total_cells_counted_in_coord
print("Number of round/flat cells in this round: {}".format(
cells_counted_in_round))
# Save to excel
cell_Data.to_excel(
os.path.join(
os.path.join(
folder,
round_num + "_" +
datetime.now().strftime("%Y-%m-%d_%H-%M-%S") +
"_CellsProperties.xlsx",
)))
return cell_Data