import sys sys.path.append("../") from utils.fileIO import Codebook, read_table from utils.imgproc import warp_image,register_stack from utils.helper_functions import performDumbExtraction, findErrorBits from .BaseReport import BaseReport import numpy as np import matplotlib.pyplot as plt import matplotlib as mpl import scipy.ndimage as ndi import skimage from sklearn.neighbors import NearestNeighbors mpl.rcParams["image.interpolation"] = "none" class DecodabilityReport(BaseReport): """This report produces graphs and figures that provide insight into the decodeability of a dataset. Args: deconvolved_img_stack (str): The path to the img stack of deconvolved images coord_info (str): The path to the coordinate information file for the fov and z codebook_file (str): The path to the codebook csv file data_org_file (str): The path to the data organization file fov (str): The fov name that belongs to the image stack z (str): The z name that belongs to the image stack out_detection_stats (str): The path that the detection stats will be printed into Raises: AssertionError: If the number of wavelenghts is not 4 in the coordinate file, then the image warping in the data organization file wont work. """ def __init__( self, deconvolved_img_stack: str, coord_info: str, codebook_file: str, data_org_file: str, fov: int, z: int, out_detection_stats: str, ): super().__init__(deconvolved_img_stack, coord_info) if not (len(self.coords["wvs"]) == 4): raise AssertionError("Image Stack must contain 4 channels") self.fov_name = fov self.z_name = z self.out_detection_stats = out_detection_stats # Load the codebook file self.codebook = Codebook(codebook_file) # Load the dataorg file data_org = read_table(data_org_file).to_dict("records") self.registered_images = register_stack(self.imgstack) # Warp the deconvolved image stack into (16,x,y) self.warped_imgs = warp_image(data_org, self.registered_images, 16) # Pass into the perform the dumb extraction ( self.filtered_warped_images, self.bright_pixel_count, self.bit_map, self.bit_vector_map, self.dist_map, self.barcode_map, self.error_bit_map, self.detection_number_map, self.codebook_bits, ) = performDumbExtraction( self.warped_imgs, self.codebook, clip_thresh=98.5 ) """ ====MAKE REPORTS===== """ def make_bit_imgs(self): """Generates the 3,4, and 5 bit detection images. These are bits that are within 1 manhattan distance to an entry in the codebook """ # make 3,4,5 bit images image_blank = np.zeros( (self.warped_imgs.shape[1], self.warped_imgs.shape[2]) ) bc3_img = np.multiply( np.reshape(self.barcode_map[3], image_blank.shape) + 1, np.reshape(self.dist_map[3] == 1, image_blank.shape), ) bc4_img = np.multiply( np.reshape(self.barcode_map[4], image_blank.shape) + 1, np.reshape(self.dist_map[4] == 0, image_blank.shape), ) bc5_img = np.multiply( np.reshape(self.barcode_map[5], image_blank.shape) + 1, np.reshape(self.dist_map[5] == 1, image_blank.shape), ) bc3_img = np.where(bc3_img > 0, 1, 0) bc4_img = np.where(bc4_img > 0, 1, 0) bc5_img = np.where(bc5_img > 0, 1, 0) f, ax = plt.subplots( nrows=1, ncols=3, sharex=True, sharey=True, figsize=(3 * 10, 10) ) ax[0].imshow( bc3_img, cmap="gray", vmin=0, vmax=1, interpolation="none" ) ax[1].imshow( bc4_img, cmap="gray", vmin=0, vmax=1, interpolation="none" ) ax[2].imshow( bc5_img, cmap="gray", vmin=0, vmax=1, interpolation="none" ) ax[0].set_title("Image of 3-bit Decodable barcodes") ax[1].set_title("Image of 4-bit Decodable barcodes") ax[2].set_title("Image of 5-bit Decodable barcodes") plt.tight_layout() self.pdf.savefig() plt.close(f) # Set the colour images colour_img = np.zeros((bc5_img.shape[0], bc5_img.shape[1], 3)) colour_img[:, :, 0] = bc3_img colour_img[:, :, 1] = bc4_img colour_img[:, :, 2] = bc5_img f, ax = plt.subplots(figsize=(40, 40)) ax.imshow(colour_img, vmin=0, vmax=1, interpolation="none") plt.title("Red: 3-bit words; Green: 4-bit words; Blue: 5-bit words") plt.tight_layout() self.pdf.savefig() plt.close(f) # For each bit in the filtered warped image, spit out an RGB image f, axs = plt.subplots(4, 4, figsize=(43, 43)) axs = axs.flatten() window_half_width = 50 while True: r, c = ( np.random.randint(0, bc3_img.shape[0]), np.random.randint(0, bc3_img.shape[1]), ) tl = [int(r - window_half_width), int(c - window_half_width)] br = [int(r + window_half_width), int(c + window_half_width)] fwi = np.moveaxis(self.filtered_warped_images, 0, -1) wi = np.moveaxis(self.warped_imgs, 0, -1) masked3 = np.multiply( fwi, np.reshape(self.dist_map[3] == 1, image_blank.shape)[ ..., None ], ) masked4 = np.multiply( fwi, np.reshape(self.dist_map[4] == 0, image_blank.shape)[ ..., None ], ) masked5 = np.multiply( fwi, np.reshape(self.dist_map[5] == 1, image_blank.shape)[ ..., None ], ) cropped_mask3 = masked3[tl[0]: br[0] + 1, tl[1]: br[1] + 1, :] cropped_mask4 = masked4[tl[0]: br[0] + 1, tl[1]: br[1] + 1, :] cropped_mask5 = masked5[tl[0]: br[0] + 1, tl[1]: br[1] + 1, :] if np.all(cropped_mask3 != 1) and np.all(cropped_mask5 != 1): pass # If the cropped area is empty, then try it all again else: break mpl.rcParams["image.interpolation"] = "none" for iax, ax in enumerate(axs): colour_image = np.stack( ( cropped_mask3[:, :, iax], cropped_mask4[:, :, iax], cropped_mask5[:, :, iax], ), axis=2, ) ax.imshow(colour_image * 255, vmin=0, vmax=1) ax.set_title(f"Bit: {iax}") plt.suptitle( f"Red: 3-bit words; Green: 4-bit words; Blue: 5-bit words \n \ Row: {r}, Column {c}, CropSize {2*window_half_width+1}" ) # plt.tight_layout() self.pdf.savefig() plt.close(f) f, axs = plt.subplots(4, 4, figsize=(43, 43)) axs = axs.flatten() masked3 = np.multiply( fwi, np.reshape(self.dist_map[3] == 1, image_blank.shape)[..., None], ) masked4 = np.multiply( fwi, np.reshape(self.dist_map[4] == 0, image_blank.shape)[..., None], ) masked5 = np.multiply( fwi, np.reshape(self.dist_map[5] == 1, image_blank.shape)[..., None], ) cropped_mask3 = masked3[tl[0]: br[0] + 1, tl[1]: br[1] + 1, :] cropped_mask4 = masked4[tl[0]: br[0] + 1, tl[1]: br[1] + 1, :] cropped_mask5 = masked5[tl[0]: br[0] + 1, tl[1]: br[1] + 1, :] row3, col3, z3 = np.where(cropped_mask3 == 1) row4, col4, z4 = np.where(cropped_mask4 == 1) row5, col5, z5 = np.where(cropped_mask5 == 1) for iax, ax in enumerate(axs): crop = wi[tl[0]: br[0] + 1, tl[1]: br[1] + 1, iax] crap_max = ndi.maximum_filter(crop, size=2, mode="constant") peak = skimage.feature.peak_local_max(crap_max, min_distance=2) # print(peak) ax.imshow(crop, cmap="gray") ax.scatter(col3, row3, s=15, color="red", marker="*") ax.scatter(col4, row4, s=15, color="green", marker="*") ax.scatter(col5, row5, s=15, color="blue", marker="*") ax.scatter( peak[:, 1], peak[:, 0], s=10, color="yellow", marker="*" ) ax.set_title(f"Bit: {iax}") plt.suptitle( "Red: 3-bit words; Green: 4-bit words; Blue: 5-bit words\n \ Overlayed on smoothed deconvolved image" ) # plt.tight_layout() self.pdf.savefig() plt.close(f) f, axs = plt.subplots(4, 4, figsize=(40, 40)) axs = axs.flatten() for iax, ax in enumerate(axs): crop = fwi[tl[0]: br[0] + 1, tl[1]: br[1] + 1, iax] crap_max = ndi.maximum_filter(crop, size=2, mode="constant") peak = skimage.feature.peak_local_max(crap_max, min_distance=2) # print(peak) ax.imshow(crop, cmap="gray") ax.scatter(col3, row3, s=15, color="red", marker="*") ax.scatter(col4, row4, s=15, color="green", marker="*") ax.scatter(col5, row5, s=15, color="blue", marker="*") ax.scatter( peak[:, 1], peak[:, 0], s=10, color="yellow", marker="*" ) ax.set_title(f"Bit: {iax}") plt.suptitle( "Red: 3-bit words; Green: 4-bit words; Blue: 5-bit words\n \ Overlayed thresholded deconvolved image" ) # plt.tight_layout() self.pdf.savefig() plt.close(f) def make_decodability_hists(self): """Make the histograms that for determining decoability based on thresholded pixels """ f, ax = plt.subplots(figsize=(10, 10)) plt.hist( self.bright_pixel_count.ravel(), bins=[i for i in range(17)], ) ax.set_xlim([1, 17]) ax.set_ylim([0, 0.6e6]) ax.vlines(x=[3, 6], ymin=0, ymax=ax.get_ylim()[1], colors=["r", "r"]) ax.set_xticks( ticks=[i + 0.5 for i in range(17)], labels=[i for i in range(17)] ) ax.set_title("Number of bright pixels stacked") plt.tight_layout() self.pdf.savefig() plt.close(f) codebook_bits = np.array(self.codebook.barcode_arrays) nbrs = NearestNeighbors( n_neighbors=1, algorithm="ball_tree", p=1 ).fit(codebook_bits) bright_pix_3bcs = np.reshape(self.bit_map[3], (16, -1)).T bright_pix_4bcs = np.reshape(self.bit_map[4], (16, -1)).T bright_pix_5bcs = np.reshape(self.bit_map[5], (16, -1)).T bright_pix_3bcs = bright_pix_3bcs[bright_pix_3bcs.sum(axis=1) > 0, :] bright_pix_4bcs = bright_pix_4bcs[bright_pix_4bcs.sum(axis=1) > 0, :] bright_pix_5bcs = bright_pix_5bcs[bright_pix_5bcs.sum(axis=1) > 0, :] # Look at distances for 3 pixel barcodes dist3, barcode3_ids = nbrs.kneighbors(bright_pix_3bcs) dist3_fraction = (dist3 == 1).sum() / (dist3 > 0).sum() f, ax = plt.subplots(figsize=(10, 10)) plt.hist(dist3.ravel(), bins=[i for i in range(17)]) plt.vlines(x=[1, 2], ymin=0, ymax=ax.get_ylim()[1], colors=["r", "r"]) ax.set_xticks( ticks=[i + 0.5 for i in range(17)], labels=[i for i in range(17)] ) ax.set_xlabel("Manhattan Distance away from entry in codebook") ax.set_ylabel("Number of Pixels") plt.title( "3 Bright Pixel Barcodes: Manhattan Distance \n Detection fraction: \ {:.02f}".format(dist3_fraction) ) plt.tight_layout() self.pdf.savefig() plt.close(f) # Look at distances for 4 pixel barcodes dist4, barcode4_ids = nbrs.kneighbors(bright_pix_4bcs) dist4_fraction = (dist4 == 0).sum() / (dist4 > -1).sum() f, ax = plt.subplots(figsize=(10, 10)) plt.hist(dist4.ravel(), bins=[i for i in range(17)]) plt.vlines(x=[0, 1], ymin=0, ymax=ax.get_ylim()[1], colors=["r", "r"]) ax.set_xticks( ticks=[i + 0.5 for i in range(17)], labels=[i for i in range(17)] ) ax.set_xlabel("Manhattan Distance away from entry in codebook") ax.set_ylabel("Number of Pixels") plt.title( "4 Bright Pixel Barcodes: Manhattan Distance \n Detection fraction: \ {:.02f}".format(dist4_fraction) ) plt.tight_layout() self.pdf.savefig() plt.close(f) # Look at distances for 5 pixel barcodes dist5, barcode5_ids = nbrs.kneighbors(bright_pix_5bcs) dist5_fraction = (dist5 == 1).sum() / (dist5 > -1).sum() f, ax = plt.subplots(figsize=(10, 10)) plt.hist(dist5.ravel(), bins=[i for i in range(17)]) plt.vlines(x=[1, 2], ymin=0, ymax=ax.get_ylim()[1], colors=["r", "r"]) ax.set_xticks( ticks=[i + 0.5 for i in range(17)], labels=[i for i in range(17)] ) ax.set_xlabel("Manhattan Distance away from entry in codebook") ax.set_ylabel("Number of Pixels") plt.title( "5 Bright Pixel Barcodes: Manhattan Distance \ \n Detection fraction: {:.02f}".format( dist5_fraction ) ) plt.tight_layout() self.pdf.savefig() plt.close(f) """ Find out what is the bit distribution of the spots that had 3-5 bits, but didnt match the codebook """ unique_dist3 = np.unique(dist3) dist3_barcode_hist = np.zeros((16, 16)) for ud3 in unique_dist3: dist3_barcode_hist[int(ud3), :] = np.sum( bright_pix_3bcs[np.squeeze(dist3 == ud3), :], axis=0 ) dist3_barcode_hist[int(ud3), :] = np.nan_to_num( dist3_barcode_hist[int(ud3), :] / dist3_barcode_hist[int(ud3), :].sum() ) unique_dist4 = np.unique(dist4) dist4_barcode_hist = np.zeros((16, 16)) for ud4 in unique_dist4: dist4_barcode_hist[int(ud4), :] = np.sum( bright_pix_4bcs[np.squeeze(dist4 == ud4), :], axis=0 ) dist4_barcode_hist[int(ud4), :] = np.nan_to_num( dist4_barcode_hist[int(ud4), :] / dist4_barcode_hist[int(ud4), :].sum() ) unique_dist5 = np.unique(dist5) dist5_barcode_hist = np.zeros((16, 16)) for ud5 in unique_dist5: dist5_barcode_hist[int(ud5), :] = np.sum( bright_pix_5bcs[np.squeeze(dist5 == ud5), :], axis=0 ) dist5_barcode_hist[int(ud5), :] = np.nan_to_num( dist5_barcode_hist[int(ud5), :] / dist5_barcode_hist[int(ud5), :].sum() ) f, ax = plt.subplots(figsize=(10, 10)) plt.imshow(dist3_barcode_hist) for ud in range(dist3_barcode_hist.shape[0]): for bp in range(dist3_barcode_hist.shape[0]): _ = ax.text( bp, ud, f"{dist3_barcode_hist[ud, bp]*100:.2f}%", ha="center", va="center", color="w", ) ax.set_ylabel("Manhattan Distance away from entry in codebook") ax.set_xlabel("Bit Number") plt.title("3 Bright Pixel bit distribution") plt.tight_layout() self.pdf.savefig() plt.close(f) f, ax = plt.subplots(figsize=(10, 10)) plt.imshow(dist4_barcode_hist) for ud in range(dist4_barcode_hist.shape[0]): for bp in range(dist4_barcode_hist.shape[0]): _ = ax.text( bp, ud, f"{dist4_barcode_hist[ud, bp]*100:.2f}%", ha="center", va="center", color="w", ) ax.set_ylabel("Manhattan Distance away from entry in codebook") ax.set_xlabel("Bit Number") plt.title("4 Bright Pixel bit distribution") plt.tight_layout() self.pdf.savefig() plt.close(f) f, ax = plt.subplots(figsize=(10, 10)) plt.imshow(dist5_barcode_hist) for ud in range(dist5_barcode_hist.shape[0]): for bp in range(dist5_barcode_hist.shape[0]): _ = ax.text( bp, ud, f"{dist5_barcode_hist[ud, bp]*100:.2f}%", ha="center", va="center", color="w", ) ax.set_ylabel("Manhattan Distance away from entry in codebook") ax.set_xlabel("Bit Number") plt.title("5 Bright Pixel bit distribution") plt.tight_layout() self.pdf.savefig() plt.close(f) errorBit3 = findErrorBits( bright_pix_3bcs, self.codebook_bits, dist3, barcode3_ids, filter_val=1, ) errorBit4 = findErrorBits( bright_pix_4bcs, self.codebook_bits, dist4, barcode4_ids, filter_val=0, ) errorBit5 = findErrorBits( bright_pix_5bcs, self.codebook_bits, dist5, barcode5_ids, filter_val=1, ) f, ax = plt.subplots(1, 3, figsize=(3 * 10, 10)) ax[0].hist( errorBit3.ravel(), bins=[i for i in range(17)], align="left" ) ax[1].hist( errorBit4.ravel(), bins=[i for i in range(17)], align="left" ) ax[2].hist( errorBit5.ravel(), bins=[i for i in range(17)], align="left" ) ax[0].set_title("Bits to correct for 3-bit detections") ax[1].set_title("Bits to correct for 4-bit detections") ax[2].set_title("Bits to correct for 5-bit detections") self.pdf.savefig() plt.close(f) # # Save the output of detected barcodes number_report = [ f"Number of 3bit Detections: {dist3[dist3==1].sum()}\n", f"Number of 4bit Detections: {(1+dist4[dist4==0]).sum()}\n", f"Number of 5bit Detections: {dist5[dist5==1].sum()}", ] with open(f"{self.out_detection_stats}", "w") as f: f.writelines(number_report) if __name__ == "__main__": print("test")