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 from itertools import cycle from .data_preprocess import analyse_traces import math import scipy.linalg as la def get_paths_from_traces_file(traces_file): """ Parses the specified traces file and extracts paths and their lengths. Args: traces_file (str): Path to the XML traces file. Returns: tuple: A tuple containing a list of paths (each path is a list of tuples representing points) and a list of corresponding path lengths. """ 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(float(length)) return all_paths, path_lengths def calculate_path_length_partials(point_list, voxel_size=(1,1,1)): """ Calculate the partial path length of a series of points. Args: point_list (list of tuple): List of points, each represented as a tuple of coordinates (x, y, z). voxel_size (tuple, optional): Size of the voxel in each dimension (x, y, z). Defaults to (1, 1, 1). Returns: numpy.ndarray: Array of cumulative partial path lengths at each point. """ # Simple calculation section_lengths = [0.0] s = np.array(voxel_size) for i in range(len(point_list)-1): # Euclidean distance between successive points section_lengths.append(la.norm(s * (np.array(point_list[i+1]) - np.array(point_list[i])))) return np.cumsum(section_lengths) def visualise_ordering(points_list, dim, wr=5, wc=5): """ Visualize the ordering of points in an image. Args: points_list (list): List of points to be visualized. dim (tuple): Dimensions of the image (rows, columns, channels). wr (int, optional): Width of the region to visualize around the point in the row direction. Defaults to 5. wc (int, optional): Width of the region to visualize around the point in the column direction. Defaults to 5. Returns: np.array: An image array with visualized points. """ # Visualizes the ordering of the points in the list on a blank image. 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, _ = map(int, p) vis[max(0,r-wr):min(rdim,r+wr+1),max(0,c-wc):min(cdim,c+wc+1)] = get_col(n) return vis # A color map for paths col_map = [(255,0,0), (0,255,0), (0,0,255), (255,255,0), (255,0,255), (0,255,255), (255,127,0), (255, 0, 127), (127, 255, 0), (0, 255, 127), (127,0,255), (0,127,255)] def draw_paths(all_paths, foci_stack, foci_index=None, r=3, screened_foci_data=None): """ Draws paths on the provided image stack and overlays markers for the foci Args: all_paths (list): List of paths where each path is a list of points. foci_stack (np.array): 3D numpy array representing the image stack. foci_index (list, optional): List of list of focus indices (along each path). Defaults to None. r (int, optional): Radius for the ellipse or line drawing around the focus. Defaults to 3. screened_foci_data (list, optional): List of RemovedPeakData for screened foci Returns: PIL.Image.Image: An image with the drawn paths. """ 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) draw = ImageDraw.Draw(im) for i, (p, col) in enumerate(zip(all_paths, cycle(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) if screened_foci_data is not None: for i, removed_peaks in enumerate(screened_foci_data): for p in removed_peaks: u = all_paths[i][p.idx] v = all_paths[p.screening_peak[0]][p.screening_peak[1]] draw.line((int(u[0]), int(u[1]), int(v[0]), int(v[1])), fill=(127,127,127), width=2) if foci_index is not None: for i, (idx, p, col) in enumerate(zip(foci_index, all_paths, cycle(col_map))): if len(idx): for j in idx: draw.line((int(p[j][0]-r), int(p[j][1]), int(p[j][0]+r), int(p[j][1])), fill=col, width=2) draw.line((int(p[j][0]), int(p[j][1]-r), int(p[j][0]), int(p[j][1]+r)), fill=col, width=2) return im def measure_from_mask(mask, measure_stack): """ Compute the sum of measure_stack values where the mask is equal to 1. Args: mask (numpy.ndarray): Binary mask where the measurement should be applied. measure_stack (numpy.ndarray): Stack of measurements. Returns: measure_stack.dtype: Sum of measure_stack values where the mask is 1. """ return np.sum(mask * measure_stack) # Max of measure_stack over region where mask==1 def max_from_mask(mask, measure_stack): """ Compute the maximum of measure_stack values where the mask is equal to 1. Args: mask (numpy.ndarray): Binary mask where the measurement should be applied. measure_stack (numpy.ndarray): Stack of measurements. Returns: measure_stack.dtype: Maximum value of measure_stack where the mask is 1. """ return np.max(mask * measure_stack) def make_mask_s(p, melem, measure_stack): """ Translate a mask to point p, ensuring correct treatment near the edges of the measure_stack. Args: p (tuple): Target point (r, c, z). melem (numpy.ndarray): Structuring element for the mask. measure_stack (numpy.ndarray): Stack of measurements. Returns: tuple: A tuple containing the translated mask and a section of the measure_stack. """ # R = [u//2 for u in melem.shape] r, c, z = p mask = np.zeros(melem.shape) m_data = np.zeros(melem.shape) s = measure_stack.shape o_1, o_2, o_3 = max(R[0]-r, 0), max(R[1]-c, 0), max(R[2]-z,0) e_1, e_2, e_3 = min(R[0]-r+s[0], 2*R[0]+1), min(R[1]-c+s[1], 2*R[1]+1), min(R[2]-z+s[2], 2*R[2]+1) m_data[o_1:e_1,o_2:e_2,o_3:e_3] = measure_stack[max(r-R[0],0):min(r+R[0]+1,s[0]),max(c-R[1],0):min(c+R[1]+1,s[1]),max(z-R[2],0):min(z+R[2]+1, s[2])] mask[o_1:e_1,o_2:e_2,o_3:e_3] = melem[o_1:e_1,o_2:e_2,o_3:e_3] return mask, m_data def measure_at_point(p, melem, measure_stack, op='mean'): """ Measure the mean or max value of measure_stack around a specific point using a structuring element. Args: p (tuple): Target point (r, c, z). melem (numpy.ndarray): Structuring element for the mask. measure_stack (numpy.ndarray): Stack of measurements. op (str, optional): Operation to be applied; either 'mean' or 'max'. Default is 'mean'. Returns: float: Measured value based on the specified operation. """ p = map(int, p) if op=='mean': mask, m_data = make_mask_s(p, melem, measure_stack) melem_size = np.sum(mask) 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): """ Generate a binary representation of a sphere in 3D space. Args: R (int, optional): Radius of the sphere. Default is 5. Centred on the centre of the middle voxel. Includes all voxels whose centre is precisely R from the middle voxel. z_scale_ratio (float, optional): Scaling factor for the z-axis. Default is 2.3. Returns: numpy.ndarray: Binary representation of the sphere. """ R_z = int(math.ceil(R/z_scale_ratio)) x, y, z = np.ogrid[-R:R+1, -R:R+1, -R_z:R_z+1] 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', R=5, z_scale_ratio=2.3): """ Measure the values of measure_stack at each point in a list using a spherical region. Args: points_list (list): List of points (r, c, z) to be measured. measure_stack (numpy.ndarray): Stack of measurements. op (str, optional): Operation to be applied; either 'mean' or 'max'. Default is 'mean'. R (int, optional): Radius of the sphere. Default is 5. z_scale_ratio (float, optional): Scaling factor for the z-axis. Default is 2.3. Returns: list: List of measured values for each point. """ melem = make_sphere(R, z_scale_ratio) 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, intensity, config): """ Measure fluorescence levels along an ordered skeleton. Args: path (list): List of ordered path points (r, c, z). intensity (numpy.ndarray): 3D fluorescence data. config (dict): Configuration dictionary containing 'z_res', 'xy_res', and 'sphere_radius' values. Returns: tuple: A tuple containing the visualization, mean measurements, and max measurements along the path. """ # Calculate size of spheroid used for measurement scale_ratio = config['z_res']/config['xy_res'] sphere_xy_radius = int(math.ceil(config['sphere_radius']/config['xy_res'])) vis = visualise_ordering(path, dim=intensity.shape, wr=sphere_xy_radius, wc=sphere_xy_radius) measurements = measure_all_with_sphere(path, intensity, op='mean', R=sphere_xy_radius, z_scale_ratio=scale_ratio) measurements_max = measure_all_with_sphere(path, intensity, op='max', R=sphere_xy_radius, z_scale_ratio=scale_ratio) return vis, measurements, measurements_max def extract_peaks(cell_id, all_paths, path_lengths, measured_traces, config): """ Extract peak information from given traces and compile them into a DataFrame. Args: - cell_id (int or str): Identifier for the cell being analyzed. - all_paths (list of lists): Contains ordered path points for multiple paths. - path_lengths (list of floats): List containing lengths of each path in all_paths. - measured_traces (list of lists): Contains fluorescence measurement values along the paths. - config (dict): Configuration dictionary containing: - 'peak_threshold': Threshold value to determine a peak in the trace. - 'sphere_radius': Radius of the sphere used in fluorescence measurement. Returns: - pd.DataFrame: DataFrame containing peak information for each path. - list of lists: Absolute intensities of the detected foci. - list of lists: Index positions of the detected foci. - list of lists: Absolute focus intensity threshold for each trace. - list of numpy.ndarray: For each trace, distances of each point from start of trace in microns """ n_paths = len(all_paths) data = [] foci_absolute_intensity, foci_position, foci_position_index, screened_foci_data, trace_median_intensities, trace_thresholds = analyse_traces(all_paths, path_lengths, measured_traces, config) # Normalize foci intensities (for quantification) using trace medians as estimates of background foci_intensities = [] for path_foci_abs_int, tmi in zip(foci_absolute_intensity, trace_median_intensities): foci_intensities.extend(list(path_foci_abs_int - tmi)) # Divide all foci intensities by the mean within the cell mean_intensity = np.mean(foci_intensities) trace_positions = [] for i in range(n_paths): # Calculate real (Euclidean) distance of each point along the traced path pl = calculate_path_length_partials(all_paths[i], (config['xy_res'], config['xy_res'], config['z_res'])) path_data = { 'Cell_ID':cell_id, 'Trace': i+1, 'SNT_trace_length(um)': path_lengths[i], 'Measured_trace_length(um)': pl[-1], 'Trace_median_intensity': trace_median_intensities[i], 'Detection_sphere_radius(um)': config['sphere_radius'], 'Screening_distance(voxels)': config['screening_distance'], 'Foci_ID_threshold': config['peak_threshold'], 'Trace_foci_number': len(foci_position_index[i]) } for j, (idx, u,v) in enumerate(zip(foci_position_index[i], foci_position[i], foci_absolute_intensity[i])): if config['use_corrected_positions']: # Use the calculated position along the traced path path_data[f'Foci_{j+1}_position(um)'] = pl[idx] else: # Use the measured trace length (from SNT), and assume all steps of path are approximately the same length path_data[f'Foci_{j+1}_position(um)'] = u # The original measured intensity (mean in spheroid around detected peak) path_data[f'Foci_{j+1}_absolute_intensity'] = v # Measure relative intensity by removing per-trace background and dividing by cell total path_data[f'Foci_{j+1}_relative_intensity'] = (v - trace_median_intensities[i])/mean_intensity data.append(path_data) trace_positions.append(pl) return pd.DataFrame(data), foci_absolute_intensity, foci_position_index, screened_foci_data, trace_thresholds, trace_positions def analyse_paths(cell_id, foci_file, traces_file, config ): """ Analyzes paths for the given cell ID using provided foci and trace files. Args: cell_id (int/str): Identifier for the cell. foci_file (str): Path to the foci image file. traces_file (str): Path to the XML traces file. config (dict): Configuration dictionary containing necessary parameters such as resolutions and thresholds. Returns: tuple: A tuple containing an overlay image of the traces, visualization images for each trace, a figure with plotted measurements, and a dataframe with extracted peaks. """ # Read stack foci_stack = tifffile.imread(foci_file) # If 2D add additional (z) dimension if foci_stack.ndim==2: foci_stack = foci_stack[None,:,:] all_paths, path_lengths = get_paths_from_traces_file(traces_file) all_trace_vis = [] # Per-path visualizations all_m = [] # Per-path measured intensities for p in all_paths: # Measure intensity along path - transpose the stack ZYX -> XYZ vis, m, _ = measure_chrom2(p,foci_stack.transpose(2,1,0), config) all_trace_vis.append(vis) all_m.append(m) # Extract all data from paths and traces extracted_peaks, foci_absolute_intensity, foci_pos_index, screened_foci_data, trace_thresholds, trace_positions = extract_peaks(cell_id, all_paths, path_lengths, all_m, config) # Plot per-path measured intensities and indicate foci n_cols = 2 n_rows = (len(all_paths)+n_cols-1)//n_cols fig, ax = plt.subplots(n_rows,n_cols, figsize=(5*n_cols, 3*n_rows)) ax = ax.flatten() for i, m in enumerate(all_m): ax[i].set_title(f'Trace {i+1}') ax[i].plot(trace_positions[i], m) if len(foci_pos_index[i]): # Plot detected foci ax[i].plot(trace_positions[i][foci_pos_index[i]], np.array(m)[foci_pos_index[i]], 'rx') if len(screened_foci_data[i]): # Indicate screened foci by gray circles on plots screened_foci_pos_index = [u.idx for u in screened_foci_data[i]] ax[i].plot(trace_positions[i][screened_foci_pos_index], np.array(m)[screened_foci_pos_index], color=(0.5,0.5,0.5), marker='o', linestyle='None') # Show per-trace intensity thresholds with red dotted lines if trace_thresholds[i] is not None: ax[i].axhline(trace_thresholds[i], c='r', ls=':') ax[i].set_xlabel('Distance from start (um)') ax[i].set_ylabel('Intensity') # Hide excess plots for i in range(len(all_m), n_cols*n_rows): ax[i].axis('off') plt.tight_layout() trace_overlay = draw_paths(all_paths, foci_stack, foci_index=foci_pos_index, screened_foci_data=screened_foci_data) return trace_overlay, all_trace_vis, fig, extracted_peaks