From 5ab7a51f86c2c75e6cedbecc8101598847014c3b Mon Sep 17 00:00:00 2001
From: Monica J Emerson <monj@dtu.dk>
Date: Wed, 29 Sep 2021 15:47:42 +0200
Subject: [PATCH] added code for sharing
---
code/image_preprocessing.py | 156 ++++
code/local_features.py | 117 +++
...feature_patch_allPatients_singleProtein.py | 645 ++++++++++++++++
..._feature_patch_allPatients_threeProtein.py | 689 ++++++++++++++++++
...ng_feature_patch_allPatients_twoProtein.py | 551 ++++++++++++++
code/lung_lif_to_png.py | 51 ++
code/microscopy_analysis.py | 624 ++++++++++++++++
7 files changed, 2833 insertions(+)
create mode 100755 code/image_preprocessing.py
create mode 100755 code/local_features.py
create mode 100755 code/lung_feature_patch_allPatients_singleProtein.py
create mode 100755 code/lung_feature_patch_allPatients_threeProtein.py
create mode 100755 code/lung_feature_patch_allPatients_twoProtein.py
create mode 100644 code/lung_lif_to_png.py
create mode 100755 code/microscopy_analysis.py
diff --git a/code/image_preprocessing.py b/code/image_preprocessing.py
new file mode 100755
index 0000000..7c50445
--- /dev/null
+++ b/code/image_preprocessing.py
@@ -0,0 +1,156 @@
+#%% #!/usr/bin/env python3
+# -*- coding: utf-8 -*-
+"""
+Preprocessing of the images
+
+1. Compute maximum projections.
+2. Explore the intensities of the maximum projection images.
+3. Correct the intensities. Normalisation across channels and images,
+to equalise the standard deviation for intensity values > th (just considering
+the pixels that contain protein, and not those represinting air, so as to avoid
+enhancing the background noise)
+
+Created on Wed Oct 7 10:34:52 2020
+
+@author: monj
+"""
+import os
+import microscopy_analysis as ma
+import matplotlib.pyplot as plt
+from datetime import datetime
+import numpy as np
+import skimage.io as io
+import copy
+
+# Turn interactive plotting off
+plt.ioff()
+
+plt.close('all')
+
+start_time_global = datetime.now()
+
+#%% I/O directories
+
+diseases = ['emphysema','uip','sarcoidosis','covid'] #I don't include sarcoidosis and uip, I manually create the group later, just so those don't count twice for the std estimation
+conditions = diseases[:]
+conditions.insert(0,'control')
+
+# Input directories - images are stored in folders starting with the name 'frame'
+base_dir = '../input_data/'
+dir_in = base_dir + '/raw/'
+base_name = 'frame'
+
+# Output directories: max projections and postprocessed
+dir_maxProj_raw = base_dir + 'maximum_projections/'
+ma.make_output_dirs(dir_maxProj_raw,conditions)
+
+dir_postprocessed = base_dir + 'postprocessed_maxProjs/'
+
+dir_maxProj_processed = dir_postprocessed + 'rgb/'
+ma.make_output_dirs(dir_maxProj_processed,conditions)
+
+dir_maxProj_processed_cmy = dir_postprocessed + 'cmy/'
+ma.make_output_dirs(dir_maxProj_processed_cmy,conditions)
+
+dir_results_intinspect = dir_postprocessed + 'intensity_inspection/'
+ma.make_output_dirs(dir_results_intinspect,conditions)
+
+#%% Get maximum projection images
+
+# Automatically decide whether to recompute or read the max. projections images
+read_flag = True
+for disease in diseases:
+ disease_flag = len(os.listdir(dir_maxProj_raw + disease + '/'))>2
+ #print(disease)
+ #print(disease_flag)
+ read_flag = disease_flag&read_flag
+
+
+# Get maximum projection images
+if not(read_flag):
+ # Compute maximum projection images (runtime ~= 135 s)
+ print('Computing maximum projection images')
+ startTime = datetime.now()
+ max_img_list = []
+ for condition in conditions:
+ max_img_list += [ma.comp_max_imgs(dir_in + condition, base_name, dir_maxProj_raw + condition)]
+ endTime = datetime.now() - startTime
+ print(endTime)
+else:
+ # Read maximum projection images (runtime ~= 20 s )
+ print('Reading maximum projection images')
+ startTime = datetime.now()
+ max_img_list = []
+ for condition in conditions:
+ max_img_list += [ma.read_max_imgs(dir_maxProj_raw + condition, base_name)]
+ endTime = datetime.now() - startTime
+ print(endTime)
+
+#%% Plot histograms of raw images (approx. 29s per condition)
+for condition, max_img_list_condition in zip(conditions, max_img_list):
+ ma.plotHist_perChannel_imgset_list(max_img_list_condition, dir_in + condition, dir_results_intinspect + condition, name_tag = 'original')
+
+#%% Measure the spread of intensities in each channel
+"""The spread of intensities is quantified by the standard deviation of the
+intensities that are over a threshold. The threshold value is set manually
+and it defines the minimum intensity associated with the presence of protein.
+Image regions with intensity values under the threshold are considered to
+image air, i.e. absence of protein."""
+
+# Protein presence/absence (air) intensity threshold
+th_int = 15 #could it be set automatically based on the data?
+
+# Compute stds per image channel and, for each sample, plot intensity histograms per channel.
+std_list = []
+for condition, max_img_list_condition in zip(conditions, max_img_list):
+ std_list += [ma.comp_std_perChannel(max_img_list_condition, th_int, dir_in + condition)]
+
+# Histogram of standard deviations (std) per image channel
+fig, axs = plt.subplots(len(conditions),4, figsize = (4*2,len(conditions)*2), sharex = True, sharey = True)
+plt.suptitle('Distribution of intensity spread across images\n Rows: ' + ', '.join(conditions))
+for ind_c,std_list_condition in enumerate(std_list):
+ for ind,channel_list in enumerate(std_list_condition):
+ axs[0][ind+1].title.set_text('Channel ' + str(ind+1))
+ axs[ind_c][ind+1].hist(channel_list, bins = 50, density = True)
+
+# Histogram of standard deviations per image
+axs[0][0].title.set_text('All channels')
+std_list_flat = []
+for ind_c,std_list_condition in enumerate(std_list):
+ std_list_condition_flat = ma.flatten_list(std_list_condition)
+ axs[ind_c][0].hist(std_list_condition_flat, bins = 50, density = True)
+ std_list_flat += std_list_condition_flat
+plt.show()
+
+# Average intesity-spread (std) across all image channels
+mean_std = sum(std_list_flat)/len(std_list_flat)
+print('The average spread of an image channel is ' + str(round(mean_std)))
+
+#%% Correct intensities (34s per condition)
+max_img_corr_list = []
+for condition, max_img_list_condition in zip(conditions, max_img_list):
+ max_img_corr_list += [ma.intensity_spread_normalisation(copy.deepcopy(max_img_list_condition), th_int, mean_std, dir_in + condition, base_name, dir_maxProj_processed + condition)]
+
+#%% Plot histograms of corrected images (29s per condition)
+for condition, max_img_corr_list_condition in zip(conditions, max_img_corr_list):
+ ma.plotHist_perChannel_imgset_list(max_img_corr_list_condition, dir_in + condition, dir_results_intinspect + condition, name_tag = 'corrected')
+
+#%% Visualise raw vs. corrected images
+for max_img_list_condition, max_img_corr_list_condition, condition in zip(max_img_list, max_img_corr_list, conditions):
+ ma.compare_imgpairs_list(max_img_list_condition, max_img_corr_list_condition , dir_in + condition, dir_results_intinspect + condition)
+
+ #%% Exchange channels: fibrillar collagen with elastin
+for max_img_list_condition in max_img_corr_list:
+ print(len(max_img_list_condition))
+ for max_img_list_sample in max_img_list_condition:
+ print(len(max_img_list_sample))
+ for nr_frame,max_img in enumerate(max_img_list_sample):
+ max_img_list_sample[nr_frame]= max_img[:,:,[0,2,1]]
+
+#%% Save images in cmy colourspace
+for max_img_corr_list_condition, condition in zip(max_img_corr_list, conditions):
+ ma.rgb2cmy_list(max_img_corr_list_condition, dir_in + condition, base_name, dir_maxProj_processed_cmy + condition)
+
+#%%% Print total computational time
+end_time_global = datetime.now() - startTime
+print(end_time_global)
\ No newline at end of file
diff --git a/code/local_features.py b/code/local_features.py
new file mode 100755
index 0000000..11b5b3b
--- /dev/null
+++ b/code/local_features.py
@@ -0,0 +1,117 @@
+"""
+Helping functions for extracting features from images
+"""
+
+import skimage.io
+import numpy as np
+import matplotlib.pyplot as plt
+import scipy.ndimage
+
+def get_gauss_feat_im(im, sigma=1, normalize=False):
+ """Gauss derivative feaures for every image pixel.
+ Arguments:
+ image: a 2D image, shape (r,c).
+ sigma: standard deviation for Gaussian derivatives.
+ normalize: flag indicating normalization of features.
+ Returns:
+ imfeat: a 3D array of size (r,c,15) with a 15-dimentional feature
+ vector for every image pixel.
+ Author: vand@dtu.dk, 2020
+ """
+
+ r,c = im.shape
+ imfeat = np.zeros((r,c,15))
+ imfeat[:,:,0] = scipy.ndimage.gaussian_filter(im,sigma,order=0)
+ imfeat[:,:,1] = scipy.ndimage.gaussian_filter(im,sigma,order=[0,1])
+ imfeat[:,:,2] = scipy.ndimage.gaussian_filter(im,sigma,order=[1,0])
+ imfeat[:,:,3] = scipy.ndimage.gaussian_filter(im,sigma,order=[0,2])
+ imfeat[:,:,4] = scipy.ndimage.gaussian_filter(im,sigma,order=[1,1])
+ imfeat[:,:,5] = scipy.ndimage.gaussian_filter(im,sigma,order=[2,0])
+ imfeat[:,:,6] = scipy.ndimage.gaussian_filter(im,sigma,order=[0,3])
+ imfeat[:,:,7] = scipy.ndimage.gaussian_filter(im,sigma,order=[1,2])
+ imfeat[:,:,8] = scipy.ndimage.gaussian_filter(im,sigma,order=[2,1])
+ imfeat[:,:,9] = scipy.ndimage.gaussian_filter(im,sigma,order=[3,0])
+ imfeat[:,:,10] = scipy.ndimage.gaussian_filter(im,sigma,order=[0,4])
+ imfeat[:,:,11] = scipy.ndimage.gaussian_filter(im,sigma,order=[1,3])
+ imfeat[:,:,12] = scipy.ndimage.gaussian_filter(im,sigma,order=[2,2])
+ imfeat[:,:,13] = scipy.ndimage.gaussian_filter(im,sigma,order=[3,1])
+ imfeat[:,:,14] = scipy.ndimage.gaussian_filter(im,sigma,order=[4,0])
+
+ if normalize:
+ imfeat -= np.mean(imfeat, axis=(0,1))
+ imfeat *= (1/np.std(imfeat, axis=(0,1)))
+
+ return imfeat
+
+def im2col(im, patch_size=[3,3], stepsize=1):
+ """Rearrange image patches into columns
+ Arguments:
+ image: a 2D image, shape (r,c).
+ patch size: size of extracted paches.
+ stepsize: patch step size.
+ Returns:
+ patches: a 2D array which in every column has a patch associated
+ with one image pixel. For stepsize 1, number of returned column
+ is (r-patch_size[0]+1)*(c-patch_size[0]+1) due to bounary. The
+ length of columns is pathc_size[0]*patch_size[1].
+ """
+
+ r,c = im.shape
+ s0, s1 = im.strides
+ nrows =r-patch_size[0]+1
+ ncols = c-patch_size[1]+1
+ shp = patch_size[0],patch_size[1],nrows,ncols
+ strd = s0,s1,s0,s1
+
+ out_view = np.lib.stride_tricks.as_strided(im, shape=shp, strides=strd)
+ return out_view.reshape(patch_size[0]*patch_size[1],-1)[:,::stepsize]
+
+
+def ndim2col(im, block_size=[3,3], stepsize=1):
+ """Rearrange image blocks into columns for N-D image (e.g. RGB image)"""""
+ if(im.ndim == 2):
+ return im2col(im, block_size, stepsize)
+ else:
+ r,c,l = im.shape
+ patches = np.zeros((l*block_size[0]*block_size[1],
+ (r-block_size[0]+1)*(c-block_size[1]+1)))
+ for i in range(l):
+ patches[i*block_size[0]*block_size[1]:(i+1)*block_size[0]*block_size[1],
+ :] = im2col(im[:,:,i],block_size,stepsize)
+ return patches
+
+#%%
+
+if __name__ == '__main__':
+
+ filename = '../example_data/training_image.png'
+ I = skimage.io.imread(filename)
+ I = I.astype(np.float)
+
+
+ # features based on gaussian derivatives
+ sigma = 1;
+ gf = get_gauss_feat_im(I, sigma)
+
+ fig,ax = plt.subplots(3,5)
+ for j in range(5):
+ for i in range(3):
+ ax[i][j].imshow(gf[:,:,5*i+j])
+ ax[i][j].set_title(f'layer {5*i+j}')
+
+
+ # features based on image patches
+ I = I[I.shape[0]//2:I.shape[0]//2+20,I.shape[1]//2:I.shape[1]//2+20] # smaller image such that we can see the difference in layers
+ pf = im2col(I,[3,3])
+ pf = pf.reshape((9,I.shape[0]-2,I.shape[1]-2))
+
+ fig, ax = plt.subplots()
+ ax.imshow(I)
+
+ fig,ax = plt.subplots(3,3)
+ for j in range(3):
+ for i in range(3):
+ ax[i][j].imshow(pf[3*i+j])
+ ax[i][j].set_title(f'layer {3*i+j}')
+
+
diff --git a/code/lung_feature_patch_allPatients_singleProtein.py b/code/lung_feature_patch_allPatients_singleProtein.py
new file mode 100755
index 0000000..a43234d
--- /dev/null
+++ b/code/lung_feature_patch_allPatients_singleProtein.py
@@ -0,0 +1,645 @@
+#!/usr/bin/env python3
+# -*- coding: utf-8 -*-
+"""Explorative analysis of protein composition and structure in lung ECM to
+study disease-induced remodeling of the three proteins that provide structural
+integrity to the lung: collagen IV, elastin and fibrillar collagen.
+
+The single-protein analyses provide the following:
+ - A 'Data Model' per protein, a dictionary of image patches representative
+ of the local protein structure found in the data (both
+ healthy and disease group) at the scale defined by the patch size.
+ - A 'Sorted Data Model' or disease signature per protein, a sorted version
+ of the dictionary above, based on how often a dictionary element (called
+ cluster centre in the publication) is found in the healthy vs. the disease.
+ - A 'Probability Map' for every image based on each protein, with pixel
+ correspondance to the input images (maximum projections), highlighting
+ regions characteristic of health or disease.
+ - An 'Agreement Map' for every image, which combines the corresponding
+ probability maps from the three proteins to visualise when structural
+ remodelling of proteins coexists in space.
+ - A 'Disease Effect' per protein, to quantify how well the control and
+ disease groups are separated after assigning a health probability score to
+ each image, computed as the average of its probability map values.
+
+by Monica J. Emerson monj@dtu.dk and Anders B. Dahl abda@dtu.dk
+
+For more information, read our article (add link and citation)"""
+
+#%% Load packages and initialise parameters
+
+import numpy as np
+import matplotlib.pyplot as plt
+import sklearn.cluster
+import microscopy_analysis as ma
+import skimage.io
+import skimage.transform
+
+import os
+from datetime import datetime
+import sys
+from matplotlib.offsetbox import OffsetImage, AnnotationBbox
+from math import ceil
+
+startTime = datetime.now()
+
+plt.close('all')
+
+sc_fac = 0.5 #25 #25 #0.5 # scaling factor of the image
+patch_size = 17
+nr_clusters = 100 # number of clusters
+protein_mode = 'single'
+colour_mode = 'cmy' #for image visualisation
+th_back = 15
+nr_runs = 10
+
+#%% Define and create directories
+
+disease = 'emphysema' #diseased (mix, 2 of each condition)' #''emphysema' 'sarcoidosis' 'ild' 'covid'
+control = 'control'
+conditions = [disease, control]
+
+# input directories - images start with the name 'frame'
+base_dir_in = '../input_data/'
+#dir_in = base_dir_in + 'maximum_projections' #uncomment to read maximum projections without contrast equalisation
+dir_in = base_dir_in + 'postprocessed_maxProjs/rgb/' #Read rgb for correct protein channel ordering, visualisation will be in cmy
+base_name = 'frame'
+
+#output directory
+dir_results = '../results/' #rerun just to make sure not to muck up the results
+os.makedirs(dir_results, exist_ok = True)
+os.makedirs(dir_results + '/statistics', exist_ok = True)
+
+ #%% Read (preprocessed) maximum corrected images
+#Read maximum projection images (runtime ~= 20 s )
+print('Reading maximum projection images')
+
+max_img_list_control = ma.read_max_imgs(dir_in + control, base_name)
+max_img_list_disease = ma.read_max_imgs(dir_in + disease, base_name)
+
+#%% Exchange channels: fibrillar collagen with elastin
+for max_img_sample_control in max_img_list_control:
+ for nr_frame, max_img_control in enumerate(max_img_sample_control):
+ max_img_sample_control[nr_frame] = max_img_control[:,:,[0,2,1]]
+
+for max_img_sample_disease in max_img_list_disease:
+ for nr_frame, max_img_disease in enumerate(max_img_sample_disease):
+ max_img_sample_disease[nr_frame] = max_img_disease[:,:,[0,2,1]]
+
+#%% Prepare maximum projection images for feature extraction
+
+#Flatten and merge conditions
+max_img_list_control_flat = ma.flatten_list(max_img_list_control)
+max_img_list_disease_flat = ma.flatten_list(max_img_list_disease)
+
+#Rescale
+max_img_list_control_processed = []
+for max_img in ma.flatten_list(max_img_list_control):
+ max_img_list_control_processed += [skimage.transform.rescale(max_img, sc_fac, preserve_range = True, multichannel=True).astype(max_img.dtype)]
+
+max_img_list_disease_processed = []
+for max_img in ma.flatten_list(max_img_list_disease) :
+ max_img_list_disease_processed += [skimage.transform.rescale(max_img, sc_fac, preserve_range = True, multichannel=True).astype('uint8')]
+
+#%% Compute patches
+patch_feat_list_control = [[],[],[]]
+for max_img in max_img_list_control_processed:
+ for channel in range(max_img.shape[2]):
+ patch_feat_list_control[channel] += [ma.ndim2col_pad(max_img[...,channel], (patch_size, patch_size),norm=False).transpose()]
+
+patch_feat_list_disease = [[],[],[]]
+for max_img in max_img_list_disease_processed:
+ for channel in range(max_img.shape[2]):
+ patch_feat_list_disease[channel] += [ma.ndim2col_pad(max_img[...,channel], (patch_size, patch_size),norm=False).transpose()]
+
+for run in range(nr_runs):
+ #directories for results from the different runs
+ dir_probs = dir_results + disease + '_' + protein_mode + '_%dclusters_%ddownscale_%dpatchsize/'%(nr_clusters,1/sc_fac,patch_size)
+ dir_probs += 'run'+str(run)+'/'
+ ma.make_output_dirs(dir_probs, conditions)
+
+ #%% Run the whole analysis for the 3 channels independently
+ prob_img_list_control_allchannels = [[],[],[]]
+ prob_img_list_disease_allchannels = [[],[],[]]
+
+ for channel in range(len(patch_feat_list_control)):
+
+ #k-means clustering
+ startTime_clustering = datetime.now()
+
+ batch_size = 10000
+ th_nr_pathesINcluster = 20
+ th_nr_weakClusters = 0 #added for covid and ild
+
+ #If cluster centers were already computed, use those
+ if os.path.exists(dir_probs + 'array_cluster_centers_channel'+str(channel)+'.npy'):
+ cluster_centers = np.load(dir_probs + 'array_cluster_centers_channel'+str(channel)+'.npy')
+ kmeans = sklearn.cluster.MiniBatchKMeans(n_clusters=nr_clusters, batch_size = batch_size)
+ kmeans.cluster_centers_ = cluster_centers
+
+ #Compute cluster centers
+ else:
+ #Get features to cluster
+ patch_feat_total = patch_feat_list_control[channel] + patch_feat_list_disease[channel]
+
+ #features for clustering
+ nr_keep = 10000 # number of features/image randomly picked for clustering
+ n_im = len(patch_feat_total)
+ feat_dim = patch_feat_total[0].shape[1]
+ patch_feat_to_cluster = np.zeros((nr_keep*n_im,feat_dim))
+
+ f = 0
+ nr_keep_control = nr_keep//(2*len(patch_feat_list_control[channel])//len(patch_feat_list_disease[channel]))
+ nr_keep_disease = nr_keep//2
+ for patch_feat in patch_feat_list_control[channel]:
+ keep_indices = np.random.permutation(np.arange(patch_feat.shape[0]))[:nr_keep_control]
+ patch_feat_to_cluster[f:f+nr_keep_control,:] = patch_feat[keep_indices,:]
+ f += nr_keep
+
+ for patch_feat in patch_feat_list_disease[channel]:
+ keep_indices = np.random.permutation(np.arange(patch_feat.shape[0]))[:nr_keep_disease]
+ patch_feat_to_cluster[f:f+nr_keep_disease,:] = patch_feat[keep_indices,:]
+ f += nr_keep
+
+ #Get feature vectors
+ feat_to_cluster = patch_feat_to_cluster
+
+ #Compute cluster centers and save if there is no weak clusters
+ nr_weakClusters = th_nr_weakClusters+1
+ repetition = 0
+ nr_weakClusters_list = []
+ while nr_weakClusters>th_nr_weakClusters: #While weak clusters is different from empty recompute
+ kmeans = sklearn.cluster.MiniBatchKMeans(n_clusters=nr_clusters, batch_size = batch_size, verbose = False, reassignment_ratio=0.0005, max_no_improvement = 100) #0.00075 was good for uip
+ kmeans.fit(feat_to_cluster)
+
+ #Nr. patches that have contributed to each cluster
+ nr_feat_in_cluster = [sum(kmeans.labels_==cluster) for cluster in range(nr_clusters)]
+
+ weak_cluster_IDs = [ind for ind,val in enumerate(nr_feat_in_cluster) if val<th_nr_pathesINcluster]
+ print('Weak cluster IDs channel '+ str(channel) + ': ',weak_cluster_IDs)
+
+ #Weak clusters are those composed by very few patches
+ nr_weakClusters = len([1 for i in nr_feat_in_cluster if i<th_nr_pathesINcluster])
+
+ #If there are weak clusters, the clustering should be recomputed
+ if nr_weakClusters>th_nr_weakClusters:
+ print(str(nr_weakClusters)+ " clusters composed of less than "+str(th_nr_pathesINcluster)+" images")
+ else:
+ np.save(dir_probs + 'array_cluster_centers_channel'+str(channel)+'.npy', kmeans.cluster_centers_) # .npy extension is added if not given
+
+ print(repetition)
+ repetition += 1
+ nr_weakClusters_list += [nr_weakClusters]
+
+
+ endTime_clustering = datetime.now() - startTime_clustering
+ print(endTime_clustering)
+
+ #%% Plot all cluster centers (grid view)
+ cluster_centers = kmeans.cluster_centers_
+ int_sum_list= ma.plot_grid_cluster_centers(cluster_centers, range(nr_clusters), patch_size, protein_mode = protein_mode, colour_mode = colour_mode, channel = channel)
+ plt.savefig(dir_probs + 'clustercenters_channel'+str(channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Histograms for background, healthy and disease
+ hist_control, assignment_list_control= ma.compute_assignment_hist(patch_feat_list_control[channel], kmeans)
+ hist_disease, assignment_list_disease = ma.compute_assignment_hist(patch_feat_list_disease[channel], kmeans)
+
+ #Show histograms (bar plot) of healthy and disease
+ fig, ax = plt.subplots(1,1)
+ ax.bar(np.array(range(0,nr_clusters))-0.25, hist_control, width = 0.5, label='Control', color = 'b')
+ ax.bar(np.array(range(0,nr_clusters))+0.25, hist_disease, width = 0.5, label='Disease', color = 'r')
+ ax.legend()
+ plt.savefig(dir_probs + 'assignmentHistograms_channel'+str(channel)+'_%dclusters_%ddownscale_%dpatchsize.pdf'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Compute populated, occurrence and importance cluster measures from the histogram
+ #grid dimensions
+
+ #OCCURRENCE RATIO: How more often does it occur in one of the conditions
+ occurrence_ratio = np.empty((nr_clusters))
+ ind_control = hist_control>hist_disease
+ ind_disease = ~ind_control
+ control_cond = (hist_disease!=0) & ind_control
+ occurrence_ratio[control_cond] = hist_control[control_cond]/hist_disease[control_cond]
+ disease_cond = (hist_control!=0) & ind_disease
+ occurrence_ratio[disease_cond] = -hist_disease[disease_cond]/hist_control[disease_cond]
+
+ #POPULATED: How trustworthy is the cluster?
+ populated = hist_control+hist_disease
+ weights = populated/populated.max()
+
+ #Cluster IMPORTANCE: occurrence weighed by POPULATED
+ def sigmoid(x,k):
+ return 2 / (1 + np.exp(-k*x)) - 1
+
+ importance = occurrence_ratio * sigmoid(weights,100)
+
+ #%% Plot cluster centers in the 2d populated/occurrence space
+ fig_control, ax_control = plt.subplots(figsize=(15,15))
+ ax_control.scatter(populated[occurrence_ratio>1], occurrence_ratio[occurrence_ratio>1])
+ ax_control.set_ylim(0.8,7)
+ ax_control.set_xlim(0,0.08)
+
+ fig_disease, ax_disease = plt.subplots(figsize=(15,15))
+ ax_disease.scatter(populated[occurrence_ratio<1], -occurrence_ratio[occurrence_ratio<1])
+ ax_disease.set_ylim(0.8,7)
+ ax_disease.set_xlim(0,0.08)
+
+ for x0, y0, cluster_nr in zip(populated, occurrence_ratio, np.arange(0,nr_clusters)):
+ cluster_centre = np.reshape(kmeans.cluster_centers_[cluster_nr,:],(patch_size,patch_size))
+ if y0>0:
+ ab = AnnotationBbox(OffsetImage(cluster_centre.astype('uint8'),cmap='gray'), (x0, y0), frameon=False)
+ ax_control.add_artist(ab)
+ else:
+ ab = AnnotationBbox(OffsetImage(cluster_centre.astype('uint8'),cmap='gray'), (x0, -y0), frameon=False)
+ ax_disease.add_artist(ab)
+ plt.figure(fig_control.number)
+ plt.savefig(dir_probs + 'controlClustercenters_2Dspace_channel'+str(channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ plt.figure(fig_disease.number)
+ plt.savefig(dir_probs + 'diseaseClustercenters_2Dspace_channel'+str(channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Plot all clusters sorted by IMPORTANCE
+ clusters_control_occW_sorted = [np.arange(0,100)[i] for i in np.argsort(importance.flatten()) if (importance.flatten())[i]>0]
+ clusters_control_occW_sorted.reverse()
+
+ clusters_disease_occW_sorted = [np.arange(0,100)[i] for i in np.argsort(importance.flatten()) if (importance.flatten())[i]<0]
+
+ clusters_occW_sorted = [np.arange(0,100)[i] for i in np.argsort(importance.flatten())]
+ clusters_occW_sorted.reverse()
+
+ occurrence_ratio_sorted = [occurrence_ratio[i] for i in np.argsort(importance.flatten())]
+ occurrence_ratio_sorted.reverse()
+
+ ma.plot_grid_cluster_centers(kmeans.cluster_centers_, clusters_occW_sorted, patch_size, protein_mode = protein_mode, colour_mode = colour_mode, channel = channel) #contrast = 'equal',
+ plt.suptitle('Cluster centers channel '+str(channel)+' \n sorted by occ*f(p) from control to disease with cluster ' + str(len(clusters_control_occW_sorted)) + ' in between')
+ plt.savefig(dir_probs + 'clustercenters_occW_sorted_channel'+str(channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+
+ #%% Compute probabilities
+ #per cluster
+ prob_control= hist_control/(hist_control+hist_disease)
+ prob_disease = hist_disease/(hist_control+hist_disease)
+
+ #per image region
+ r,c,_ = max_img_list_control_flat[0].shape
+ img_shape = (int(r*sc_fac),int(c*sc_fac))
+ prob_img_list_control = ma.comp_prob_imgs(prob_control,assignment_list_control,img_shape)
+ prob_img_list_disease = ma.comp_prob_imgs(prob_control,assignment_list_disease,img_shape)
+ prob_img_list_control_allchannels[channel] = prob_img_list_control
+ prob_img_list_disease_allchannels[channel] = prob_img_list_disease
+
+ #per image and sample
+ #control
+ sample_names_control = ma.listdir_custom(dir_in + control, string_start_pos = -4, dir_flag = True, )
+
+ nr_list = 0
+ prob_list_control= []
+ for max_img_list_sample,sample_name in zip(max_img_list_control, sample_names_control): #in general, in max_img_list_condition
+ #print(sample_name)
+ nr_frames = len(max_img_list_sample)
+ prob_img_list_sample = prob_img_list_control[nr_list:nr_list+nr_frames]
+ nr_list += nr_frames
+
+ prob_sample = np.zeros((nr_frames,1))
+ for prob_img, frame_nr in zip(prob_img_list_sample,range(nr_frames)):
+ #compute accumulated probabilities
+ prob_frame = sum(sum(prob_img))/prob_img.size
+ #print(prob_frame)
+ prob_sample[frame_nr] = prob_frame
+ prob_list_control += [prob_sample]
+
+ #disease
+ sample_names_disease = ma.listdir_custom(dir_in + disease, string_start_pos = -4, dir_flag = True)
+
+ nr_list = 0
+ prob_list_disease = []
+ for max_img_list_sample,sample_name in zip(max_img_list_disease, sample_names_disease): #in general, in max_img_list_condition
+ #print(sample_name)
+ nr_frames = len(max_img_list_sample)
+ prob_img_list_sample = prob_img_list_disease[nr_list:nr_list+nr_frames]
+ nr_list += nr_frames
+
+ prob_sample = np.zeros((nr_frames,1))
+ for prob_img, frame_nr in zip(prob_img_list_sample,range(nr_frames)):
+ #compute accumulated probabilities
+ prob_frame = sum(sum(prob_img))/prob_img.size
+ #print(prob_frame)
+ prob_sample[frame_nr] = prob_frame
+ prob_list_disease += [prob_sample]
+
+ prob_list_samples = prob_list_control + prob_list_disease
+
+
+ #%% Box plots
+ fig, ax = plt.subplots(figsize = (15,5))
+ ax.set_title('Image health scores')
+
+ sample_sep = 0.85
+ patient_sep = 2.35
+ condition_sep = 2.5
+ positions_control = np.tile([0,0+sample_sep],int(len(max_img_list_control)/2)) + np.repeat(np.arange(0,len(max_img_list_control)/2*patient_sep,patient_sep),2)
+ positions_disease = np.tile([len(max_img_list_control)+condition_sep,len(max_img_list_control)+condition_sep+sample_sep],int(len(max_img_list_disease)/2)) + np.repeat(np.arange(0,len(max_img_list_disease)/2*patient_sep,patient_sep),2)
+ array_probs = np.squeeze(np.asarray(prob_list_samples))
+ bp_control = ax.boxplot((array_probs[:len(max_img_list_control),...]).T, positions = positions_control, patch_artist = True)
+ bp_disease = ax.boxplot(array_probs[len(max_img_list_control):,...].T, positions = positions_disease, patch_artist = True)
+
+ for element in ['boxes', 'whiskers', 'fliers', 'means', 'medians', 'caps']:
+ plt.setp(bp_control[element], color='blue')
+ plt.setp(bp_control[element], linewidth = 2)
+
+ for patch in bp_control['boxes']:
+ patch.set(facecolor='white')
+ patch.set(linewidth = 2)
+
+ for element in ['boxes','whiskers', 'fliers', 'means', 'medians', 'caps']:
+ plt.setp(bp_disease[element], color='red')
+ plt.setp(bp_disease[element], linewidth = 2)
+
+ for patch in bp_disease['boxes']:
+ patch.set(facecolor= 'white')
+ patch.set(linewidth = 2)
+ #patch.set(alpha=0.2)
+
+ ax.set_xticklabels([i[-4:] for i in sample_names_control]+[i[-4:] for i in sample_names_disease])
+ ax.set_ylim(0.3,0.7)
+ ax.set_ylabel('Probability health')
+ #ax.set_ylim(0,1)
+
+ ax.tick_params(
+ axis='both', # changes apply to the x-axis
+ which='both', # both major and minor ticks are affected
+ #labelleft = False, # ticks along the top edge are off
+ #labelbottom=False
+ )
+
+ #plot points
+ positions = list(positions_control) + list(positions_disease)
+ for prob_sample, position in zip(prob_list_samples, positions):
+ print(prob_sample)
+ print(5*[position])
+ # Add some random "jitter" to the x-axis
+ x = np.random.normal(5*[position], 0)
+ #plot(x, y, 'r.', alpha=0.2)
+ ax.plot(x, prob_sample,'k.', markersize = 6, zorder = 3)
+
+ #horizontal line
+ x = np.linspace(0,position.max())
+ y = 0.5*np.ones(x.shape)
+ ax.plot(x,y,'k',linewidth = 1, linestyle = 'dashed')
+
+ plt.savefig(dir_probs + 'scatterBoxplots_channel'+str(channel) +'_%dclusters_%ddownscale_%dpatchsize.pdf'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ fig2, ax2 = plt.subplots(figsize = (15,5))
+ #plot points
+ for prob_sample, position in zip(prob_list_control, positions_control):
+ ax2.plot(5*[position], prob_sample,'b.', markersize = 4)
+
+ for prob_sample, position in zip(prob_list_disease, positions_disease):
+ ax2.plot(5*[position], prob_sample,'r.', markersize = 4)
+
+ #horizontal line
+ x = np.linspace(0,position.max())
+ y = 0.5*np.ones(x.shape)
+ ax2.plot(x,y,'k',linewidth = 1, linestyle = 'dashed')
+
+ ax2.set_xticks(ax.get_xticks())
+ ax2.set_xticklabels([i[-4:] for i in sample_names_control]+[i[-4:] for i in sample_names_disease])
+ ax2.set_ylim(0.3,0.7)
+ ax2.set_ylabel('Probability health')
+
+ plt.savefig(dir_probs + 'samplewiseScatterPlots_channel'+str(channel) +'_%dclusters_%ddownscale_%dpatchsize.pdf'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Create agreement maps
+ prob_img_rgb_list_control = []
+ for prob_img_c0, prob_img_c1, prob_img_c2 in zip(prob_img_list_control_allchannels[0],prob_img_list_control_allchannels[1],prob_img_list_control_allchannels[2]):
+ prob_img_rgb = np.stack((prob_img_c0,prob_img_c1, prob_img_c2), axis = 2)
+ prob_img_rgb_list_control += [prob_img_rgb]
+
+ prob_img_rgb_list_disease = []
+ for prob_img_c0, prob_img_c1, prob_img_c2 in zip(prob_img_list_disease_allchannels[0],prob_img_list_disease_allchannels[1],prob_img_list_disease_allchannels[2]):
+ prob_img_rgb = np.stack((prob_img_c0,prob_img_c1, prob_img_c2), axis = 2)
+ prob_img_rgb_list_disease += [prob_img_rgb]
+
+ #%% Plot per channel prob images, and agreement maps
+ nr_frames = len(max_img_list_control[0])
+
+ min_prob_img = []
+ max_prob_img = []
+
+ #control
+ sample_names_control = ma.listdir_custom(dir_in + control, string_start_pos = -4, dir_flag = True)
+ nr_list = 0
+ fig_amaps, ax_amaps = plt.subplots(nr_frames, len(sample_names_control),figsize = (len(sample_names_control),nr_frames), sharex=True, sharey=True)
+ fig_maximgs, ax_maximgs = plt.subplots(nr_frames, len(sample_names_control),figsize = (len(sample_names_control),nr_frames), sharex=True, sharey=True)
+ for max_img_list_sample,sample_name, sample_nr in zip(max_img_list_control, sample_names_control,range(len(sample_names_control))): #in general, in max_img_list_condition
+ print(sample_name)
+ prob_img_list_sample = prob_img_rgb_list_control[nr_list:nr_list+nr_frames]
+ nr_list += nr_frames
+
+ fig, axs = plt.subplots(5,nr_frames, figsize = (nr_frames*2,5*2), sharex=True, sharey=True)
+ for max_img,prob_img, frame_nr in zip(max_img_list_sample,prob_img_list_sample,range(nr_frames)):
+ max_img_ds = skimage.transform.rescale(max_img, sc_fac, preserve_range = True, multichannel=True).astype('uint8')
+
+ #set probs as titles
+ for channel in range(prob_img.shape[2]):
+ prob_frame = sum(sum(prob_img[...,channel]))/prob_img[...,channel].size
+ axs[channel+2][frame_nr].set_title("%.2f" % np.round(prob_frame,2))
+
+ #remove background
+ ind_back = (max_img_ds[:,:,0]<th_back)&(max_img_ds[:,:,1]<th_back)&(max_img_ds[:,:,2]<th_back)
+ prob_img[ind_back] = 0.5
+
+ #plot
+ max_img_ds = ma.rgb2cmy(max_img_ds)
+ axs[0][frame_nr].imshow(max_img_ds)
+ axs[0][frame_nr].xaxis.set_ticklabels([])
+ axs[0][frame_nr].xaxis.set_ticks_position('none')
+ axs[0][frame_nr].yaxis.set_ticklabels([])
+ axs[0][frame_nr].yaxis.set_ticks_position('none')
+
+ axs[2][frame_nr].imshow(1-prob_img[...,0],cmap = plt.cm.bwr, vmin = 0, vmax = 1)
+ axs[2][frame_nr].xaxis.set_ticklabels([])
+ axs[2][frame_nr].xaxis.set_ticks_position('none')
+ axs[2][frame_nr].yaxis.set_ticklabels([])
+ axs[2][frame_nr].yaxis.set_ticks_position('none')
+
+ axs[3][frame_nr].imshow(1-prob_img[...,1],cmap = plt.cm.bwr, vmin = 0, vmax = 1)
+ axs[3][frame_nr].xaxis.set_ticklabels([])
+ axs[3][frame_nr].xaxis.set_ticks_position('none')
+ axs[3][frame_nr].yaxis.set_ticklabels([])
+ axs[3][frame_nr].yaxis.set_ticks_position('none')
+
+ axs[4][frame_nr].imshow(1-prob_img[...,2],cmap = plt.cm.bwr, vmin = 0, vmax = 1)
+ axs[4][frame_nr].xaxis.set_ticklabels([])
+ axs[4][frame_nr].xaxis.set_ticks_position('none')
+ axs[4][frame_nr].yaxis.set_ticklabels([])
+ axs[4][frame_nr].yaxis.set_ticks_position('none')
+
+ #normalise range of agreement map so that global max and min values are 0 and 1
+ prob_img_norm = (prob_img-0.2)/0.6
+ min_prob_img += [np.min(prob_img)]
+ max_prob_img += [np.max(prob_img)]
+
+ axs[1][frame_nr].imshow(prob_img_norm)
+ axs[1][frame_nr].xaxis.set_ticklabels([])
+ axs[1][frame_nr].xaxis.set_ticks_position('none')
+ axs[1][frame_nr].yaxis.set_ticklabels([])
+ axs[1][frame_nr].yaxis.set_ticks_position('none')
+
+ ax_amaps[frame_nr][sample_nr].imshow(prob_img_norm)
+ ax_amaps[frame_nr][sample_nr].xaxis.set_ticklabels([])
+ ax_amaps[frame_nr][sample_nr].xaxis.set_ticks_position('none')
+ ax_amaps[frame_nr][sample_nr].yaxis.set_ticklabels([])
+ ax_amaps[frame_nr][sample_nr].yaxis.set_ticks_position('none')
+
+ ax_maximgs[frame_nr][sample_nr].imshow(max_img_ds)
+ ax_maximgs[frame_nr][sample_nr].xaxis.set_ticklabels([])
+ ax_maximgs[frame_nr][sample_nr].xaxis.set_ticks_position('none')
+ ax_maximgs[frame_nr][sample_nr].yaxis.set_ticklabels([])
+ ax_maximgs[frame_nr][sample_nr].yaxis.set_ticks_position('none')
+
+ plt.suptitle('Per-channel health probabilities for sample '+ sample_name +', avg:' + str(round(prob_sample.mean(),2))+ ' std:' + str(round(prob_sample.std(),2)))
+ plt.figure(fig.number),plt.savefig(dir_probs + '/' + control + '/' + 'channelWise_probImHealth_'+sample_name+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=1000)
+ #plt.show()
+ plt.figure(fig_amaps.number),plt.savefig(dir_probs + '/' + control + '/' + 'agreementMaps_control_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+ plt.figure(fig_maximgs.number),plt.savefig(dir_probs + '/' + control + '/' + 'maxIMGs_control_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+
+ #disease
+ sample_names_disease = ma.listdir_custom(dir_in + disease, string_start_pos = -4, dir_flag = True)
+
+ nr_list = 0
+ fig_amaps, ax_amaps = plt.subplots(nr_frames, len(sample_names_disease),figsize = (len(sample_names_disease),nr_frames), sharex=True, sharey=True)
+ fig_maximgs, ax_maximgs = plt.subplots(nr_frames, len(sample_names_disease),figsize = (len(sample_names_disease),nr_frames), sharex=True, sharey=True)
+ for max_img_list_sample,sample_name, sample_nr in zip(max_img_list_disease, sample_names_disease, range(len(sample_names_disease))): #in general, in max_img_list_condition
+ print(sample_name)
+ prob_img_list_sample = prob_img_rgb_list_disease[nr_list:nr_list+nr_frames]
+ nr_list += nr_frames
+
+ fig, axs = plt.subplots(5,nr_frames, figsize = (nr_frames*2,5*2), sharex=True, sharey=True)
+ for max_img, prob_img, frame_nr in zip(max_img_list_sample, prob_img_list_sample,range(nr_frames)):
+ max_img_ds = skimage.transform.rescale(max_img, sc_fac, preserve_range = True, multichannel=True).astype('uint8')
+
+ #set probs as titles
+ for channel in range(prob_img.shape[2]):
+ prob_frame = sum(sum(prob_img[...,channel]))/prob_img[...,channel].size
+ axs[channel+2][frame_nr].set_title("%.2f" % np.round(prob_frame,2))
+
+ #remove background
+ ind_back = (max_img_ds[:,:,0]<th_back)&(max_img_ds[:,:,1]<th_back)&(max_img_ds[:,:,2]<th_back)
+ prob_img[ind_back] = 0.5
+
+ #plot
+ max_img_ds = ma.rgb2cmy(max_img_ds)
+ axs[0][frame_nr].imshow(max_img_ds)
+ axs[0][frame_nr].xaxis.set_ticklabels([])
+ axs[0][frame_nr].xaxis.set_ticks_position('none')
+ axs[0][frame_nr].yaxis.set_ticklabels([])
+ axs[0][frame_nr].yaxis.set_ticks_position('none')
+
+ axs[2][frame_nr].imshow(1-prob_img[...,0],cmap = plt.cm.bwr, vmin = 0, vmax = 1)
+ axs[2][frame_nr].xaxis.set_ticklabels([])
+ axs[2][frame_nr].xaxis.set_ticks_position('none')
+ axs[2][frame_nr].yaxis.set_ticklabels([])
+ axs[2][frame_nr].yaxis.set_ticks_position('none')
+
+ axs[3][frame_nr].imshow(1-prob_img[...,1],cmap = plt.cm.bwr, vmin = 0, vmax = 1)
+ axs[3][frame_nr].xaxis.set_ticklabels([])
+ axs[3][frame_nr].xaxis.set_ticks_position('none')
+ axs[3][frame_nr].yaxis.set_ticklabels([])
+ axs[3][frame_nr].yaxis.set_ticks_position('none')
+
+ axs[4][frame_nr].imshow(1-prob_img[...,2],cmap = plt.cm.bwr, vmin = 0, vmax = 1)
+ axs[4][frame_nr].xaxis.set_ticklabels([])
+ axs[4][frame_nr].xaxis.set_ticks_position('none')
+ axs[4][frame_nr].yaxis.set_ticklabels([])
+ axs[4][frame_nr].yaxis.set_ticks_position('none')
+
+ #normalise range of agreement map so that global max and min values are 0 and 1
+ prob_img_norm = (prob_img-0.2)/0.6
+ min_prob_img += [np.min(prob_img)]
+ max_prob_img += [np.max(prob_img)]
+
+ axs[1][frame_nr].imshow(prob_img_norm)
+ axs[1][frame_nr].xaxis.set_ticklabels([])
+ axs[1][frame_nr].xaxis.set_ticks_position('none')
+ axs[1][frame_nr].yaxis.set_ticklabels([])
+ axs[1][frame_nr].yaxis.set_ticks_position('none')
+
+ ax_amaps[frame_nr][sample_nr].imshow(prob_img_norm)
+ ax_amaps[frame_nr][sample_nr].xaxis.set_ticklabels([])
+ ax_amaps[frame_nr][sample_nr].xaxis.set_ticks_position('none')
+ ax_amaps[frame_nr][sample_nr].yaxis.set_ticklabels([])
+ ax_amaps[frame_nr][sample_nr].yaxis.set_ticks_position('none')
+
+ ax_maximgs[frame_nr][sample_nr].imshow(max_img_ds)
+ ax_maximgs[frame_nr][sample_nr].xaxis.set_ticklabels([])
+ ax_maximgs[frame_nr][sample_nr].xaxis.set_ticks_position('none')
+ ax_maximgs[frame_nr][sample_nr].yaxis.set_ticklabels([])
+ ax_maximgs[frame_nr][sample_nr].yaxis.set_ticks_position('none')
+
+ plt.suptitle('Per-channel health probabilities for sample '+ sample_name +', avg:' + str(round(prob_sample.mean(),2))+ ' std:' + str(round(prob_sample.std(),2)))
+ plt.figure(fig.number), plt.savefig(dir_probs + '/' + disease + '/' + 'channelWise_probImHealth_'+sample_name+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=1000)
+ #plt.show()
+ plt.figure(fig_amaps.number),plt.savefig(dir_probs + '/' + disease + '/' + 'agreementMaps_'+disease+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+ plt.figure(fig_maximgs.number),plt.savefig(dir_probs + '/' + disease + '/' + 'maxIMGs_'+disease+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+
+ print('Agreement map minimum ',sum(min_prob_img)/len(min_prob_img))
+ print('Agreement map maximum ',sum(max_prob_img)/len(max_prob_img))
+
+ #%% Load packages to Testt the statistical significance of the mean differences
+ from statsmodels.multivariate.manova import MANOVA
+ from statsmodels.formula.api import ols
+ from statsmodels.stats.anova import anova_lm
+ import pandas as pd
+ from patsy import ModelDesc
+
+ nr_frames_patient = 10
+
+ #%%Image probabilities
+ img_probs_control = []
+ for prob_img_list in prob_img_list_control_allchannels:
+ img_probs_control += [[np.mean(prob_img) for prob_img in prob_img_list]]
+
+ img_probs_disease = []
+ for prob_img_list in prob_img_list_disease_allchannels:
+ img_probs_disease += [np.array([np.mean(prob_img) for prob_img in prob_img_list])]
+
+ img_probs = [np.concatenate((probs_control,probs_disease)) for probs_control,probs_disease in zip(img_probs_control,img_probs_disease)]
+
+ #%%ANOVA SETUP
+
+ img_nr = np.tile(np.arange(nr_frames),img_probs[0].size//nr_frames)
+ sample_nr = np.tile(np.repeat(np.arange(nr_frames_patient//nr_frames),nr_frames),img_probs[0].size//nr_frames_patient)
+ patient_nr = np.concatenate([np.repeat(np.arange(len(max_img_list_control_flat)//nr_frames_patient),nr_frames_patient),np.repeat(np.arange(len(max_img_list_disease_flat)//nr_frames_patient),nr_frames_patient)])
+ condition_nr = np.concatenate([np.zeros(len(max_img_list_control_flat)), np.ones(len(max_img_list_disease_flat))])
+
+ for channel in range(len(img_probs)):
+ dep_var = (img_probs[channel]).reshape(img_probs[0].size,1)
+
+ print(np.mean(dep_var))
+ print(np.mean(dep_var[:len(dep_var)//2]))
+ print(np.mean(dep_var[len(dep_var)//2:]))
+
+ data_dict = {'Image':img_nr,
+ 'Patient':patient_nr,
+ 'Sample':sample_nr,
+ 'Condition':condition_nr,
+ 'S':dep_var}
+
+ formula = 'S ~ C(Condition)/C(Patient)/C(Sample)'
+
+ desc = ModelDesc.from_formula(formula)
+ print(desc.describe())
+
+ lm = ols(formula, data_dict).fit()
+ print(lm.summary())
+
+ anova_table = anova_lm(lm)
+ print(anova_table)
+ print(disease + ' on channel ' + str(channel))
+ writer_anova = pd.ExcelWriter(dir_results + 'statistics/statistics_anova_' + disease + '_' + protein_mode + '_channel' + str(channel) + '_run' + str(run) + '.xlsx', engine='xlsxwriter')
+ anova_table.to_excel(writer_anova,sheet_name = disease + ' on channel ' + str(channel))
+ writer_anova.save()
+
\ No newline at end of file
diff --git a/code/lung_feature_patch_allPatients_threeProtein.py b/code/lung_feature_patch_allPatients_threeProtein.py
new file mode 100755
index 0000000..0e7ae7a
--- /dev/null
+++ b/code/lung_feature_patch_allPatients_threeProtein.py
@@ -0,0 +1,689 @@
+#!/usr/bin/env python3
+# -*- coding: utf-8 -*-
+"""Explorative analysis of protein composition and structure in lung ECM to
+study disease-induced remodeling of the three proteins that provide structural
+integrity to the lung: collagen IV, elastin and fibrillar collagen.
+
+The three-protein analysis provides the following:
+ - A 'Data Model', a dictionary of image patches representative of the local
+ protein structure and composition found in the data (both healthy and
+ disease group) at the scale defined by the patch size.
+ - A 'Sorted Data Model' or disease signature, a sorted version of the
+ dictionary above, based on how often a dictionary element (called
+ cluster centre in the publication) is found in the healthy vs. the disease.
+ - A 'Probability Map' for every image based on the three proteins, with
+ pixel correspondance to the input images (maximum projections),
+ highlighting regions characteristic of health or disease.
+ - A 'Disease Effect', to quantify how well the control and
+ disease groups are separated after assigning a health probability score to
+ each image, computed as the average of its probability map values.
+
+by Monica J. Emerson monj@dtu.dk and Anders B. Dahl abda@dtu.dk
+
+For more information, read our article (add link and citation)"""
+
+#%% Load packages and initialise parameters
+
+import numpy as np
+import matplotlib.pyplot as plt
+import sklearn.cluster
+import microscopy_analysis as ma
+import skimage.io
+import skimage.transform
+
+import os
+from datetime import datetime
+import sys
+from matplotlib.offsetbox import OffsetImage, AnnotationBbox
+from math import ceil
+
+startTime = datetime.now()
+
+plt.close('all')
+
+sc_fac = 0.5 #25 #25 #0.5 # scaling factor of the image
+patch_size = 17
+nr_clusters = 100 #49#100 # number of clusters, choose a value with an integer as a square root
+protein_mode = 'triple'
+colour_mode = 'cmy' #'bnw' #colour
+th_back = 15
+nr_runs = 10 #5
+
+#%% Define and create directories
+
+disease = 'covid' #diseased (mix, 2 of each condition)' #''emphysema' 'sarcoidosis' 'ild' 'covid'
+control = 'control'
+conditions = [disease, control]
+
+# input directories - images start with the name 'frame'
+base_dir_in = '../input_data/'
+#dir_in = base_dir_in + 'maximum_projections' #uncomment to read maximum projections without contrast equalisation
+dir_in = base_dir_in + 'postprocessed_maxProjs/rgb/' #Read rgb for correct protein channel ordering, visualisation will be in cmy
+base_name = 'frame'
+
+#output directory
+dir_results = '../results/' #rerun just to make sure not to muck up the results
+os.makedirs(dir_results, exist_ok = True)
+os.makedirs(dir_results + '/statistics', exist_ok = True)
+
+#%% Read (preprocessed) maximum corrected images
+#Read maximum projection images (runtime ~= 20 s )
+print('Reading maximum projection images')
+
+max_img_list_control = ma.read_max_imgs(dir_in + control, base_name)
+max_img_list_disease = ma.read_max_imgs(dir_in + disease, base_name)
+
+#%% Exchange channels: fibrillar collagen with elastin
+for max_img_sample_control in max_img_list_control:
+ for nr_frame, max_img_control in enumerate(max_img_sample_control):
+ max_img_sample_control[nr_frame] = max_img_control[:,:,[0,2,1]]
+
+for max_img_sample_disease in max_img_list_disease:
+ for nr_frame, max_img_disease in enumerate(max_img_sample_disease):
+ max_img_sample_disease[nr_frame] = max_img_disease[:,:,[0,2,1]]
+
+#%% Prepare maximum projection images for feature extraction
+#Flatten and merge conditions
+max_img_list_control_flat = ma.flatten_list(max_img_list_control)
+max_img_list_disease_flat = ma.flatten_list(max_img_list_disease)
+
+#Rescale
+max_img_list_control_processed = []
+for max_img in ma.flatten_list(max_img_list_control) :
+ max_img_list_control_processed += [skimage.transform.rescale(max_img, sc_fac, preserve_range = True, multichannel=True).astype(max_img.dtype)]
+
+max_img_list_disease_processed = []
+for max_img in ma.flatten_list(max_img_list_disease) :
+ max_img_list_disease_processed += [skimage.transform.rescale(max_img, sc_fac, preserve_range = True, multichannel=True).astype(max_img.dtype)]
+
+#%% Compute patches
+patch_feat_list_control = []
+for max_img in max_img_list_control_processed:
+ patch_feat_list_control += [ma.ndim2col_pad(max_img, (patch_size, patch_size),norm=False).transpose()]
+
+patch_feat_list_disease = []
+for max_img in max_img_list_disease_processed:
+ patch_feat_list_disease += [ma.ndim2col_pad(max_img, (patch_size, patch_size),norm=False).transpose()]
+
+patch_feat_total = patch_feat_list_control + patch_feat_list_disease
+
+for run in range(nr_runs):
+
+ #directories for results from the different runs
+ dir_probs = dir_results + disease + '_' + protein_mode + '_%dclusters_%ddownscale_%dpatchsize/'%(nr_clusters,1/sc_fac,patch_size)
+ dir_probs += 'run'+str(run)+'/'
+ ma.make_output_dirs(dir_probs, conditions)
+ dir_probs_noBack = dir_probs + 'noBackground/'
+ os.makedirs(dir_probs_noBack, exist_ok = True)
+
+ #%% k-means clustering
+ startTime_clustering = datetime.now()
+
+ batch_size = 10000
+ th_nr_pathesINcluster = 20
+ th_nr_weakClusters = 0 #added for covid and ild
+
+ #If cluster centers were already computed, use those
+ if os.path.exists(dir_probs + 'array_cluster_centers_colour.npy'):
+ cluster_centers = np.load(dir_probs + 'array_cluster_centers_colour.npy')
+ kmeans = sklearn.cluster.MiniBatchKMeans(n_clusters=nr_clusters, batch_size = batch_size)
+ kmeans.cluster_centers_ = cluster_centers
+
+ #Compute cluster centres
+ else:
+ #features for clustering
+ nr_keep = 10000 # number of features/image randomly picked for clustering
+ n_im = len(patch_feat_total)
+ feat_dim = patch_feat_total[0].shape[1]
+ patch_feat_to_cluster = np.zeros((nr_keep*n_im,feat_dim),dtype = max_img.dtype)
+
+ f = 0
+ nr_keep_control = nr_keep//(2*len(patch_feat_list_control)//len(patch_feat_list_disease))
+ nr_keep_disease = nr_keep//2
+ for patch_feat in patch_feat_list_control:
+ keep_indices = np.random.permutation(np.arange(patch_feat.shape[0]))[:nr_keep_control]
+ patch_feat_to_cluster[f:f+nr_keep_control,:] = patch_feat[keep_indices,:]
+ f += nr_keep
+
+ for patch_feat in patch_feat_list_disease:
+ keep_indices = np.random.permutation(np.arange(patch_feat.shape[0]))[:nr_keep_disease]
+ patch_feat_to_cluster[f:f+nr_keep_disease,:] = patch_feat[keep_indices,:]
+ f += nr_keep
+
+ #Get feature vectors
+ feat_to_cluster = patch_feat_to_cluster
+
+ #Compute cluster centers and save if there is no weak clusters
+ nr_weakClusters = th_nr_weakClusters+1
+ repetition = 0
+ nr_weakClusters_list = []
+ while nr_weakClusters>th_nr_weakClusters: #While weak clusters is different from empty recompute
+ kmeans = sklearn.cluster.MiniBatchKMeans(n_clusters=nr_clusters, batch_size = batch_size, verbose = False, reassignment_ratio=0.0005, max_no_improvement = 100) #0.00075 was good for uip
+ kmeans.fit(feat_to_cluster)
+
+ #Nr. patches that have contributed to each cluster
+ nr_feat_in_cluster = [sum(kmeans.labels_==cluster) for cluster in range(nr_clusters)]
+
+ weak_cluster_IDs = [ind for ind,val in enumerate(nr_feat_in_cluster) if val<th_nr_pathesINcluster]
+ print('Weak cluster IDs: ',weak_cluster_IDs)
+
+ #Weak clusters are those composed by very few patches
+ nr_weakClusters = len([1 for i in nr_feat_in_cluster if i<th_nr_pathesINcluster])
+
+ #If there are weak clusters, the clustering should be recomputed
+ if nr_weakClusters>th_nr_weakClusters:
+ print(str(nr_weakClusters)+ " clusters composed of less than "+str(th_nr_pathesINcluster)+" images")
+ else:
+ np.save(dir_probs + 'array_cluster_centers_colour.npy', kmeans.cluster_centers_) # .npy extension is added if not given
+
+ print(repetition)
+ repetition += 1
+ nr_weakClusters_list += [nr_weakClusters]
+
+
+ endTime_clustering = datetime.now() - startTime_clustering
+ print(endTime_clustering)
+
+ #%% Plot all cluster centers (grid view)
+ cluster_centers = kmeans.cluster_centers_
+ int_sum_list = ma.plot_grid_cluster_centers(cluster_centers, range(nr_clusters), patch_size, colour_mode = colour_mode)
+ plt.savefig(dir_probs + 'clustercenters_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Histograms for background, control and disease
+ hist_control, assignment_list_control= ma.compute_assignment_hist(patch_feat_list_control, kmeans)
+ hist_disease, assignment_list_disease = ma.compute_assignment_hist(patch_feat_list_disease, kmeans)
+
+ #%% Show histograms (bar plot) of control and disease
+ fig, ax = plt.subplots(1,1)
+ ax.bar(np.array(range(0,nr_clusters))-0.25, hist_control, width = 0.5, label='Control', color = 'b')
+ ax.bar(np.array(range(0,nr_clusters))+0.25, hist_disease, width = 0.5, label='Disease', color = 'r')
+ ax.legend()
+ plt.savefig(dir_probs + 'assignmentHistograms_%dclusters_%ddownscale_%dpatchsize.pdf'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Compute populated, occurrence and importance cluster measures from the histogram
+
+ #OCCURRENCE RATIO: How more often does it occur in one of the conditions
+ occurrence_ratio = np.empty((nr_clusters))
+ ind_control = hist_control>hist_disease
+ ind_disease = ~ind_control
+ control_cond = (hist_disease!=0) & ind_control
+ occurrence_ratio[control_cond] = hist_control[control_cond]/hist_disease[control_cond]
+ disease_cond = (hist_control!=0) & ind_disease
+ occurrence_ratio[disease_cond] = -hist_disease[disease_cond]/hist_control[disease_cond]
+
+ #POPULATED: How trustworthy is the cluster?
+ populated = hist_control+hist_disease
+ weights = populated/populated.max()
+
+ #Cluster IMPORTANCE: occurrence weighed by POPULATED
+ def sigmoid(x,k):
+ return 2 / (1 + np.exp(-k*x)) - 1
+
+ x = np.linspace(0,1,100)
+ plt.plot(x,sigmoid(x,100))
+
+ importance = occurrence_ratio * sigmoid(weights,100)
+
+ #%% Plot all control and disease cluster centers in the 2d populated/occurrence space
+ fig_control, ax_control = plt.subplots(figsize=(15,15))
+ ax_control.scatter(populated[occurrence_ratio>1], occurrence_ratio[occurrence_ratio>1])
+ ax_control.set_ylim(0.8,7)
+ ax_control.set_xlim(0,0.08)
+
+ fig_disease, ax_disease = plt.subplots(figsize=(15,15))
+ ax_disease.scatter(populated[occurrence_ratio<1], -occurrence_ratio[occurrence_ratio<1])
+ ax_disease.set_ylim(0.8,7)
+ ax_disease.set_xlim(0,0.08)
+
+ for x0, y0, cluster_nr in zip(populated, occurrence_ratio, np.arange(0,nr_clusters)):
+ cluster_centre = np.transpose((np.reshape(kmeans.cluster_centers_[cluster_nr,:],(3,patch_size,patch_size))),(1,2,0))
+ cluster_centre = ma.rgb2cmy(cluster_centre)
+ if y0>0:
+ ab = AnnotationBbox(OffsetImage(cluster_centre.astype(max_img.dtype)), (x0, y0), frameon=False)
+ ax_control.add_artist(ab)
+ else:
+ ab = AnnotationBbox(OffsetImage(cluster_centre.astype(max_img.dtype)), (x0, -y0), frameon=False)
+ ax_disease.add_artist(ab)
+
+ plt.figure(fig_control.number)
+ plt.savefig(dir_probs + 'controlClustercenters_2Dspace_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ plt.figure(fig_disease.number)
+ plt.savefig(dir_probs + 'diseaseClustercenters_2Dspace_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Plot all clusters sorted by IMPORTANCE
+
+ #According to occurence
+ clusters_control_occ_sorted = [np.arange(0,100)[i] for i in np.argsort(occurrence_ratio.flatten()) if (occurrence_ratio.flatten())[i]>0]
+ clusters_control_occ_sorted.reverse()
+
+ clusters_disease_occ_sorted = [np.arange(0,100)[i] for i in np.argsort(occurrence_ratio.flatten()) if (occurrence_ratio.flatten())[i]<0]
+
+ clusters_occ_sorted = [np.arange(0,100)[i] for i in np.argsort(occurrence_ratio.flatten())]
+ clusters_occ_sorted.reverse()
+
+ occurrence_ratio_sorted = [occurrence_ratio[i] for i in np.argsort(occurrence_ratio.flatten())]
+ occurrence_ratio_sorted.reverse()
+
+ ma.plot_grid_cluster_centers(kmeans.cluster_centers_, clusters_occ_sorted, patch_size, colour_mode = colour_mode)
+ plt.suptitle('Cluster centers \n sorted by occ from control to disease with cluster ' + str(len(clusters_control_occ_sorted)) + ' in between')
+ plt.savefig(dir_probs + 'clustercenters_occ_sorted_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #According to occurence weighed by populated
+ clusters_control_occW_sorted = [np.arange(0,100)[i] for i in np.argsort(importance.flatten()) if (importance.flatten())[i]>0]
+ clusters_control_occW_sorted.reverse()
+
+ clusters_disease_occW_sorted = [np.arange(0,100)[i] for i in np.argsort(importance.flatten()) if (importance.flatten())[i]<0]
+
+ clusters_occW_sorted = [np.arange(0,100)[i] for i in np.argsort(importance.flatten())]
+ clusters_occW_sorted.reverse()
+
+ occurrence_ratio_sorted = [occurrence_ratio[i] for i in np.argsort(importance.flatten())]
+ occurrence_ratio_sorted.reverse()
+
+ cluster_nums = np.arange(0,nr_clusters)
+ cluster_nums_sorted = [cluster_nums[i] for i in np.argsort(importance.flatten())]
+ cluster_nums_sorted.reverse()
+
+ prob_control = hist_control/(hist_control+hist_disease)
+ prob_control_sorted = [prob_control[i] for i in np.argsort(importance.flatten())]
+ prob_control_sorted.reverse()
+
+
+ ma.plot_grid_cluster_centers(kmeans.cluster_centers_, clusters_occW_sorted, patch_size, colour_mode = colour_mode)
+ plt.suptitle('Cluster centers \n sorted by occ*f(p) from control to disease with cluster ' + str(len(clusters_control_occW_sorted)) + ' in between')
+ plt.savefig(dir_probs + 'clustercenters_occW_sorted_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #with probabilities
+ ma.plot_grid_cluster_centers(kmeans.cluster_centers_, clusters_occW_sorted, patch_size, colour_mode = colour_mode, titles = True, occurrence = prob_control_sorted)
+ plt.suptitle('Cluster centers \n sorted by occ*f(p) from control to disease with cluster ' + str(len(clusters_control_occW_sorted)) + ' in between')
+ plt.savefig(dir_probs + 'clustercenters_occW_sorted_wProbs_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #with cluster numbers
+ ma.plot_grid_cluster_centers(kmeans.cluster_centers_, clusters_occW_sorted, patch_size, colour_mode = colour_mode, titles = True, occurrence = cluster_nums_sorted)
+ plt.suptitle('Cluster centers \n sorted by occ*f(p) from control to disease with cluster ' + str(len(clusters_control_occW_sorted)) + ' in between')
+ plt.savefig(dir_probs + 'clustercenters_occW_sorted_wClusternums_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Plot all clusters sorted - split channelwise view
+ ma.plot_grid_cluster_centers(np.copy(kmeans.cluster_centers_), clusters_occW_sorted, patch_size, protein_mode = protein_mode, colour_mode = 'cmy', channel = 0)
+ plt.savefig(dir_probs + 'clustercenters_occW_sorted_split_channel1_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ ma.plot_grid_cluster_centers(np.copy(kmeans.cluster_centers_), clusters_occW_sorted, patch_size, protein_mode = protein_mode, colour_mode = 'cmy', channel = 1)
+ plt.savefig(dir_probs + 'clustercenters_occW_sorted_split_channel2_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ ma.plot_grid_cluster_centers(np.copy(kmeans.cluster_centers_), clusters_occW_sorted, patch_size, protein_mode = protein_mode, colour_mode = 'cmy', channel = 2)
+ plt.savefig(dir_probs + 'clustercenters_occW_sorted_split_channel3_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+
+ #%% Find background clusters
+ clusters_background_intBased = [i for i in range(len(int_sum_list)) if int_sum_list[i] < th_back*patch_size**2]
+
+ clusters_background = clusters_background_intBased
+ print('Background clusters'+str(clusters_background))
+
+ #%% Plot grid of ordered, non-background clusters that are populated over a thresh
+ th_populated = 0.5/nr_clusters
+ th_occurr = 1.25
+
+ #control
+ clusters_control_occW_sorted_noBackground = [i for i in clusters_control_occW_sorted if (i not in clusters_background)&(populated[i]>th_populated)&(occurrence_ratio[i]>th_occurr)]
+ ma.plot_grid_cluster_centers(kmeans.cluster_centers_, clusters_control_occW_sorted_noBackground, patch_size, colour_mode = colour_mode)
+ #plt.suptitle('Characteristic clusters for control, excluding background, \n from most important to least')
+ plt.savefig(dir_probs_noBack + 'clustercenters_control_occW_sorted_noBack%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #disease
+ clusters_disease_occW_sorted_noBackground = [i for i in clusters_disease_occW_sorted if (i not in clusters_background)&(populated[i]>th_populated)&(abs(occurrence_ratio[i])>th_occurr)]
+ ma.plot_grid_cluster_centers(kmeans.cluster_centers_, clusters_disease_occW_sorted_noBackground, patch_size, colour_mode = colour_mode)
+ #plt.suptitle('Charactertistic clusters for ' + disease + ', excluding background, \n from most important to least')
+ plt.savefig(dir_probs_noBack + 'clustercenters_disease_occW_sorted_noBack%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Plot channelwise split characteristic clusters
+ nr_clusters_display = 10
+ clusters_control = clusters_control_occW_sorted_noBackground[:nr_clusters_display]
+ clusters_disease = clusters_disease_occW_sorted_noBackground[:nr_clusters_display]
+
+ #control clusters
+ cluster_centers_control = kmeans.cluster_centers_[clusters_control]
+ fig, axs = plt.subplots(1,len(clusters_control), figsize=(len(clusters_control)*3,3), sharex=True, sharey=True)
+ fig.suptitle('Cluster centers for control')
+
+ fig_split, axs_split = plt.subplots(3,len(clusters_control), figsize=(len(clusters_control)*3,9), sharex=True, sharey=True)
+ fig_split.suptitle('Cluster centers for control - split')
+ for l in np.arange(0,len(clusters_control)):
+
+ cluster_centre = np.transpose((np.reshape(cluster_centers_control[l,:],(3,patch_size,patch_size))),(1,2,0))
+
+ #channel 1 - collagen IV
+ im = np.zeros(cluster_centre.shape)
+ im[...,0] = cluster_centre[...,0]
+ im = ma.rgb2cmy(im)
+ axs_split[0][l].imshow(im.astype(np.uint8))
+ axs_split[0][l].axis('off')
+ axs_split[0][l].set_title(clusters_control[l])
+
+ #channel 2 - elastin
+ im = np.zeros(cluster_centre.shape)
+ im[...,1] = cluster_centre[...,1]
+ im = ma.rgb2cmy(im)
+ axs_split[1][l].imshow(im.astype(np.uint8))
+ axs_split[1][l].axis('off')
+
+ #channel 3 - SHG
+ im = np.zeros(cluster_centre.shape)
+ im[...,2] = cluster_centre[...,2]
+ im = ma.rgb2cmy(im)
+ axs_split[2][l].imshow(im.astype(np.uint8))
+ axs_split[2][l].axis('off')
+
+ plt.savefig(dir_probs_noBack + 'clustercenters_control_split_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #cmy merge
+ cluster_centre = ma.rgb2cmy(cluster_centre)
+ axs[l].imshow(cluster_centre.astype(np.uint8))
+ axs[l].axis('off')
+ axs[l].set_title(clusters_control[l])
+
+ plt.figure(fig.number),
+ plt.savefig(dir_probs_noBack + 'clustercenters_control_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #disease clusters
+ cluster_centers_disease = kmeans.cluster_centers_[clusters_disease]
+ fig, axs = plt.subplots(1,len(clusters_disease), figsize=(len(clusters_disease)*3,3), sharex=True, sharey=True)
+ fig.suptitle('Cluster centers for disease')
+
+ fig_split, axs_split = plt.subplots(3,len(clusters_disease), figsize=(len(clusters_disease)*3,9), sharex=True, sharey=True)
+ fig_split.suptitle('Cluster centers for disease - split')
+ for l in np.arange(0,len(clusters_disease)):
+
+ cluster_centre = np.transpose((np.reshape(cluster_centers_disease[l,:],(3,patch_size,patch_size))),(1,2,0))
+
+ #channel 1 - collagen IV
+ im = np.zeros(cluster_centre.shape)
+ im[...,0] = cluster_centre[...,0]
+ im = ma.rgb2cmy(im)
+ axs_split[0][l].imshow(im.astype(np.uint8))
+ axs_split[0][l].axis('off')
+ axs_split[0][l].set_title(clusters_disease[l])
+
+ #channel 2 - elastin
+ im = np.zeros(cluster_centre.shape)
+ im[...,1] = cluster_centre[...,1]
+ im = ma.rgb2cmy(im)
+ axs_split[1][l].imshow(im.astype(np.uint8))
+ axs_split[1][l].axis('off')
+
+ #channel 3 - SHG
+ im = np.zeros(cluster_centre.shape)
+ im[...,2] = cluster_centre[...,2]
+ im = ma.rgb2cmy(im)
+ axs_split[2][l].imshow(im.astype(np.uint8))
+ axs_split[2][l].axis('off')
+
+ plt.savefig(dir_probs_noBack + 'clustercenters_disease_split_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #cmy merge
+ cluster_centre = ma.rgb2cmy(cluster_centre)
+ axs[l].imshow(cluster_centre.astype(np.uint8))
+ axs[l].axis('off')
+ axs[l].set_title(clusters_disease[l])
+
+ plt.figure(fig.number),
+ plt.savefig(dir_probs_noBack + 'clustercenters_disease_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Compute probability images
+ startTime_probs = datetime.now()
+
+ #Assign probabilities of control and disease to clusters
+ prob_control= hist_control/(hist_control+hist_disease)
+ prob_disease = hist_disease/(hist_control+hist_disease)
+
+ #Compute probability images
+ r,c,_ = max_img_list_control_flat[0].shape
+ img_shape = (int(r*sc_fac),int(c*sc_fac))
+ prob_img_list_control = ma.comp_prob_imgs(prob_control,assignment_list_control,img_shape)
+ prob_img_list_disease = ma.comp_prob_imgs(prob_control,assignment_list_disease,img_shape)
+
+ #%% Display probability images and accumulate probs per frame and sample
+ nr_frames = len(max_img_list_control[0])
+
+ #control
+ sample_names_control = ma.listdir_custom(dir_in + control, string_start_pos = -4, dir_flag = True, )
+
+ nr_list = 0
+ prob_list_control= []
+ fig_probmaps, ax_probmaps = plt.subplots(nr_frames, len(sample_names_control),figsize = (len(sample_names_control),nr_frames), sharex=True, sharey=True)
+ for max_img_list_sample,sample_name,sample_nr in zip(max_img_list_control, sample_names_control, range(len(sample_names_control))): #in general, in max_img_list_condition
+ print(sample_name)
+ nr_frames = len(max_img_list_sample)
+ prob_img_list_sample = prob_img_list_control[nr_list:nr_list+nr_frames]
+ nr_list += nr_frames
+
+ prob_sample = np.zeros((nr_frames,1))
+ fig, axs = plt.subplots(2,nr_frames, figsize = (nr_frames*2,2*2), sharex=True, sharey=True)
+ for prob_img, max_img, frame_nr in zip(prob_img_list_sample,max_img_list_sample,range(nr_frames)):
+ max_img_ds = skimage.transform.rescale(max_img, sc_fac, multichannel = True, preserve_range = True).astype(max_img.dtype)
+
+ #compute image probability
+ prob_frame = sum(sum(prob_img))/prob_img.size
+
+ #remove background from prob imgs
+ ind_back = (max_img_ds[:,:,0]<th_back)&(max_img_ds[:,:,1]<th_back)&(max_img_ds[:,:,2]<th_back)
+ prob_img[ind_back] = 0.5
+
+ #plot
+ ax_probmaps[frame_nr][sample_nr].imshow(1-prob_img, cmap=plt.cm.bwr, vmin = 0, vmax = 1)
+ ax_probmaps[frame_nr][sample_nr].xaxis.set_ticklabels([])
+ ax_probmaps[frame_nr][sample_nr].xaxis.set_ticks_position('none')
+ ax_probmaps[frame_nr][sample_nr].yaxis.set_ticklabels([])
+ ax_probmaps[frame_nr][sample_nr].yaxis.set_ticks_position('none')
+
+ axs[1][frame_nr].imshow(1-prob_img, cmap=plt.cm.bwr, vmin = 0, vmax = 1)
+ axs[1][frame_nr].set_title("%.2f" % np.round(prob_frame,2))
+ axs[1][frame_nr].xaxis.set_ticklabels([])
+ axs[1][frame_nr].xaxis.set_ticks_position('none')
+ axs[1][frame_nr].yaxis.set_ticklabels([])
+ axs[1][frame_nr].yaxis.set_ticks_position('none')
+
+ if colour_mode == 'cmy':
+ max_img_ds = ma.rgb2cmy(max_img_ds)
+ axs[0][frame_nr].imshow(max_img_ds)
+ axs[0][frame_nr].xaxis.set_ticklabels([])
+ axs[0][frame_nr].xaxis.set_ticks_position('none')
+ axs[0][frame_nr].yaxis.set_ticklabels([])
+ axs[0][frame_nr].yaxis.set_ticks_position('none')
+
+ #save img probs
+ prob_sample[frame_nr] = prob_frame
+ prob_list_control += [prob_sample]
+
+ plt.suptitle('Health probability of sample '+ sample_name +', avg:' + str(round(prob_sample.mean(),2))+ ' std:' + str(round(prob_sample.std(),2)))
+ plt.savefig(dir_probs + '/' + control + '/' + 'probImHealth_'+sample_name+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+ plt.figure(fig_probmaps.number),plt.savefig(dir_probs + '/' + control + '/' + 'probMaps_'+control+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+
+ #disease
+ sample_names_disease = ma.listdir_custom(dir_in + disease, string_start_pos = -4, dir_flag = True)
+
+ nr_list = 0
+ prob_list_disease = []
+ fig_probmaps, ax_probmaps = plt.subplots(nr_frames, len(sample_names_disease),figsize = (len(sample_names_disease),nr_frames), sharex=True, sharey=True)
+ for max_img_list_sample,sample_name,sample_nr in zip(max_img_list_disease, sample_names_disease, range(len(sample_names_disease))): #in general, in max_img_list_condition
+ print(sample_name)
+ nr_frames = len(max_img_list_sample)
+ prob_img_list_sample = prob_img_list_disease[nr_list:nr_list+nr_frames]
+ nr_list += nr_frames
+
+ prob_sample = np.zeros((nr_frames,1))
+ fig, axs = plt.subplots(2,nr_frames, figsize = (nr_frames*2,2*2), sharex=True, sharey=True)
+ for prob_img, max_img, frame_nr in zip(prob_img_list_sample,max_img_list_sample,range(nr_frames)):
+ max_img_ds = skimage.transform.rescale(max_img, sc_fac, multichannel = True, preserve_range = True).astype(max_img.dtype)
+
+ #compute image probability
+ prob_frame = sum(sum(prob_img))/prob_img.size
+
+ #remove background from prob imgs
+ ind_back = (max_img_ds[:,:,0]<th_back)&(max_img_ds[:,:,1]<th_back)&(max_img_ds[:,:,2]<th_back)
+ prob_img[ind_back] = 0.5
+
+ #plot
+ ax_probmaps[frame_nr][sample_nr].imshow(1-prob_img, cmap=plt.cm.bwr, vmin = 0, vmax = 1)
+ ax_probmaps[frame_nr][sample_nr].xaxis.set_ticklabels([])
+ ax_probmaps[frame_nr][sample_nr].xaxis.set_ticks_position('none')
+ ax_probmaps[frame_nr][sample_nr].yaxis.set_ticklabels([])
+ ax_probmaps[frame_nr][sample_nr].yaxis.set_ticks_position('none')
+
+ axs[1][frame_nr].imshow(1-prob_img, cmap=plt.cm.bwr, vmin = 0, vmax = 1)
+ axs[1][frame_nr].set_title("%.2f" % np.round(prob_frame,2))
+ axs[1][frame_nr].xaxis.set_ticklabels([])
+ axs[1][frame_nr].xaxis.set_ticks_position('none')
+ axs[1][frame_nr].yaxis.set_ticklabels([])
+ axs[1][frame_nr].yaxis.set_ticks_position('none')
+
+ if colour_mode == 'cmy':
+ max_img_ds = ma.rgb2cmy(max_img_ds)
+ axs[0][frame_nr].imshow(max_img_ds)
+ axs[0][frame_nr].xaxis.set_ticklabels([])
+ axs[0][frame_nr].xaxis.set_ticks_position('none')
+ axs[0][frame_nr].yaxis.set_ticklabels([])
+ axs[0][frame_nr].yaxis.set_ticks_position('none')
+
+ #save img probs
+ prob_sample[frame_nr] = prob_frame
+ prob_list_disease += [prob_sample]
+
+ plt.suptitle('Health probability of sample '+ sample_name +', avg:' + str(round(prob_sample.mean(),2))+ ' std:' + str(round(prob_sample.std(),2)))
+ plt.savefig(dir_probs + '/' + disease + '/' + 'probImHealth_'+sample_name+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+ plt.figure(fig_probmaps.number),plt.savefig(dir_probs + '/' + disease + '/' + 'probMaps_'+disease+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+
+ prob_list_samples = prob_list_control + prob_list_disease
+
+ #%% Box plots
+ fig, ax = plt.subplots(figsize = (15,5))
+ ax.set_title('Image health scores ')
+
+ sample_sep = 0.85
+ patient_sep = 2.35
+ condition_sep = 2.5
+ positions_control = np.tile([0,0+sample_sep],int(len(max_img_list_control)/2)) + np.repeat(np.arange(0,len(max_img_list_control)/2*patient_sep,patient_sep),2)
+ positions_disease = np.tile([len(max_img_list_control)+condition_sep,len(max_img_list_control)+condition_sep+sample_sep],int(len(max_img_list_disease)/2)) + np.repeat(np.arange(0,len(max_img_list_disease)/2*patient_sep,patient_sep),2)
+ array_probs = np.squeeze(np.asarray(prob_list_samples))
+ bp_control = ax.boxplot((array_probs[:len(max_img_list_control),...]).T, positions = positions_control, patch_artist = True)
+ bp_disease = ax.boxplot(array_probs[len(max_img_list_control):,...].T, positions = positions_disease, patch_artist = True)
+
+ for element in ['boxes', 'whiskers', 'fliers', 'means', 'medians', 'caps']:
+ plt.setp(bp_control[element], color='blue')
+ plt.setp(bp_control[element], linewidth = 2)
+
+ for patch in bp_control['boxes']:
+ patch.set(facecolor='white')
+ patch.set(linewidth = 2)
+
+ for element in ['boxes','whiskers', 'fliers', 'means', 'medians', 'caps']:
+ plt.setp(bp_disease[element], color='red')
+ plt.setp(bp_disease[element], linewidth = 2)
+
+ for patch in bp_disease['boxes']:
+ patch.set(facecolor= 'white')
+ patch.set(linewidth = 2)
+ #patch.set(alpha=0.2)
+
+ ax.set_xticklabels([i[-4:] for i in sample_names_control]+[i[-4:] for i in sample_names_disease])
+ ax.set_ylim(0.3,0.7)
+ ax.set_ylabel('Probability health')
+ #ax.set_ylim(0,1)
+
+ ax.tick_params(
+ axis='both', # changes apply to the x-axis
+ which='both', # both major and minor ticks are affected
+ #labelleft = False, # ticks along the top edge are off
+ #labelbottom=False
+ )
+
+ #plot points
+ positions = list(positions_control) + list(positions_disease)
+ for prob_sample, position in zip(prob_list_samples, positions):
+ print(prob_sample)
+ print(5*[position])
+ # Add some random "jitter" to the x-axis
+ x = np.random.normal(5*[position], 0)
+ #plot(x, y, 'r.', alpha=0.2)
+ ax.plot(x, prob_sample,'k.', markersize = 6, zorder = 3)
+
+ #horizontal line
+ x = np.linspace(0,position.max())
+ y = 0.5*np.ones(x.shape)
+ ax.plot(x,y,'k',linewidth = 1, linestyle = 'dashed')
+
+ plt.savefig(dir_probs + 'scatterBoxplots_%dclusters_%ddownscale_%dpatchsize.pdf'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ fig2, ax2 = plt.subplots(figsize = (15,5))
+ #plot points
+ for prob_sample, position in zip(prob_list_control, positions_control):
+ ax2.plot(5*[position], prob_sample,'b.', markersize = 4)
+
+ for prob_sample, position in zip(prob_list_disease, positions_disease):
+ ax2.plot(5*[position], prob_sample,'r.', markersize = 4)
+
+ #horizontal line
+ x = np.linspace(0,position.max())
+ y = 0.5*np.ones(x.shape)
+ ax2.plot(x,y,'k',linewidth = 1, linestyle = 'dashed')
+
+ ax2.set_xticks(ax.get_xticks())
+ ax2.set_xticklabels([i[-4:] for i in sample_names_control]+[i[-4:] for i in sample_names_disease])
+ ax2.set_ylim(0.3,0.7)
+ ax2.set_ylabel('Probability health')
+
+ plt.savefig(dir_probs + 'samplewiseScatterPlots_%dclusters_%ddownscale_%dpatchsize.pdf'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+
+ #%% Print computational time
+ endTime = datetime.now() - startTime
+ print(endTime)
+
+
+ #%%ANOVA SETUP
+ os.makedirs(dir_results + 'statistics/', exist_ok = True)
+
+ from statsmodels.formula.api import ols
+ from statsmodels.stats.anova import anova_lm
+ import pandas as pd
+ from patsy import ModelDesc
+
+ nr_frames_patient = 10
+ img_nr = np.tile(np.arange(nr_frames),array_probs.size//nr_frames)
+ sample_nr = np.tile(np.repeat(np.arange(nr_frames_patient//nr_frames),nr_frames),array_probs.size//nr_frames_patient)
+ #patient_nr = np.tile(np.repeat(np.arange(array_probs.size//nr_frames_patient//len(conditions)),nr_frames_patient),len(conditions))
+ patient_nr = np.concatenate([np.repeat(np.arange(len(max_img_list_control_flat)//nr_frames_patient),nr_frames_patient),np.repeat(np.arange(len(max_img_list_disease_flat)//nr_frames_patient),nr_frames_patient)])
+ condition_nr = np.concatenate([np.zeros(len(max_img_list_control_flat)), np.ones(len(max_img_list_disease_flat))])
+
+
+ dep_var = array_probs.flatten()
+
+ data_dict = {'Image':img_nr,
+ 'Sample':sample_nr,
+ 'Patient':patient_nr,
+ 'Condition': condition_nr,
+ 'S':dep_var}
+
+ formula = 'S ~ C(Condition)/C(Patient)/C(Sample)'
+
+ desc = ModelDesc.from_formula(formula)
+ print(desc.describe())
+
+ lm = ols(formula, data_dict).fit()
+ print(lm.summary())
+
+ anova_table = anova_lm(lm)
+ print(anova_table)
+ writer_anova = pd.ExcelWriter(dir_results + 'statistics/statistics_anova_' + disease + '_' + protein_mode + '_ds'+ str(1/sc_fac) +'_run' + str(run) +'.xlsx', engine='xlsxwriter')
+ anova_table.to_excel(writer_anova,sheet_name = disease)
+ writer_anova.save()
+
+
+
+
+
+
+
diff --git a/code/lung_feature_patch_allPatients_twoProtein.py b/code/lung_feature_patch_allPatients_twoProtein.py
new file mode 100755
index 0000000..debe53f
--- /dev/null
+++ b/code/lung_feature_patch_allPatients_twoProtein.py
@@ -0,0 +1,551 @@
+#!/usr/bin/env python3
+# -*- coding: utf-8 -*-
+"""Explorative analysis of protein composition and structure in lung ECM to
+study disease-induced remodeling of the three proteins that provide structural
+integrity to the lung: collagen IV, elastin and fibrillar collagen.
+
+The two-protein analyses provide the following:
+ - A 'Data Model' per combination of proteins, a dictionary of image patches
+ representative of the local protein structure and composition found in the
+ data (both healthy and disease group) at the scale defined by the patch
+ size.
+ - A 'Sorted Data Model' or disease signature per combination of proteins,
+ a sorted version of the dictionary above, based on how often a dictionary
+ element (called cluster centre in the publication) is found in the healthy
+ vs. the disease.
+ - A 'Probability Map' for every image based on each combination of
+ proteins, with pixel correspondance to the input images (maximum
+ projections), highlighting regions characteristic of health or disease.
+ - A 'Disease Effect' per combination of proteins, to quantify how well the
+ control and disease groups are separated after assigning a health
+ probability score to each image, computed as the average of its probability
+ map values.
+
+by Monica J. Emerson monj@dtu.dk and Anders B. Dahl abda@dtu.dk
+
+For more information, read our article (add link and citation)"""
+
+#%% Load packages and initialise parameters
+
+import numpy as np
+import matplotlib.pyplot as plt
+import sklearn.cluster
+import microscopy_analysis as ma
+import skimage.io
+import skimage.transform
+
+import os
+from datetime import datetime
+import sys
+from matplotlib.offsetbox import OffsetImage, AnnotationBbox
+from math import ceil
+import copy
+
+startTime = datetime.now()
+
+plt.close('all')
+
+sc_fac = 0.5 #25 #25 #0.5 # scaling factor of the image
+patch_size = 17
+nr_clusters = 100 #49#100 # number of clusters, choose a value with an integer as a square root
+th_back = 15
+nr_runs = 10
+
+protein_mode = 'dropout' #'single', 'triple'
+colour_mode = 'cmy'
+
+# drop_channel = 0
+# keep_channels = [x for x in range(3) if x!=drop_channel]
+
+#%% Define and create directories
+
+disease = 'emphysema' #diseased (mix, 2 of each condition)' #''emphysema' 'sarcoidosis' 'ild' 'covid'
+control = 'control'
+conditions = [disease, control]
+
+# input directories - images start with the name 'frame'
+base_dir_in = '../input_data/'
+#dir_in = base_dir_in + 'maximum_projections' #uncomment to read maximum projections without contrast equalisation
+dir_in = base_dir_in + 'postprocessed_maxProjs/rgb/' #Read rgb for correct protein channel ordering, visualisation will be in cmy
+base_name = 'frame'
+
+#output directory
+dir_results = '../results/' #rerun just to make sure not to muck up the results
+os.makedirs(dir_results, exist_ok = True)
+os.makedirs(dir_results + '/statistics', exist_ok = True)
+
+#%% Read (preprocessed) maximum corrected images
+#Read maximum projection images (runtime ~= 20 s )
+print('Reading maximum projection images')
+
+max_img_list_control_3c = ma.read_max_imgs(dir_in + control, base_name)
+max_img_list_disease_3c = ma.read_max_imgs(dir_in + disease, base_name)
+
+for drop_channel in range(3):
+ keep_channels = [x for x in range(3) if x!=drop_channel]
+ #%% Exchange channels: fibrillar collagen with elastin
+ max_img_list_control = copy.deepcopy(max_img_list_control_3c)
+ for max_img_sample_control in max_img_list_control:
+ for nr_frame, max_img_control in enumerate(max_img_sample_control):
+ img = max_img_control[:,:,[0,2,1]]
+ max_img_sample_control[nr_frame] = img[:,:,keep_channels]
+
+ max_img_list_disease = copy.deepcopy(max_img_list_disease_3c)
+ for max_img_sample_disease in max_img_list_disease:
+ for nr_frame, max_img_disease in enumerate(max_img_sample_disease):
+ img = max_img_disease[:,:,[0,2,1]]
+ max_img_sample_disease[nr_frame] = img[:,:,keep_channels]
+
+ #%% Prepare maximum projection images for feature extraction
+ #Flatten and merge conditions
+ max_img_list_control_flat = ma.flatten_list(max_img_list_control)
+ max_img_list_disease_flat = ma.flatten_list(max_img_list_disease)
+
+ #Rescale
+ max_img_list_control_processed = []
+ for max_img in ma.flatten_list(max_img_list_control) :
+ max_img_list_control_processed += [skimage.transform.rescale(max_img, sc_fac, preserve_range = True, multichannel=True).astype(max_img.dtype)]
+
+ max_img_list_disease_processed = []
+ for max_img in ma.flatten_list(max_img_list_disease) :
+ max_img_list_disease_processed += [skimage.transform.rescale(max_img, sc_fac, preserve_range = True, multichannel=True).astype(max_img.dtype)]
+
+ #%% Compute patches
+ patch_feat_list_control = []
+ for max_img in max_img_list_control_processed:
+ patch_feat_list_control += [ma.ndim2col_pad(max_img, (patch_size, patch_size),norm=False).transpose()]
+
+ patch_feat_list_disease = []
+ for max_img in max_img_list_disease_processed:
+ patch_feat_list_disease += [ma.ndim2col_pad(max_img, (patch_size, patch_size),norm=False).transpose()]
+
+ patch_feat_total = patch_feat_list_control + patch_feat_list_disease
+
+ for run in range(nr_runs):
+
+ #directories for results from the different runs
+ dir_probs = dir_results + disease + '_' + protein_mode + '_%dclusters_%ddownscale_%dpatchsize/'%(nr_clusters,1/sc_fac,patch_size)
+ dir_probs += 'run'+str(run)+'/'
+ ma.make_output_dirs(dir_probs, conditions)
+
+ #%% k-means clustering
+ startTime_clustering = datetime.now()
+
+ batch_size = 10000
+ th_nr_pathesINcluster = 20
+ th_nr_weakClusters = 0 #added for covid and ild
+
+ #If cluster centers were already computed, use those
+ if os.path.exists(dir_probs + 'array_cluster_centers_'+protein_mode+'_channel'+str(drop_channel)+'.npy'):
+ cluster_centers = np.load(dir_probs + 'array_cluster_centers_'+protein_mode+'_channel'+str(drop_channel)+'.npy')
+ kmeans = sklearn.cluster.MiniBatchKMeans(n_clusters=nr_clusters, batch_size = batch_size)
+ kmeans.cluster_centers_ = cluster_centers
+
+ #Compute cluster centres
+ else:
+ #features for clustering
+ nr_keep = 10000 # number of features/image randomly picked for clustering
+ n_im = len(patch_feat_total)
+ feat_dim = patch_feat_total[0].shape[1]
+ patch_feat_to_cluster = np.zeros((nr_keep*n_im,feat_dim),dtype = max_img.dtype)
+
+ f = 0
+ nr_keep_control = nr_keep//(2*len(patch_feat_list_control)//len(patch_feat_list_disease))
+ nr_keep_disease = nr_keep//2
+ for patch_feat in patch_feat_list_control:
+ keep_indices = np.random.permutation(np.arange(patch_feat.shape[0]))[:nr_keep_control]
+ patch_feat_to_cluster[f:f+nr_keep_control,:] = patch_feat[keep_indices,:]
+ f += nr_keep
+
+ for patch_feat in patch_feat_list_disease:
+ keep_indices = np.random.permutation(np.arange(patch_feat.shape[0]))[:nr_keep_disease]
+ patch_feat_to_cluster[f:f+nr_keep_disease,:] = patch_feat[keep_indices,:]
+ f += nr_keep
+
+ #Get feature vectors
+ feat_to_cluster = patch_feat_to_cluster
+
+ #Compute cluster centers and save if there is no weak clusters
+ nr_weakClusters = th_nr_weakClusters+1
+ repetition = 0
+ nr_weakClusters_list = []
+ while nr_weakClusters>th_nr_weakClusters: #While weak clusters is different from empty recompute
+ kmeans = sklearn.cluster.MiniBatchKMeans(n_clusters=nr_clusters, batch_size = batch_size, verbose = False, reassignment_ratio=0.0005, max_no_improvement = 100) #0.00075 was good for uip
+ kmeans.fit(feat_to_cluster)
+
+ #Nr. patches that have contributed to each cluster
+ nr_feat_in_cluster = [sum(kmeans.labels_==cluster) for cluster in range(nr_clusters)]
+
+ weak_cluster_IDs = [ind for ind,val in enumerate(nr_feat_in_cluster) if val<th_nr_pathesINcluster]
+ print('Weak cluster IDs: ',weak_cluster_IDs)
+
+ #Weak clusters are those composed by very few patches
+ nr_weakClusters = len([1 for i in nr_feat_in_cluster if i<th_nr_pathesINcluster])
+
+ #If there are weak clusters, the clustering should be recomputed
+ if nr_weakClusters>th_nr_weakClusters:
+ print(str(nr_weakClusters)+ " clusters composed of less than "+str(th_nr_pathesINcluster)+" images")
+ else:
+ np.save(dir_probs + 'array_cluster_centers_'+protein_mode+'_channel'+str(drop_channel)+'.npy', kmeans.cluster_centers_) # .npy extension is added if not given
+
+ print(repetition)
+ repetition += 1
+ nr_weakClusters_list += [nr_weakClusters]
+
+
+ endTime_clustering = datetime.now() - startTime_clustering
+ print(endTime_clustering)
+
+ #%% Plot all cluster centers (grid view)
+ cluster_centers = kmeans.cluster_centers_
+ int_sum_list = ma.plot_grid_cluster_centers(cluster_centers, range(nr_clusters), patch_size, protein_mode, colour_mode, channel = drop_channel)
+ plt.savefig(dir_probs + 'clustercenters_'+protein_mode+'_channel'+str(drop_channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Histograms for background, healthy and disease
+ hist_control, assignment_list_control= ma.compute_assignment_hist(patch_feat_list_control, kmeans)
+ hist_disease, assignment_list_disease = ma.compute_assignment_hist(patch_feat_list_disease, kmeans)
+
+ # Show histograms (bar plot) of healthy and disease
+ fig, ax = plt.subplots(1,1)
+ ax.bar(np.array(range(0,nr_clusters))-0.25, hist_control, width = 0.5, label='Control', color = 'b')
+ ax.bar(np.array(range(0,nr_clusters))+0.25, hist_disease, width = 0.5, label='Disease', color = 'r')
+ ax.legend()
+ plt.savefig(dir_probs + 'assignmentHistograms_'+protein_mode+'_channel'+str(drop_channel)+'_%dclusters_%ddownscale_%dpatchsize.pdf'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Compute populated, occurrence and importance cluster measures from the histogram
+ #grid dimensions
+
+ #OCCURRENCE RATIO: How more often does it occur in one of the conditions
+ occurrence_ratio = np.empty((nr_clusters))
+ ind_control = hist_control>hist_disease
+ ind_disease = ~ind_control
+ control_cond = (hist_disease!=0) & ind_control
+ occurrence_ratio[control_cond] = hist_control[control_cond]/hist_disease[control_cond]
+ disease_cond = (hist_control!=0) & ind_disease
+ occurrence_ratio[disease_cond] = -hist_disease[disease_cond]/hist_control[disease_cond]
+
+ #POPULATED How trustworthy is the cluster?
+ populated = hist_control+hist_disease
+ weights = populated/populated.max()
+
+ #Cluster IMPORTANCE: occurrence weighed by POPULATED
+ def sigmoid(x,k):
+ return 2 / (1 + np.exp(-k*x)) - 1
+
+ importance = occurrence_ratio * sigmoid(weights,100)
+
+ #%% Plot cluster centers in the 2d populated/occurrence space
+ fig_control, ax_control = plt.subplots(figsize=(15,15))
+ ax_control.scatter(populated[occurrence_ratio>1], occurrence_ratio[occurrence_ratio>1])
+ ax_control.set_ylim(0.8,7)
+ ax_control.set_xlim(0,0.08)
+
+ fig_disease, ax_disease = plt.subplots(figsize=(15,15))
+ ax_disease.scatter(populated[occurrence_ratio<1], -occurrence_ratio[occurrence_ratio<1])
+ ax_disease.set_ylim(0.8,7)
+ ax_disease.set_xlim(0,0.08)
+
+ for x0, y0, cluster_nr in zip(populated, occurrence_ratio, np.arange(0,nr_clusters)):
+ if protein_mode == 'dropout':
+ cluster_centre = np.zeros((patch_size,patch_size,3),dtype = cluster_centers.dtype)
+ cluster_centre[:,:,keep_channels] = np.transpose(np.reshape(kmeans.cluster_centers_[cluster_nr,:],(2,patch_size,patch_size)),(1,2,0))
+ else:
+ cluster_centre = np.transpose((np.reshape(kmeans.cluster_centers_[cluster_nr,:],(3,patch_size,patch_size))),(1,2,0))
+ cluster_centre = ma.rgb2cmy(cluster_centre)
+ if y0>0:
+ ab = AnnotationBbox(OffsetImage(cluster_centre.astype(max_img.dtype)), (x0, y0), frameon=False)
+ ax_control.add_artist(ab)
+ else:
+ ab = AnnotationBbox(OffsetImage(cluster_centre.astype(max_img.dtype)), (x0, -y0), frameon=False)
+ ax_disease.add_artist(ab)
+
+ plt.figure(fig_control.number)
+ plt.savefig(dir_probs + 'controlClustercenters_2Dspace_'+protein_mode+'_channel'+str(drop_channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+
+ plt.figure(fig_disease.number)
+ plt.savefig(dir_probs + 'diseaseClustercenters_2Dspace_'+protein_mode+'_channel'+str(drop_channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Plot all clusters sorted by IMPORTANCE
+ clusters_control_occW_sorted = [np.arange(0,100)[i] for i in np.argsort(importance.flatten()) if (importance.flatten())[i]>0]
+ clusters_control_occW_sorted.reverse()
+
+ clusters_disease_occW_sorted = [np.arange(0,100)[i] for i in np.argsort(importance.flatten()) if (importance.flatten())[i]<0]
+
+ clusters_occW_sorted = [np.arange(0,100)[i] for i in np.argsort(importance.flatten())]
+ clusters_occW_sorted.reverse()
+
+ occurrence_ratio_sorted = [occurrence_ratio[i] for i in np.argsort(importance.flatten())]
+ occurrence_ratio_sorted.reverse()
+
+ ma.plot_grid_cluster_centers(kmeans.cluster_centers_, clusters_occW_sorted, patch_size, protein_mode, colour_mode, drop_channel)
+ plt.suptitle('Cluster centers \n sorted by occ*f(p) from control to disease with cluster ' + str(len(clusters_control_occW_sorted)) + ' in between')
+ plt.savefig(dir_probs + 'clustercenters_occW_sorted_'+protein_mode+'_channel'+str(drop_channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Compute probability images
+
+ startTime_probs = datetime.now()
+
+ #Assign probabilities of control and disease to clusters
+ prob_control= hist_control/(hist_control+hist_disease)
+ prob_disease = hist_disease/(hist_control+hist_disease)
+
+ #Compute probability images
+ r,c,_ = max_img_list_control_flat[0].shape
+ img_shape = (int(r*sc_fac),int(c*sc_fac))
+ prob_img_list_control = ma.comp_prob_imgs(prob_control,assignment_list_control,img_shape)
+ prob_img_list_disease = ma.comp_prob_imgs(prob_control,assignment_list_disease,img_shape)
+
+ #%% Display probability images and accumulate probs per frame and sample
+ nr_frames = len(max_img_list_control[0])
+
+ #control
+ sample_names_control = ma.listdir_custom(dir_in + control, string_start_pos = -4, dir_flag = True, )
+
+ nr_list = 0
+ prob_list_control= []
+ fig_probmaps, ax_probmaps = plt.subplots(nr_frames, len(sample_names_control),figsize = (len(sample_names_control),nr_frames), sharex=True, sharey=True)
+ for max_img_list_sample,sample_name,sample_nr in zip(max_img_list_control, sample_names_control, range(len(sample_names_control))): #in general, in max_img_list_condition
+ print(sample_name)
+ nr_frames = len(max_img_list_sample)
+ prob_img_list_sample = prob_img_list_control[nr_list:nr_list+nr_frames]
+ nr_list += nr_frames
+
+ prob_sample = np.zeros((nr_frames,1))
+ fig, axs = plt.subplots(2,nr_frames, figsize = (nr_frames*2,2*2), sharex=True, sharey=True)
+ for prob_img, max_img, frame_nr in zip(prob_img_list_sample,max_img_list_sample,range(nr_frames)):
+ max_img_ds = skimage.transform.rescale(max_img, sc_fac, multichannel = True, preserve_range = True).astype(max_img.dtype)
+
+ #compute image probability
+ prob_frame = sum(sum(prob_img))/prob_img.size
+
+ #background
+ ind_back = np.ones((max_img_ds.shape[0],max_img_ds.shape[1]), dtype = bool)
+ for channel in range(max_img_ds.shape[2]):
+ ind_back = ind_back & (max_img_ds[:,:,channel]<th_back)
+ prob_img[ind_back] = 0.5
+
+ if protein_mode == 'dropout':
+ max_img_ds_3c = np.zeros((max_img_ds.shape[0],max_img_ds.shape[1],3))
+ max_img_ds_3c[:,:,keep_channels] = max_img_ds
+ max_img_ds = max_img_ds_3c
+
+ #plot
+ ax_probmaps[frame_nr][sample_nr].imshow(1-prob_img, cmap=plt.cm.bwr, vmin = 0, vmax = 1)
+ ax_probmaps[frame_nr][sample_nr].xaxis.set_ticklabels([])
+ ax_probmaps[frame_nr][sample_nr].xaxis.set_ticks_position('none')
+ ax_probmaps[frame_nr][sample_nr].yaxis.set_ticklabels([])
+ ax_probmaps[frame_nr][sample_nr].yaxis.set_ticks_position('none')
+
+ axs[1][frame_nr].imshow(1-prob_img, cmap=plt.cm.bwr, vmin = 0, vmax = 1)
+ axs[1][frame_nr].set_title("%.2f" % np.round(prob_frame,2))
+ axs[1][frame_nr].xaxis.set_ticklabels([])
+ axs[1][frame_nr].xaxis.set_ticks_position('none')
+ axs[1][frame_nr].yaxis.set_ticklabels([])
+ axs[1][frame_nr].yaxis.set_ticks_position('none')
+
+ if colour_mode == 'cmy':
+ max_img_ds = ma.rgb2cmy(max_img_ds)
+ axs[0][frame_nr].imshow(max_img_ds)
+ axs[0][frame_nr].xaxis.set_ticklabels([])
+ axs[0][frame_nr].xaxis.set_ticks_position('none')
+ axs[0][frame_nr].yaxis.set_ticklabels([])
+ axs[0][frame_nr].yaxis.set_ticks_position('none')
+
+ #compute accumulated probabilities:part 2
+ prob_sample[frame_nr] = prob_frame
+ prob_list_control += [prob_sample]
+
+ plt.suptitle('Health probability of sample '+ sample_name +', avg:' + str(round(prob_sample.mean(),2))+ ' std:' + str(round(prob_sample.std(),2)))
+ plt.savefig(dir_probs + '/' + control + '/' + 'probImHealth_'+sample_name+'_'+protein_mode+'_channel'+str(drop_channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+ plt.figure(fig_probmaps.number),plt.savefig(dir_probs + '/' + control + '/' + 'probMaps_'+control+'_'+protein_mode+'_channel'+str(drop_channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+
+ #disease
+ sample_names_disease = ma.listdir_custom(dir_in + disease, string_start_pos = -4, dir_flag = True)
+
+ nr_list = 0
+ prob_list_disease = []
+ fig_probmaps, ax_probmaps = plt.subplots(nr_frames, len(sample_names_disease),figsize = (len(sample_names_disease),nr_frames), sharex=True, sharey=True)
+ for max_img_list_sample,sample_name,sample_nr in zip(max_img_list_disease, sample_names_disease, range(len(sample_names_disease))): #in general, in max_img_list_condition
+ print(sample_name)
+ nr_frames = len(max_img_list_sample)
+ prob_img_list_sample = prob_img_list_disease[nr_list:nr_list+nr_frames]
+ nr_list += nr_frames
+
+ prob_sample = np.zeros((nr_frames,1))
+ fig, axs = plt.subplots(2,nr_frames, figsize = (nr_frames*2,2*2), sharex=True, sharey=True)
+ for prob_img, max_img, frame_nr in zip(prob_img_list_sample,max_img_list_sample,range(nr_frames)):
+ max_img_ds = skimage.transform.rescale(max_img, sc_fac, multichannel = True, preserve_range = True).astype(max_img.dtype)
+
+ #compute image probability
+ prob_frame = sum(sum(prob_img))/prob_img.size
+
+ #background
+ ind_back = np.ones((max_img_ds.shape[0],max_img_ds.shape[1]), dtype = bool)
+ for channel in range(max_img_ds.shape[2]):
+ ind_back = ind_back & (max_img_ds[:,:,channel]<th_back)
+ prob_img[ind_back] = 0.5
+
+ if protein_mode == 'dropout':
+ max_img_ds_3c = np.zeros((max_img_ds.shape[0],max_img_ds.shape[1],3))
+ max_img_ds_3c[:,:,keep_channels] = max_img_ds
+ max_img_ds = max_img_ds_3c
+
+ #plot
+ ax_probmaps[frame_nr][sample_nr].imshow(1-prob_img, cmap=plt.cm.bwr, vmin = 0, vmax = 1)
+ ax_probmaps[frame_nr][sample_nr].xaxis.set_ticklabels([])
+ ax_probmaps[frame_nr][sample_nr].xaxis.set_ticks_position('none')
+ ax_probmaps[frame_nr][sample_nr].yaxis.set_ticklabels([])
+ ax_probmaps[frame_nr][sample_nr].yaxis.set_ticks_position('none')
+
+ axs[1][frame_nr].imshow(1-prob_img, cmap=plt.cm.bwr, vmin = 0, vmax = 1)
+ axs[1][frame_nr].set_title("%.2f" % np.round(prob_frame,2))
+ axs[1][frame_nr].xaxis.set_ticklabels([])
+ axs[1][frame_nr].xaxis.set_ticks_position('none')
+ axs[1][frame_nr].yaxis.set_ticklabels([])
+ axs[1][frame_nr].yaxis.set_ticks_position('none')
+
+ if colour_mode == 'cmy':
+ max_img_ds = ma.rgb2cmy(max_img_ds)
+ axs[0][frame_nr].imshow(max_img_ds)
+ axs[0][frame_nr].xaxis.set_ticklabels([])
+ axs[0][frame_nr].xaxis.set_ticks_position('none')
+ axs[0][frame_nr].yaxis.set_ticklabels([])
+ axs[0][frame_nr].yaxis.set_ticks_position('none')
+
+ #compute accumulated probabilities:part 2
+ prob_sample[frame_nr] = prob_frame
+ prob_list_disease += [prob_sample]
+
+ plt.suptitle('Health probability of sample '+ sample_name +', avg:' + str(round(prob_sample.mean(),2))+ ' std:' + str(round(prob_sample.std(),2)))
+ plt.savefig(dir_probs + '/' + disease + '/' + 'probImHealth_'+sample_name+'_'+protein_mode+'_channel'+str(drop_channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+ plt.figure(fig_probmaps.number),plt.savefig(dir_probs + '/' + disease + '/' + 'probMaps_'+disease+'_'+protein_mode+'_channel'+str(drop_channel)+'_%dclusters_%ddownscale_%dpatchsize.png'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+ #plt.show()
+
+ prob_list_samples = prob_list_control + prob_list_disease
+
+ #%% Box plots
+ fig, ax = plt.subplots(figsize = (15,5))
+ ax.set_title('Image health scores ')
+
+ sample_sep = 0.85
+ patient_sep = 2.35
+ condition_sep = 2.5
+ positions_control = np.tile([0,0+sample_sep],int(len(max_img_list_control)/2)) + np.repeat(np.arange(0,len(max_img_list_control)/2*patient_sep,patient_sep),2)
+ positions_disease = np.tile([len(max_img_list_control)+condition_sep,len(max_img_list_control)+condition_sep+sample_sep],int(len(max_img_list_disease)/2)) + np.repeat(np.arange(0,len(max_img_list_disease)/2*patient_sep,patient_sep),2)
+ array_probs = np.squeeze(np.asarray(prob_list_samples))
+ bp_control = ax.boxplot((array_probs[:len(max_img_list_control),...]).T, positions = positions_control, patch_artist = True)
+ bp_disease = ax.boxplot(array_probs[len(max_img_list_control):,...].T, positions = positions_disease, patch_artist = True)
+
+ for element in ['boxes', 'whiskers', 'fliers', 'means', 'medians', 'caps']:
+ plt.setp(bp_control[element], color='blue')
+ plt.setp(bp_control[element], linewidth = 2)
+
+ for patch in bp_control['boxes']:
+ patch.set(facecolor='white')
+ patch.set(linewidth = 2)
+
+ for element in ['boxes','whiskers', 'fliers', 'means', 'medians', 'caps']:
+ plt.setp(bp_disease[element], color='red')
+ plt.setp(bp_disease[element], linewidth = 2)
+
+ for patch in bp_disease['boxes']:
+ patch.set(facecolor= 'white')
+ patch.set(linewidth = 2)
+ #patch.set(alpha=0.2)
+
+ ax.set_xticklabels([i[-4:] for i in sample_names_control]+[i[-4:] for i in sample_names_disease])
+ ax.set_ylim(0.3,0.7)
+ ax.set_ylabel('Probability health')
+ #ax.set_ylim(0,1)
+
+ ax.tick_params(
+ axis='both', # changes apply to the x-axis
+ which='both', # both major and minor ticks are affected
+ #labelleft = False, # ticks along the top edge are off
+ #labelbottom=False
+ )
+
+ #plot points
+ positions = list(positions_control) + list(positions_disease)
+ for prob_sample, position in zip(prob_list_samples, positions):
+ print(prob_sample)
+ print(5*[position])
+ # Add some random "jitter" to the x-axis
+ x = np.random.normal(5*[position], 0)
+ #plot(x, y, 'r.', alpha=0.2)
+ ax.plot(x, prob_sample,'k.', markersize = 6, zorder = 3)
+
+ #horizontal line
+ x = np.linspace(0,position.max())
+ y = 0.5*np.ones(x.shape)
+ ax.plot(x,y,'k',linewidth = 1, linestyle = 'dashed')
+
+ plt.savefig(dir_probs + 'scatterBoxplots_'+protein_mode+'_channel'+str(drop_channel) +'_%dclusters_%ddownscale_%dpatchsize.pdf'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ fig2, ax2 = plt.subplots(figsize = (15,5))
+ #plot points
+ for prob_sample, position in zip(prob_list_control, positions_control):
+ ax2.plot(5*[position], prob_sample,'b.', markersize = 5)
+
+ for prob_sample, position in zip(prob_list_disease, positions_disease):
+ ax2.plot(5*[position], prob_sample,'r.', markersize = 5)
+
+ #horizontal line
+ x = np.linspace(0,position.max())
+ y = 0.5*np.ones(x.shape)
+ ax2.plot(x,y,'k',linewidth = 1, linestyle = 'dashed')
+
+ ax2.set_xticks(ax.get_xticks())
+ ax2.set_xticklabels([i[-4:] for i in sample_names_control]+[i[-4:] for i in sample_names_disease])
+ ax2.set_ylim(0.3,0.7)
+ ax2.set_ylabel('Probability health')
+
+ plt.savefig(dir_probs + 'samplewiseScatterPlots_'+protein_mode+'_channel'+str(drop_channel) +'_%dclusters_%ddownscale_%dpatchsize.pdf'%(nr_clusters,1/sc_fac,patch_size), dpi=300)
+
+ #%% Print computational time
+ endTime = datetime.now() - startTime
+ print(endTime)
+
+
+ #%%ANOVA SETUP
+ os.makedirs(dir_results + 'statistics/', exist_ok = True)
+
+ from statsmodels.formula.api import ols
+ from statsmodels.stats.anova import anova_lm
+ import pandas as pd
+ from patsy import ModelDesc
+
+ nr_frames_patient = 10
+ img_nr = np.tile(np.arange(nr_frames),array_probs.size//nr_frames)
+ sample_nr = np.tile(np.repeat(np.arange(nr_frames_patient//nr_frames),nr_frames),array_probs.size//nr_frames_patient)
+ patient_nr = np.concatenate([np.repeat(np.arange(len(max_img_list_control_flat)//nr_frames_patient),nr_frames_patient),np.repeat(np.arange(len(max_img_list_disease_flat)//nr_frames_patient),nr_frames_patient)])
+ condition_nr = np.concatenate([np.zeros(len(max_img_list_control_flat)), np.ones(len(max_img_list_disease_flat))])
+
+
+ dep_var = array_probs.flatten()
+
+ data_dict = {'Image':img_nr,
+ 'Sample':sample_nr,
+ 'Patient':patient_nr,
+ 'Condition': condition_nr,
+ 'S':dep_var}
+
+ formula = 'S ~ C(Condition)/C(Patient)/C(Sample)'
+
+ desc = ModelDesc.from_formula(formula)
+ print(desc.describe())
+
+ lm = ols(formula, data_dict).fit()
+ print(lm.summary())
+
+ anova_table = anova_lm(lm)
+ print(anova_table)
+ writer_anova = pd.ExcelWriter(dir_results + 'statistics/statistics_anova_' + disease + '_'+protein_mode+'_channel'+str(drop_channel)+'_ds'+ str(1/sc_fac) +'_run' + str(run) +'.xlsx', engine='xlsxwriter')
+ anova_table.to_excel(writer_anova,sheet_name = disease)
+ writer_anova.save()
+
+
diff --git a/code/lung_lif_to_png.py b/code/lung_lif_to_png.py
new file mode 100644
index 0000000..d61120c
--- /dev/null
+++ b/code/lung_lif_to_png.py
@@ -0,0 +1,51 @@
+#!/usr/bin/env python3
+# -*- coding: utf-8 -*-
+"""
+Transform lif files to png for easy access and visualization
+"""
+
+
+import numpy as np
+from readlif.reader import LifFile
+import skimage.io
+import os
+import microscopy_analysis as ma
+
+#%%
+# folder containing lif files
+in_dir = '../../../covidPatients/scans/' #data101220/'# data_October2020/sarcoidosis/' #'../../5April2020_control/' #healthy #'../../24December19_emphysema/' #sick
+# output folder
+out_dir = '../data_corrected_bis/covid/' #control/ #/emphysema/
+file_names = ma.listdir_custom(in_dir, string_start_pos = -7, ext = 'lif')
+
+# make output directory if it does not exist
+if not os.path.exists(out_dir):
+ os.mkdir(out_dir)
+
+# run through all lif files and save as png for easy access and visualization
+for file in file_names:
+ print(file)
+ lif_in = LifFile(in_dir + file)
+ n_im = lif_in.num_images
+ dir_name_out = (out_dir + (file.split('.')[0]).replace(' ', '_') + '/')
+ if not os.path.exists(dir_name_out):
+ os.mkdir(dir_name_out)
+ for n in range(0,n_im):
+ print(n)
+ im_in = lif_in.get_image(n)
+ r,c,n_frames = im_in.dims[:3]
+ n_ch = im_in.channels
+ if (n_ch == 3)|(n_ch==4): #TODO: Fix support for removing channel 1 in covids. Could be generalised to select channels of interest, but we don't need this functionality now
+ dir_name_out_im = dir_name_out + ('frame_%02d' % n)
+ if not os.path.exists(dir_name_out_im):
+ os.mkdir(dir_name_out_im)
+ im_out = np.zeros((r,c,3), dtype='uint8')
+ for i in range(0,n_frames):
+ im_out[:,:,0] = np.array(im_in.get_frame(i,0,0)).transpose()
+ for j in range(1,3):
+ if n_ch == 4: #support for removing channel 1 in covids.
+ im_out[:,:,j] = np.array(im_in.get_frame(i,0,j+1)).transpose()
+ else:
+ im_out[:,:,j] = np.array(im_in.get_frame(i,0,j)).transpose()
+ skimage.io.imsave(dir_name_out_im + '/image_%02d.png' % i, im_out)
+
diff --git a/code/microscopy_analysis.py b/code/microscopy_analysis.py
new file mode 100755
index 0000000..394ea5f
--- /dev/null
+++ b/code/microscopy_analysis.py
@@ -0,0 +1,624 @@
+#!/usr/bin/env python3
+# -*- coding: utf-8 -*-
+"""
+Helping functions for analysis of lif microscopy images
+
+Developed by monj@dtu.dk and abda@dtu.dk
+"""
+
+import os
+import skimage.io as io
+import skimage.transform
+import numpy as np
+import local_features as lf
+
+import matplotlib.pyplot as plt
+from math import ceil
+
+#%% General functions
+
+# sort list of directories from selected string position
+def sort_strings_customStart(strings, string_start_pos = 0):
+ """Sorts list of strings
+
+ Input(s)
+ strings: list of strings
+ string_start_pos (default 0): defines the section of the string on which the ordering is based.
+
+ Output(s)
+ strings_sortered: list of sorted strings
+
+ Author: Monica Jane Emerson (monj@dtu.dk)"""
+
+ strings_roi = [i[string_start_pos:] for i in strings]
+ #key indicates that the indices (k) are sorted according to subjects_list[k]
+ ind = np.array(sorted(range(len(strings_roi)), key=lambda k: strings_roi[k]))
+ strings_sorted = [strings[i] for i in ind]
+
+ return strings_sorted
+
+#Provide the contents of a directory sorted
+def listdir_custom(directory, string_start_pos = 0, ext = '', dir_flag = False, base_name = False):
+ 'monj@dtu.dk'
+
+ if dir_flag:
+ if base_name:
+ list_dirs = [dI for dI in os.listdir(directory) if (os.path.isdir(os.path.join(directory,dI)) & dI[0:len(base_name)]==base_name)]
+ else:
+ list_dirs = [dI for dI in os.listdir(directory) if os.path.isdir(os.path.join(directory,dI))]
+ else:
+ if base_name:
+ list_dirs = [dI for dI in os.listdir(directory)if dI[0:len(base_name)]==base_name]
+ else:
+ if ext == '':
+ list_dirs = [f for f in os.listdir(directory) if f.endswith(ext)]
+ else:
+ list_dirs = [dI for dI in os.listdir(directory)]
+
+ listdirs_sorted = sort_strings_customStart(list_dirs,string_start_pos)
+
+ return listdirs_sorted
+
+def flatten_list(list):
+ 'monj@dtu.dk'
+
+ list_flat = [item for sublist in list for item in sublist]
+
+ return list_flat
+
+#%% IO functions
+
+#make directory, and subdirectories within, if they don't exist
+def make_output_dirs(directory,subdirectories = False):
+ 'monj@dtu.dk'
+
+ os.makedirs(directory, exist_ok = True)
+
+ if subdirectories:
+ for subdir in subdirectories:
+ os.makedirs(directory + subdir + '/', exist_ok = True)
+
+ # def make_output_dirs(directory,subdirectories):
+ # 'monj@dtu.dk'
+
+ # os.makedirs(directory, exist_ok = True)
+ # os.makedirs(directory + 'control/', exist_ok = True)
+
+ # for disease in diseases:
+ # os.makedirs(directory + disease + '/', exist_ok = True)
+
+
+#Reads images starting with base_name from the subdirectories of the input directory.
+#Option for reading scaled down versions and in bnw or colour
+def read_max_imgs(dir_condition, base_name, sc_fac = 1, colour_mode = 'colour'):
+ 'monj@dtu.dk'
+
+ sample_list = listdir_custom(dir_condition, string_start_pos = -4, dir_flag = True)
+ #print(sample_list)
+
+ max_img_list = []
+ for sample in sample_list:
+ sample_dir = dir_condition + '/' + sample + '/'
+ frame_list = listdir_custom(sample_dir, base_name = base_name)
+ #print(frame_list)
+
+ frame_img_list = []
+ for frame in frame_list:
+ frame_path = sample_dir + frame
+
+ #Option to load in bnw or colour
+ if colour_mode == 'bnw':
+ img = io.imread(frame_path, as_gray = True).astype('uint8')
+ if sc_fac ==1:
+ frame_img_list += [img]
+ else:
+ frame_img_list += [skimage.transform.rescale(img, sc_fac, preserve_range = True).astype('uint8')]
+ else:
+ img = io.imread(frame_path).astype('uint8')
+ if sc_fac == 1:
+ frame_img_list += [img]
+ else:
+ frame_img_list += [skimage.transform.rescale(img, sc_fac, preserve_range = True, multichannel=True).astype('uint8')]
+
+ max_img_list += [frame_img_list]
+ #print(frame_img_list[0].dtype)
+
+ return max_img_list
+
+#%% Functions for intensity inspection and image preprocessing
+
+# computes the maximum projection image from a directory of images
+def get_max_img(in_dir, ext = '.png', n_img = 0):
+ file_names = [f for f in os.listdir(in_dir) if f.endswith(ext)]
+ file_names.sort()
+ if ( n_img < 1 ):
+ n_img = len(file_names)
+ img_in = io.imread(in_dir + file_names[0])
+ for i in range(1,n_img):
+ img_in = np.maximum(img_in, io.imread(in_dir + file_names[i]))
+ return img_in
+
+# computes a list of maximum projection images from a list of directories
+def compute_max_img_list(in_dir, ext, base_name, dir_out = ''):
+ """by abda
+ Modified by monj"""
+
+ dir_list = [dI for dI in os.listdir(in_dir) if (os.path.isdir(os.path.join(in_dir,dI)) and dI[0:len(base_name)]==base_name)]
+ dir_list.sort()
+
+ max_img_list = []
+ for d in dir_list:
+ image_dir_in = in_dir + d + '/'
+ max_img = get_max_img(image_dir_in, ext)
+ if dir_out!='':
+ os.makedirs(dir_out, exist_ok = True)
+ io.imsave(dir_out + d + '.png', max_img.astype('uint8'))
+
+ max_img_list += [max_img]
+
+ return max_img_list
+
+# One more level up from compute_max_img_list. Computes a list of lists of maximum
+#projection images from a list of directories, each containing a list of directories
+#with the set of images that should be combined into a maximum projection.
+def comp_max_imgs(dir_condition, base_name, dir_out = ''):
+ 'monj@dtu.dk'
+
+ dir_list_condition = listdir_custom(dir_condition, string_start_pos = -4, dir_flag = True)
+
+ max_img_list_condition = []
+ for directory in dir_list_condition:
+ dir_in = dir_condition + '/' + directory + '/'
+
+ if dir_out!= '':
+ subdir_out = dir_in.replace(dir_condition,dir_out)
+ else:
+ subdir_out = ''
+
+ max_img_condition = compute_max_img_list(dir_in, '.png', base_name, subdir_out)
+ max_img_list_condition+= [max_img_condition]
+
+ return max_img_list_condition
+
+
+def comp_std_perChannel(max_im_list, th_int, dir_condition):
+ 'monj@dtu.dk'
+
+ sample_list = listdir_custom(dir_condition, string_start_pos = -4, dir_flag = True)
+
+ std_list = [[],[],[]]
+ for sample, frame_img_list in zip(sample_list, max_im_list):
+
+ for ind,img in enumerate(frame_img_list):
+ h, w, channels = img.shape
+
+ for channel in range(0,channels):
+ intensities = img[:,:,channel].ravel()
+ std_list[channel] += [(intensities[intensities>th_int]).std()]
+
+ return std_list
+
+def intensity_spread_normalisation(img_list, th_int, mean_std, dir_condition, base_name, dir_results):
+ 'monj@dtu.dk'
+ sample_list = listdir_custom(dir_condition, string_start_pos = -4, dir_flag = True)
+
+ img_corr_list = []
+ for sample, sample_imgs in zip(sample_list, img_list):
+ sample_dir = dir_condition + '/' + sample + '/'
+ #print(sample_dir)
+ frame_list = listdir_custom(sample_dir, base_name = base_name)
+ #print(frame_list)
+ frame_img_corr_list = []
+ for frame,img in zip(frame_list,sample_imgs):
+ h, w, channels = img.shape
+ img_corr = img
+ for channel in range(0,channels):
+ img_channel = img_corr[:,:,channel]
+ img_channel[img_channel>th_int] = img_channel[img_channel>th_int]*(mean_std/img_channel.std())
+ img_corr[:,:,channel] = img_channel
+
+ frame_img_corr_list += [img_corr]
+ os.makedirs(dir_results + '/' + sample, exist_ok = True)
+ io.imsave(dir_results + '/' + sample + '/' + frame +'.png', img_corr)
+
+ img_corr_list += [frame_img_corr_list]
+
+ return img_corr_list
+
+def rgb2cmy_list(img_list, dir_condition, base_name, dir_results):
+ 'monj@dtu.dk'
+ sample_list = listdir_custom(dir_condition, string_start_pos = -4, dir_flag = True)
+
+ for sample, sample_imgs in zip(sample_list, img_list):
+ sample_dir = dir_condition + '/' + sample + '/'
+ #print(sample_dir)
+ frame_list = listdir_custom(sample_dir, base_name = base_name)
+ #print(frame_list)
+ for frame,img in zip(frame_list,sample_imgs):
+ img_cmy = rgb2cmy(img)
+
+ os.makedirs(dir_results + '/' + sample, exist_ok = True)
+ io.imsave(dir_results + '/' + sample + '/' + frame +'.png', img_cmy)
+
+
+#def plotHist_perChannel_imgset
+def plotHist_perChannel_imgset_list(max_img_list, dir_condition, dir_results = '', name_tag = 'original'):
+ 'monj@dtu.dk'
+
+ sample_list = listdir_custom(dir_condition, string_start_pos = -4, dir_flag = True)
+
+ for sample, frame_img_list in zip(sample_list, max_img_list):
+ fig, axs = plt.subplots(4,len(frame_img_list), figsize = (len(frame_img_list)*2,4*2))
+ plt.suptitle('Sample '+sample[-4:] + ', acq. date: ' + sample[:6])
+
+ for ind,img in enumerate(frame_img_list):
+ h, w, channels = img.shape
+ axs[0][ind].imshow(img)
+
+ for channel in range(0,channels):
+ intensities = img[:,:,channel].ravel()
+ axs[channel+1][ind].hist(intensities, bins = 50)
+ axs[channel+1][ind].set_aspect(1.0/axs[channel+1][ind].get_data_ratio())
+
+ if dir_results!= '':
+ plt.savefig(dir_results + '/' + sample[-4:] + '_' + name_tag + '_perChannelHistograms.png', dpi = 300)
+ plt.close(fig)
+
+#def compare_imgpairs
+def compare_imgpairs_list(list1_imgsets, list2_imgsets, dir_condition, dir_results = ''):
+ 'monj@dtu.dk'
+
+ sample_list = listdir_custom(dir_condition, string_start_pos = -4, dir_flag = True)
+
+ for imgset1, imgset2, sample in zip(list1_imgsets, list2_imgsets, sample_list):
+ fig, axs = plt.subplots(2,len(imgset1), figsize = (2*len(imgset1),2*2),sharex=True,sharey=True)
+ plt.suptitle('Sample '+sample[-4:] + ', acq. date: ' + sample[:6])
+
+ for ind, img1, img2 in zip(range(0,len(imgset1)), imgset1, imgset2):
+ axs[0][ind].imshow(img1)
+ axs[1][ind].imshow(img2)
+
+ if dir_results!= '':
+ plt.savefig(dir_results + '/' + sample[-4:] + '_originalVScorrected.png', dpi=300)
+ plt.close(fig)
+
+def black2white_background(im_n):
+ """
+
+ Parameters
+ ----------
+ im_n : Normalized RGB image (intensities in range [0,1] of type float)
+
+ Returns
+ -------
+ im_conv : Normalized RGB image where black has been converted to white (or visa versa of type float)
+
+ abda@dtu.dk
+ """
+ im_conv = np.zeros((im_n.shape[:2]+(3,)))
+ im_conv[:,:,0] = (1-im_n[:,:,1])*(1-im_n[:,:,2]) + im_n[:,:,0]*np.maximum(im_n[:,:,1],im_n[:,:,2])
+ im_conv[:,:,1] = (1-im_n[:,:,0])*(1-im_n[:,:,2]) + im_n[:,:,1]*np.maximum(im_n[:,:,0],im_n[:,:,2])
+ im_conv[:,:,2] = (1-im_n[:,:,0])*(1-im_n[:,:,1]) + im_n[:,:,2]*np.maximum(im_n[:,:,0],im_n[:,:,1])
+ return im_conv
+
+def rgb2cmy(img):
+ 'monj@dtu.dk'
+ img_n = img.astype(float)/255 #normalise
+ img_conv_n = black2white_background(img_n)
+ img_conv = (img_conv_n*255).astype('uint8') #denormalise
+ return 255-img_conv
+
+#%%Functions for feature analysis and visualisation
+
+# computes the max projection image and the features into a list
+def compute_max_img_feat(in_dir, ext, base_name, sigma, sc_fac, save = False, abs_intensity = True):
+ dir_list = [dI for dI in os.listdir(in_dir) if (os.path.isdir(os.path.join(in_dir,dI)) and dI[0:len(base_name)]==base_name)]
+ dir_list.sort()
+ max_img_list = []
+ for d in dir_list:
+ image_dir = in_dir + d + '/'
+ max_img = get_max_img(image_dir, ext)
+
+ if save:
+ dir_name_out_img = in_dir.replace('data','maxProjImages')
+ os.makedirs(dir_name_out_img, exist_ok = True)
+
+ io.imsave( dir_name_out_img + d + '.png', max_img.astype('uint8'))
+
+ max_img_list += [skimage.transform.rescale(max_img, sc_fac, multichannel=True)]
+
+ feat_list = []
+ for max_img in max_img_list:
+ r,c = max_img.shape[:2]
+ feat = np.zeros((r*c,45))
+ for i in range(0,3):
+ feat_tmp = lf.get_gauss_feat_im(max_img[:,:,i], sigma)
+ feat[:,i*15:(i+1)*15] = feat_tmp.reshape(-1,feat_tmp.shape[2])
+ if not(abs_intensity):
+ feat_list += [np.concatenate((feat[:,1:15],feat[:,16:30],feat[:,31:45]),axis = 1)]
+ else:
+ feat_list += [feat]
+ return max_img_list, feat_list
+
+
+# computes a histogram of features from a kmeans object
+def compute_assignment_hist(feat_list, kmeans, background_feat = None):
+ assignment_list = []
+ for feat in feat_list:
+ assignment_list += [kmeans.predict(feat)]
+ edges = np.arange(kmeans.n_clusters+1)-0.5 # histogram edges halfway between integers
+ hist = np.zeros(kmeans.n_clusters)
+ for a in assignment_list:
+ hist += np.histogram(a,bins=edges)[0]
+
+ sum_hist = np.sum(hist)
+ hist/= sum_hist
+
+ if background_feat is not None:
+ assignment_back = kmeans.predict(background_feat)
+ hist_back = np.histogram(assignment_back,bins=edges)[0]
+ return hist, assignment_list, hist_back, assignment_back
+ else:
+ return hist, assignment_list
+
+
+# image to array of patches
+def im2col(A, BSZ, stepsize=1, norm=False):
+ # Parameters
+ m,n = A.shape
+ s0, s1 = A.strides
+ nrows = m-BSZ[0]+1
+ ncols = n-BSZ[1]+1
+ shp = BSZ[0],BSZ[1],nrows,ncols
+ strd = s0,s1,s0,s1
+
+ out_view = np.lib.stride_tricks.as_strided(A, shape=shp, strides=strd)
+ out_view_shaped = out_view.reshape(BSZ[0]*BSZ[1],-1)[:,::stepsize]
+ if norm:
+ one_norm = np.sum(out_view_shaped,axis=0)
+ ind_zero_norm = np.where(one_norm !=0)
+ out_view_shaped[:,ind_zero_norm] = 255*out_view_shaped[:,ind_zero_norm]/one_norm[ind_zero_norm]
+ return out_view_shaped
+
+# nd image to array of patches
+def ndim2col(A, BSZ, stepsize=1, norm=False):
+ if(A.ndim == 2):
+ return im2col(A, BSZ, stepsize, norm)
+ else:
+ r,c,l = A.shape
+ patches = np.zeros((l*BSZ[0]*BSZ[1],(r-BSZ[0]+1)*(c-BSZ[1]+1)),dtype=A.dtype)
+ for i in range(l):
+ patches[i*BSZ[0]*BSZ[1]:(i+1)*BSZ[0]*BSZ[1],:] = im2col(A[:,:,i],BSZ,stepsize,norm)
+ return patches
+
+# nd image to array of patches with mirror padding along boundaries
+def ndim2col_pad(A, BSZ, stepsize=1, norm=False):
+ r,c = A.shape[:2]
+ if (A.ndim == 2):
+ l = 1
+ else:
+ l = A.shape[2]
+ tmp = np.zeros((r+BSZ[0]-1,c+BSZ[1]-1,l),dtype = A.dtype)
+ fhr = int(np.floor(BSZ[0]/2))
+ fhc = int(np.floor(BSZ[1]/2))
+ thr = int(BSZ[0]-fhr-1)
+ thc = int(BSZ[1]-fhc-1)
+
+ tmp[fhr:fhr+r,fhc:fhc+c,:] = A.reshape((r,c,l))
+ tmp[:fhr,:] = np.flip(tmp[fhr:fhr*2,:], axis=0)
+ tmp[fhr+r:,:] = np.flip(tmp[r:r+thr,:], axis=0)
+ tmp[:,:fhc] = np.flip(tmp[:,fhc:fhc*2], axis=1)
+ tmp[:,fhc+c:] = np.flip(tmp[:,c:c+thc], axis=1)
+ tmp = np.squeeze(tmp)
+ return ndim2col(tmp,BSZ,stepsize,norm)
+
+
+def patch2featvector(patches, colour_mode = 'bnw', colour_weight = 1):
+ patch_size = int((patches.shape[1]/3)**0.5)
+ patches_reshaped = patches.reshape(patches.shape[0],3,patch_size,patch_size)
+ patches_bnw = np.mean(patches_reshaped,axis = 1)
+
+ if colour_mode == 'bnw':
+ features = patches_bnw.reshape(patches.shape[0],patch_size**2)
+ else:
+ int_perchannel = np.mean(np.mean(patches_reshaped,axis = 2),axis = 2)
+ features = [patches_bnw.reshape(patches.shape[0],patch_size**2), colour_weight*int_perchannel]
+ return features
+
+def patch2featvector_list(list_patches, colour_mode = 'bnw', colour_weight = 1):
+ list_features = []
+ for patches in list_patches:
+ features = patch2featvector(patches, colour_mode, colour_weight)
+ list_features += [features]
+
+ return list_features
+
+def comp_prob_imgs(prob_control,assignment_list_condition, prob_img_shape):
+ #Compute probability images for condition
+ r,c = prob_img_shape
+ prob_img_list_condition = []
+ for assignment in assignment_list_condition:
+ prob = prob_control[assignment.astype(int)]
+ prob_img_list_condition += [prob.reshape(r,c)]
+ return prob_img_list_condition
+
+#%% Functions for visualisation of learnt features
+def plot_grid_cluster_centers(cluster_centers, cluster_order, patch_size, protein_mode = 'triple', colour_mode = 'rgb', channel = 'all', titles = False, occurrence = ''):
+ #grid dimensions
+ size_x = round(len(cluster_order)**(1/2))
+ size_y = ceil(len(cluster_order)/size_x)
+
+ #figure format
+ overhead = 0
+ if titles:
+ overhead = 1
+ w, h = plt.figaspect(size_x/size_y)
+ fig, axs = plt.subplots(size_x,size_y, figsize=(1.3*w,1.3*h*(1+overhead/2)), sharex=True, sharey=True)
+ #print('Grid size: ', grid_size[1], grid_size[2], 'Figure size: ', w, h)
+
+ int_sum_list = []
+ ax_list = axs.ravel()
+ for ind, cluster in enumerate(cluster_order):
+ #print(ind)
+ if colour_mode =='bnw':
+ cluster_centre = np.reshape(cluster_centers[cluster,:],(patch_size,patch_size))
+ ax_list[ind].imshow(cluster_centre.astype('uint8'),cmap='gray')
+ else:
+ if protein_mode == 'single': #in bnw + colour give the clusters a uniform colour
+ cluster_centre = np.zeros((patch_size,patch_size,3),dtype = cluster_centers.dtype)
+ cluster_centre[:,:,channel] = np.reshape(cluster_centers[cluster,:],(patch_size,patch_size))
+
+ elif protein_mode == 'dropout':
+ cluster_centre = np.zeros((patch_size,patch_size,3),dtype = cluster_centers.dtype)
+ keep_channels = [x for x in range(3) if x!=channel]
+ cluster_centre[:,:,keep_channels] = np.transpose(np.reshape(cluster_centers[cluster,:],(2,patch_size,patch_size)),(1,2,0))
+
+ else:
+ cluster_centre = np.transpose(np.reshape(cluster_centers[cluster,:],(3,patch_size,patch_size)),(1,2,0))
+ if channel!='all':
+ remove_channels = [x for x in range(3) if x!=channel]
+ cluster_centre[:,:,remove_channels] = np.zeros((patch_size,patch_size,2),dtype = cluster_centers.dtype)
+
+ if colour_mode == 'cmy':
+ cluster_centre = rgb2cmy(cluster_centre)
+ ax_list[ind].imshow(cluster_centre.astype('uint8'))
+
+ if titles:
+ if occurrence !='':
+ ax_list[ind].set_title(round(occurrence[ind],2))
+ else:
+ ax_list[ind].set_title(cluster)
+
+ if colour_mode =='bnw':
+ int_sum_list += [sum((cluster_centre).ravel())]
+ else:
+ int_sum_list += [sum((np.max(cluster_centre,2)).ravel())]
+
+ plt.setp(axs, xticks=[], yticks=[])
+ #plt.show()
+
+ return int_sum_list
+
+#%% Functions for channel statistics
+def smooth(y, box_pts):
+ box = np.ones(box_pts)/box_pts
+ y_smooth = np.convolve(y, box, mode='same')
+ return y_smooth
+
+def hist_outline(density, bins, axs, color = 'k', w = 1):
+ y,binEdges=np.histogram(density,bins=bins, density = True)
+ bincenters = 0.5*(binEdges[1:]+binEdges[:-1])
+ axs.plot(bincenters,smooth(y,w), color = color)
+
+def channel_corr_scatter(pixel_probs, axs, bins, lims = (0,1), w = 1, edgecolors='b', s=5):
+
+ hist_outline(pixel_probs[0],bins,axs[0][0], color = edgecolors, w = w)
+ axs[0][0].set_xlim(lims[0],lims[1])
+ axs[0][0].xaxis.set_label_position('top')
+ axs[0][0].set_xlabel('Colagen 4'),axs[0][0].set_ylabel('Colagen 4'),
+ axs[1][0].scatter(pixel_probs[0],pixel_probs[1], s=s, facecolors='none', edgecolors=edgecolors)
+ axs[1][0].set_xlim(lims[0],lims[1]), axs[1][0].set_ylim(lims[0],lims[1])
+ axs[1][0].set_ylabel('Fibrilar colagen')
+ axs[2][0].scatter(pixel_probs[0],pixel_probs[2], s=s, facecolors='none', edgecolors=edgecolors)
+ axs[2][0].set_xlim(lims[0],lims[1]), axs[2][0].set_ylim(lims[0],lims[1])
+ axs[2][0].set_ylabel('Elastin')
+
+ axs[0][1].scatter(pixel_probs[1],pixel_probs[0], s=s, facecolors='none', edgecolors=edgecolors)
+ axs[0][1].set_xlim(lims[0],lims[1]), axs[0][1].set_ylim(lims[0],lims[1])
+ axs[0][1].xaxis.set_label_position('top'), axs[0][1].set_xlabel('Fibrilar Colagen')
+ hist_outline(pixel_probs[1],bins,axs[1][1], color = edgecolors, w = w)
+ axs[1][1].set_xlim(lims[0],lims[1])
+ axs[2][1].scatter(pixel_probs[1],pixel_probs[2], s=s, facecolors='none', edgecolors=edgecolors)
+ axs[2][1].set_xlim(lims[0],lims[1]), axs[2][1].set_ylim(lims[0],lims[1])
+
+
+ axs[0][2].scatter(pixel_probs[2],pixel_probs[0], s=s, facecolors='none', edgecolors=edgecolors)
+ axs[0][2].set_xlim(lims[0],lims[1]), axs[0][2].set_ylim(lims[0],lims[1])
+ axs[0][2].xaxis.set_label_position('top'), axs[0][2].set_xlabel('Elastin')
+ axs[1][2].scatter(pixel_probs[2],pixel_probs[1], s=s, facecolors='none', edgecolors=edgecolors)
+ axs[1][2].set_xlim(lims[0],lims[1]), axs[1][2].set_ylim(lims[0],lims[1])
+ hist_outline(pixel_probs[2],bins,axs[2][2], color = edgecolors, w = w)
+ axs[2][2].set_xlim(lims[0],lims[1])
+
+def channel_corr_density(pixel_probs, lims = (0,1)):
+ fig, axs = plt.subplots(3,3, figsize = (6,6))
+
+ axs[0][0].hist(pixel_probs[0],density = True),
+ axs[0][0].set_xlim(lims[0],lims[1])
+ axs[0][0].xaxis.set_label_position('top')
+ axs[0][0].set_xlabel('Colagen 4'),axs[0][0].set_ylabel('Colagen 4'),
+ axs[1][0].hist2d(pixel_probs[0],pixel_probs[1])
+ axs[1][0].set_xlim(lims[0],lims[1]), axs[1][0].set_ylim(lims[0],lims[1])
+ axs[1][0].set_ylabel('Fibrilar colagen')
+ axs[2][0].hist2d(pixel_probs[0],pixel_probs[2])
+ axs[2][0].set_xlim(lims[0],lims[1]), axs[2][0].set_ylim(lims[0],lims[1])
+ axs[2][0].set_ylabel('Elastin')
+
+ axs[0][1].hist2d(pixel_probs[1],pixel_probs[0])
+ axs[0][1].set_xlim(lims[0],lims[1]), axs[0][1].set_ylim(lims[0],lims[1])
+ axs[0][1].xaxis.set_label_position('top'), axs[0][1].set_xlabel('Fibrilar Colagen')
+ axs[1][1].hist(pixel_probs[1],density = True)
+ axs[1][1].set_xlim(lims[0],lims[1])
+ axs[2][1].hist2d(pixel_probs[1],pixel_probs[2])
+ axs[2][1].set_xlim(lims[0],lims[1]), axs[2][1].set_ylim(lims[0],lims[1])
+
+ axs[0][2].hist2d(pixel_probs[2],pixel_probs[0])
+ axs[0][2].set_xlim(lims[0],lims[1]), axs[0][2].set_ylim(lims[0],lims[1])
+ axs[0][2].xaxis.set_label_position('top'), axs[0][2].set_xlabel('Elastin')
+ axs[1][2].hist2d(pixel_probs[2],pixel_probs[1])
+ axs[1][2].set_xlim(lims[0],lims[1]), axs[1][2].set_ylim(lims[0],lims[1])
+ axs[2][2].hist(pixel_probs[2],density = True)
+ axs[2][2].set_xlim(lims[0],lims[1])
+
+def get_patient_probs(img_probs):
+ nr_frames_patient = 10
+ patient_probs_allchannels= []
+ for img_channel in img_probs:
+ nr_patients = len(img_channel)//nr_frames_patient
+ patient_probs = []
+ for patient in range(nr_patients):
+ patient_img_probs = img_channel[patient*nr_frames_patient: (patient+1)*nr_frames_patient]
+ patient_probs += [sum(patient_img_probs)/len(patient_img_probs)]
+ patient_probs_allchannels += [patient_probs]
+
+ return patient_probs_allchannels
+
+# def plot_mapsAndimages(dir_condition, directory_list, map_img, max_img_list, base_name = 'frame', r = 1024, c = 1024):
+# nr_list = 0
+# for directory in directory_list:
+# in_dir = dir_condition + directory + '/'
+# dir_list = [dI for dI in os.listdir(in_dir) if (os.path.isdir(os.path.join(in_dir ,dI)) and dI[0:len(base_name)]==base_name)]
+# print(dir_list)
+# img_all_control = []
+# img_all_control += map_img[nr_list:nr_list+len(dir_list)]
+# img_all_control += max_img_list[nr_list:nr_list+len(dir_list)]
+# fig, axs = plt.subplots(2,len(dir_list), sharex=True, sharey=True)
+# nr = 0
+# prob_sample = np.zeros((len(dir_list),1))
+# for ax, img in zip(axs.ravel(), img_all_control):
+# print(nr)
+# if nr<len(dir_list):
+# prob_img = sum(sum(img))/(r*c)
+# ax.set_title('Pc '+str(round(prob_img,2)))
+# prob_sample[nr] = prob_img
+# #print(prob_sample)
+# nr += 1
+# if ( img.ndimg == 2 ):
+# ax.imshow(skimage.transform.resize(img, (1024,1024)), cmap=plt.cm.bwr, vmin = 0, vmax = 1)
+# else:
+# ax.imshow(skimage.transform.resize(img.astype(np.uint8), (1024,1024)))
+# plt.suptitle('Probability control of sample '+str(directory)+', avg:' + str(round(prob_sample.mean(),2))+ ' std:' + str(round(prob_sample.std(),2)))
+# plt.savefig(dir_probs + '/control/' + '/probImControl_'+str(directory)+'_%dclusters_%dsigma%ddownscale_absInt%s.png'%(nr_clusters,sigma,1/sc_fac,abs_intensity), dpi=1000)
+# ##plt.show()
+# nr_list += len(dir_list)
+
+
+
+
+
+
+
+
+
+
+
--
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