import lxml.etree as ET import gzip import tifffile import matplotlib.pyplot as plt import numpy as np from PIL import Image, ImageDraw import pandas as pd def get_paths_from_traces_file(traces_file): tree = ET.parse(traces_file) root = tree.getroot() all_paths = [] path_lengths = [] for path in root.findall('path'): length=path.get('reallength') path_points = [] for point in path: path_points.append((int(point.get('x')), int(point.get('y')), int(point.get('z')))) all_paths.append(path_points) path_lengths.append(length) return all_paths, path_lengths def visualise_ordering(points_list, dim): rdim, cdim, _ = dim vis = np.zeros((rdim, cdim, 3), dtype=np.uint8) def get_col(i): r = int(255 * i/len(points_list)) g = 255 - r return r, g, 0 for n, p in enumerate(points_list): c, r, _ = p wr, wc = 5, 5 vis[max(0,r-wr):min(rdim,r+wr),max(0,c-wc):min(cdim,c+wc)] = get_col(n) return vis col_map = [(255,0,0), (0,255,0), (0,0,255), (255,255,0), (255,0,255), (0,255,255)] def draw_paths(all_paths, foci_stack): im = np.max(foci_stack, axis=0) im = (im/np.max(im)*255).astype(np.uint8) im = np.dstack((im,)*3) im = Image.fromarray(im) #.convert('RGB') draw = ImageDraw.Draw(im) for i, (p, col) in enumerate(zip(all_paths, col_map)): draw.line([(u[0], u[1]) for u in p], fill=col) draw.text((p[0][0], p[0][1]), str(i+1), fill=col) return im # Sum of measure_stack over regin where mask==1 def measure_from_mask(mask, measure_stack): return np.sum(mask * measure_stack) # Max of measure_stack over region where mask==1 def max_from_mask(mask, measure_stack): return np.max(mask * measure_stack) # Translate mask to point p, treating makss near stack edges correctly def make_mask_s(p, melem, measure_stack): mask = melem R = melem.shape[0] // 2 r, c, z = p m_data = np.zeros(melem.shape) s = measure_stack.shape o_1, o_2, o_3 = max(R-r, 0), max(R-c, 0), max(R-z,0) e_1, e_2, e_3 = min(R-r+s[0], 2*R), min(R-c+s[1], 2*R), min(R-z+s[2], 2*R) m_data[o_1:e_1,o_2:e_2,o_3:e_3] = measure_stack[max(r-R,0):min(r+R,s[0]),max(c-R,0):min(c+R,s[1]),max(z-R,0):min(z+R, s[2])] return mask, m_data # Measure the (mean/max) value of measure_stack about the point p, using # the structuring element melem. op indicates the appropriate measurement (mean/max) def measure_at_point(p, melem, measure_stack, op='mean'): if op=='mean': mask, m_data = make_mask_s(p, melem, measure_stack) melem_size = np.sum(melem) return float(measure_from_mask(mask, m_data) / melem_size) else: mask, m_data = make_mask_s(p, melem, measure_stack) return float(max_from_mask(mask, m_data)) # Generate spherical region def make_sphere(R=5, z_scale_ratio=2.3): x, y, z = np.ogrid[-R:R, -R:R, -R:R] sphere = x**2 + y**2 + (z_scale_ratio * z)**2 < R**2 return sphere # Measure the values of measure_stack at each of the points of points_list in turn. # Measurement is the mean / max (specified by op) on the spherical region about each point def measure_all_with_sphere(points_list, measure_stack, op='mean'): melem = make_sphere() measure_func = lambda p: measure_at_point(p, melem, measure_stack, op) return list(map(measure_func, points_list)) # Measure fluorescence levels along ordered skeleton def measure_chrom2(path, hei10): # single chrom - structure containing skeleton (single_chrom.skel) and # fluorecence levels (single_chrom.hei10) as Image3D objects (equivalent to ndarray) # Returns list of coordinates in skeleton, the ordered path vis = visualise_ordering(path, dim=hei10.shape) measurements = measure_all_with_sphere(path, hei10, op='mean') measurements_max = measure_all_with_sphere(path, hei10, op='max') return vis, measurements, measurements_max def extract_peaks(cell_id, all_paths, path_lengths, measured_traces): n = len(all_paths) #headers = ['Cell_ID', 'Trace', 'Trace_length(um)', 'detection_sphere_radius(um)', 'Foci_ID_threshold', 'Foci_per_trace'] #for i in range(max_n): # headers += [f'Foci{i}_relative_intensity', f'Foci_{i}_position(um)'] data_dict = {} data_dict['Cell_ID'] = [cell_id]*n data_dict['Trace'] = range(1, n+1) data_dict['Trace_length(um)'] = path_lengths data_dict['Detection_sphere_radius(um)'] = [0.2]*n data_dict['Foci_ID_threshold'] = [0.4]*n return pd.DataFrame(data_dict) def analyse_paths(cell_id, foci_file, traces_file): foci_stack = tifffile.imread(foci_file) all_paths, path_lengths = get_paths_from_traces_file(traces_file) all_trace_vis = [] all_m = [] for p in all_paths: vis, m, _ = measure_chrom2(p,foci_stack.transpose(2,1,0)) all_trace_vis.append(vis) all_m.append(m) trace_overlay = draw_paths(all_paths, foci_stack) fig, ax = plt.subplots(len(all_paths),1) for i, m in enumerate(all_m): ax[i].plot(m) extracted_peaks = extract_peaks(cell_id, all_paths, path_lengths, all_m) return trace_overlay, all_trace_vis, fig, extracted_peaks