microlive 1.0.11__py3-none-any.whl

microlive/pipelines/pipeline_particle_tracking.py

@@ -0,0 +1,489 @@
+"""Pipeline module for MicroLive.
+
+This module is part of the microlive package.
+"""
+from microlive.imports import *
+
+def pipeline_particle_tracking(data_folder_path, selected_image, channels_spots, max_spots_for_threshold=100000,
+                               show_plot=True, channels_cytosol=None, channels_nucleus=None,memory=1,
+                               min_length_trajectory=5, yx_spot_size_in_px=5, z_spot_size_in_px=2, maximum_spots_cluster=4,cluster_radius_nm =500,
+                               MINIMAL_SNR=0.5, diameter_cytosol=300, diameter_nucleus=200,
+                               segmentation_selection_metric='max_area',recalculate_mask=False,optimization_segmentation_method='diameter_segmentation',
+                               pretrained_model_cyto_segmentation=None,use_watershed=False,list_images_to_process=None,save_3d_visualization=False,
+                               max_percentage_empty_data_in_trajectory=0.1,particle_detection_threshold=None,save_croparray=False,
+                               apply_photobleaching_correction=False,photobleaching_mode='inside_cell',use_maximum_projection=False,
+                               max_lag_for_MSD=30,step_size_in_sec=1,separate_clusters_and_spots=False,maximum_range_search_pixels=10,results_folder_path=None,
+                               calculate_MSD=True,calculate_correlations=True):
+    # Read images and metadata
+    # detect if the data is a lif file
+    list_images, list_names, pixel_xy_um, voxel_z_um, channel_names, number_color_channels, list_time_intervals, bit_depth = \
+        mi.ReadLif(data_folder_path, show_metadata=False, save_tif=False, save_png=False, format='TZYXC').read()
+    # Prepare full list of images and set up indices to process
+    list_images_complete = list_images.copy()
+    if list_images_to_process is not None:
+        selected_indices = [i for i in range(len(list_names)) if list_names[i] in list_images_to_process]
+        if use_maximum_projection:
+            # use the maximum projection in Z. but keep the image shape with only one Z slice
+            list_images = [np.max(list_images[i], axis=1, keepdims=True) for i in selected_indices]
+        else:
+            list_images = [list_images[i] for i in selected_indices]
+    else:
+        selected_indices = range(len(list_names))
+        if use_maximum_projection:
+            list_images = [np.max(img, axis=1, keepdims=True) for img in list_images]
+
+
+
+    # If selected_image is None, process all images
+    if selected_image is None:
+        list_df, list_masks, list_images_tested, list_diffusion_coefficient = [], [], [], []
+        
+        #for idx in range(len(list_images)):
+        for idx in range(len(list_images_complete)):
+            if idx not in selected_indices:
+                continue
+            df, masks,image,diffusion_coefficient = process_single_image(
+                data_folder_path=data_folder_path,
+                selected_image=idx,
+                channels_spots=channels_spots,
+                max_spots_for_threshold=max_spots_for_threshold,
+                show_plot=show_plot,
+                channels_cytosol=channels_cytosol,
+                channels_nucleus=channels_nucleus,
+                min_length_trajectory=min_length_trajectory,
+                yx_spot_size_in_px=yx_spot_size_in_px,
+                z_spot_size_in_px = z_spot_size_in_px,
+                maximum_spots_cluster=maximum_spots_cluster,
+                cluster_radius_nm=cluster_radius_nm,
+                MINIMAL_SNR=MINIMAL_SNR,
+                diameter_cytosol=diameter_cytosol,
+                diameter_nucleus=diameter_nucleus,
+                segmentation_selection_metric=segmentation_selection_metric,
+                list_images=list_images_complete,
+                list_names=list_names,
+                pixel_xy_um=pixel_xy_um,
+                voxel_z_um=voxel_z_um,
+                channel_names=channel_names,
+                image_time_interval=list_time_intervals[idx],
+                recalculate_mask=recalculate_mask,
+                optimization_segmentation_method=optimization_segmentation_method,
+                pretrained_model_cyto_segmentation=pretrained_model_cyto_segmentation,
+                use_watershed=use_watershed,
+                save_3d_visualization=save_3d_visualization,
+                apply_photobleaching_correction=apply_photobleaching_correction,
+                photobleaching_mode=photobleaching_mode,
+                use_maximum_projection=use_maximum_projection,
+                max_lag_for_MSD = max_lag_for_MSD,
+                step_size_in_sec=step_size_in_sec,
+                separate_clusters_and_spots=separate_clusters_and_spots,
+                maximum_range_search_pixels=maximum_range_search_pixels,
+                max_percentage_empty_data_in_trajectory=max_percentage_empty_data_in_trajectory,
+                memory=memory,
+                particle_detection_threshold=particle_detection_threshold,
+                results_folder_path=results_folder_path,
+                calculate_MSD=calculate_MSD,
+                calculate_correlations=calculate_correlations,
+                save_croparray=save_croparray,
+            )
+            #if df is None:
+            # if the df is None or empty, continue to the next image
+            if df.empty:
+                continue
+            # rename the field image_id to idx
+            df['image_id'] = idx
+            list_df.append(df)
+            list_masks.append(masks)
+            list_images_tested.append(image)
+            list_diffusion_coefficient.append(diffusion_coefficient)
+
+        if len(list_df) >1 :
+            final_df = pd.concat(list_df, ignore_index=True)
+        else:
+            final_df = df
+        return final_df, list_df, list_masks, list_images_tested, list_diffusion_coefficient
+    else:
+        # Process single image
+        df,masks,image, diffusion_coefficient= process_single_image(
+            data_folder_path=data_folder_path,
+            selected_image=selected_image,
+            channels_spots=channels_spots,
+            max_spots_for_threshold=max_spots_for_threshold,
+            show_plot=show_plot,
+            channels_cytosol=channels_cytosol,
+            channels_nucleus=channels_nucleus,
+            min_length_trajectory=min_length_trajectory,
+            yx_spot_size_in_px=yx_spot_size_in_px,
+            z_spot_size_in_px = z_spot_size_in_px,
+            maximum_spots_cluster=maximum_spots_cluster,
+            cluster_radius_nm=cluster_radius_nm,
+            MINIMAL_SNR=MINIMAL_SNR,
+            diameter_cytosol=diameter_cytosol,
+            diameter_nucleus=diameter_nucleus,
+            segmentation_selection_metric=segmentation_selection_metric,
+            list_images=list_images,
+            list_names=list_names[idx],
+            pixel_xy_um=pixel_xy_um,
+            voxel_z_um=voxel_z_um,
+            channel_names=channel_names,
+            image_time_interval = list_time_intervals[selected_image],
+            recalculate_mask=recalculate_mask,
+            optimization_segmentation_method=optimization_segmentation_method,
+            pretrained_model_cyto_segmentation=pretrained_model_cyto_segmentation,
+            use_watershed=use_watershed,
+            save_3d_visualization=save_3d_visualization,
+            apply_photobleaching_correction=apply_photobleaching_correction,
+            photobleaching_mode=photobleaching_mode,
+            use_maximum_projection=use_maximum_projection,
+            max_lag_for_MSD = max_lag_for_MSD,
+            step_size_in_sec=step_size_in_sec,
+            separate_clusters_and_spots=separate_clusters_and_spots,
+            maximum_range_search_pixels=maximum_range_search_pixels,
+            max_percentage_empty_data_in_trajectory=max_percentage_empty_data_in_trajectory,
+            memory=memory,
+            particle_detection_threshold=particle_detection_threshold,
+            results_folder_path=results_folder_path,
+            calculate_MSD=calculate_MSD,
+            calculate_correlations=calculate_correlations,
+            save_croparray=save_croparray,
+        )
+        return df, [df], [masks], [image], [diffusion_coefficient]
+
+
+
+
+
+
+@mi.Utilities().metadata_decorator(metadata_folder_func=mi.Utilities().get_metadata_folder,exclude_args=['list_images',]) # exclude_args=['list_images', 'list_names' ]
+def process_single_image(data_folder_path, selected_image, channels_spots, max_spots_for_threshold=100000,
+                         show_plot=True, channels_cytosol=None, channels_nucleus=None,memory=1,
+                         min_length_trajectory=5, yx_spot_size_in_px=5, z_spot_size_in_px=2 , maximum_spots_cluster=4,cluster_radius_nm=500,
+                         MINIMAL_SNR=0.5, diameter_cytosol=300, diameter_nucleus=200, segmentation_selection_metric='area',
+                         list_images=None, list_names=None, pixel_xy_um=None, voxel_z_um=None,
+                         channel_names=None, optimization_segmentation_method='diameter_segmentation',
+                         recalculate_mask=False,use_watershed=False, pretrained_model_cyto_segmentation=None,particle_detection_threshold=None,save_croparray=False,
+                         image_time_interval=None,save_3d_visualization=False,apply_photobleaching_correction=False,photobleaching_mode='inside_cell',max_percentage_empty_data_in_trajectory=0.1,
+                         use_maximum_projection=False,max_lag_for_MSD=30,step_size_in_sec=1,separate_clusters_and_spots=False,maximum_range_search_pixels=10,results_folder_path=None,
+                         calculate_MSD=True,calculate_correlations=True):
+    # Ensure lists are properly formatted
+    channels_spots = [channels_spots] if not isinstance(channels_spots, list) else channels_spots
+    channels_cytosol = [channels_cytosol] if not isinstance(channels_cytosol, list) else channels_cytosol
+    channels_nucleus = [channels_nucleus] if not isinstance(channels_nucleus, list) else channels_nucleus
+
+    # Convert pixel and voxel sizes to nm
+    pixel_xy_nm = int(pixel_xy_um * 1000)
+    voxel_z_nm = int(voxel_z_um * 1000)
+    list_voxels = [voxel_z_nm, pixel_xy_nm]
+    list_spot_size_px = [z_spot_size_in_px, yx_spot_size_in_px]
+    # print a line
+    print('--------------------------------------------------')
+    print(f'Processing image: {list_names[selected_image]}')
+    tested_image = list_images[selected_image]  # TZYXC
+    original_tested_image = tested_image.copy()
+    # Creating the results folder
+    results_name = 'results_' + data_folder_path.stem + '_cell_id_' + str(selected_image)
+    current_dir = pathlib.Path().absolute()
+    
+    
+    if results_folder_path is not None:
+        # ensure that results_folder_path is a Path object
+        if not isinstance(results_folder_path, pathlib.Path):
+            results_folder_path = pathlib.Path(results_folder_path)
+        results_folder = results_folder_path.joinpath(results_name)
+    else:
+        results_folder = current_dir.joinpath('results_live_cell', results_name)
+    results_folder.mkdir(parents=True, exist_ok=True)
+    mi.Utilities().clear_folder_except_substring(results_folder, 'mask')
+    # Plot the original image
+    plot_name_original = results_folder.joinpath('original_image.png')
+    suptitle=f'Image: {data_folder_path.stem[:16]} - {list_names[selected_image]} - Cell_ID: {selected_image}'
+        
+    
+    mi.Plots().plot_images(
+        image_ZYXC=tested_image[0],
+        figsize=(12, 5),
+        show_plot=show_plot,
+        use_maximum_projection=True,
+        use_gaussian_filter=True,
+        cmap='binary',
+        min_max_percentile=[0.5, 99.9],
+        show_gird=False,
+        save_plots=True,
+        plot_name=plot_name_original,
+        suptitle=suptitle
+    )
+    # Read or create masks
+    mask_file_name = 'mask_' + data_folder_path.stem + '_image_' + str(selected_image) + '.tif'
+    mask_file_path = results_folder.joinpath(mask_file_name)
+    path_mask_exist = os.path.exists(str(mask_file_path))
+    if path_mask_exist and recalculate_mask is False:
+        masks = imread(str(mask_file_path)).astype(bool)
+    else:
+        # Use Cellpose to create masks
+        if use_watershed:
+            masks_complete_cells = mi.CellSegmentationWatershed(np.max(tested_image[:,:,:,:,channels_cytosol[0]],
+                                                                       axis=(0,1)), footprint_size=2, ).apply_watershed()
+        else:
+            masks_complete_cells, _, _ = mi.CellSegmentation(
+                    tested_image[0],
+                    channels_cytosol=channels_cytosol,
+                    channels_nucleus=channels_nucleus,
+                    diameter_cytosol=diameter_cytosol,
+                    diameter_nucleus=diameter_nucleus,
+                    optimization_segmentation_method=optimization_segmentation_method,
+                    remove_fragmented_cells=False,
+                    show_plot=show_plot,
+                    image_name=None,
+                    NUMBER_OF_CORES=1,
+                    selection_metric=segmentation_selection_metric,
+                    pretrained_model_cyto_segmentation = pretrained_model_cyto_segmentation
+                    ).calculate_masks()
+        # Selecting the mask that is in the center of the image
+        center_y = masks_complete_cells.shape[0] // 2
+        center_x = masks_complete_cells.shape[1] // 2
+        selected_mask_id = masks_complete_cells[center_y, center_x]
+        if selected_mask_id > 0:
+            masks = masks_complete_cells == selected_mask_id
+        else:
+            # Select the largest mask that is not the background mask (0)
+            mask_labels = np.unique(masks_complete_cells)
+            mask_sizes = [(label, np.sum(masks_complete_cells == label)) for label in mask_labels if label != 0]
+            if mask_sizes:
+                selected_mask_id = max(mask_sizes, key=lambda x: x[1])[0]
+                masks = masks_complete_cells == selected_mask_id
+            else:
+                masks = np.zeros_like(masks_complete_cells, dtype=bool)
+        # Save the mask
+        masks = masks.astype(np.uint8)
+        tifffile.imwrite(str(mask_file_path), masks, dtype='uint8')
+
+    if apply_photobleaching_correction:
+        file_path_photobleacing = results_folder.joinpath('photobleaching.png')
+        corrected_image =  mi.Photobleaching(image_TZYXC=tested_image,mask_YX=masks, show_plot=False, mode= photobleaching_mode,plot_name=file_path_photobleacing).apply_photobleaching_correction() #mi.PhotobleachingCorrection(tested_image).apply_correction()
+
+    else:
+        corrected_image = tested_image
+    # Calculate the threshold for spot detection
+    plot_name_threshold = results_folder.joinpath('threshold_spot_detection.png')
+    
+    if particle_detection_threshold is None:
+        starting_threshold = mi.Utilities().calculate_threshold_for_spot_detection(
+            corrected_image, list_spot_size_px, list_voxels, channels_spots,
+            max_spots_for_threshold=max_spots_for_threshold,
+            show_plot=True,plot_name=plot_name_threshold
+        )
+    else:
+        starting_threshold =  [particle_detection_threshold]*len(channels_spots)
+    
+    # Run the particle tracking
+    try:
+        list_dataframes_trajectories, _ = mi.ParticleTracking(
+            image=corrected_image,
+            channels_spots=channels_spots,
+            masks=masks,
+            list_voxels=list_voxels,
+            memory=memory,
+            channels_cytosol=channels_cytosol,
+            channels_nucleus=channels_nucleus,
+            min_length_trajectory=min_length_trajectory,
+            threshold_for_spot_detection=starting_threshold,
+            yx_spot_size_in_px=yx_spot_size_in_px,
+            z_spot_size_in_px=z_spot_size_in_px,
+            maximum_spots_cluster=maximum_spots_cluster,
+            cluster_radius_nm = cluster_radius_nm,
+            separate_clusters_and_spots=separate_clusters_and_spots,
+            maximum_range_search_pixels=maximum_range_search_pixels,
+        ).run()
+    except Exception as e:
+        print(f'Error: {e}')
+        return pd.DataFrame(), masks, original_tested_image, None
+    #df_tracking = list_dataframes_trajectories[0]
+    df_tracking = list_dataframes_trajectories[0]
+
+    if len(df_tracking)==0:
+        return pd.DataFrame(), masks, original_tested_image, None
+
+
+    if len(list_dataframes_trajectories) > 1:
+        for i in range(1, len(list_dataframes_trajectories)):
+            df_tracking = pd.concat([df_tracking, list_dataframes_trajectories[i]], ignore_index=True)
+    df_tracking = df_tracking.reset_index(drop=True)    
+    #print(df_tracking)
+    # Plot histograms for the SNR
+    selected_field = 'snr'  # options are: psf_sigma, snr, 'spot_int'
+    plot_name_snr = results_folder.joinpath('spots_' + selected_field + '.png')
+    mean_snr = mi.Plots().plot_histograms_from_df(
+        df_tracking,
+        selected_field=selected_field,
+        figsize=(8, 2),
+        plot_name=plot_name_snr,
+        bin_count=60,
+        save_plot=True,
+        list_colors=channel_names,
+        remove_outliers=True
+    )
+    # Plot histograms for the spot intensity
+    selected_field = 'spot_int'
+    plot_name_int = results_folder.joinpath('spots_' + selected_field + '.png')
+    mean_int = mi.Plots().plot_histograms_from_df(
+        df_tracking,
+        selected_field=selected_field,
+        figsize=(8, 2),
+        plot_name=plot_name_int,
+        bin_count=60,
+        save_plot=True,
+        list_colors=channel_names,
+        remove_outliers=True
+    )
+    # Remove tracks with low SNR in the tracking channel
+    if MINIMAL_SNR is not None:
+        array_selected_field = mi.Utilities().df_trajectories_to_array(
+            dataframe=df_tracking,
+            selected_field=selected_field + '_ch_' + str(channels_spots[0]),
+            fill_value='nans'
+        )
+        mean_snr = np.nanmean(array_selected_field, axis=1)
+        indices_low_quality_tracks = np.where(mean_snr < MINIMAL_SNR)[0]
+        df_tracking = df_tracking[~df_tracking['particle'].isin(indices_low_quality_tracks)]
+        df_tracking = df_tracking.reset_index(drop=True)
+        df_tracking['particle'] = df_tracking.groupby('particle').ngroup()
+    # Plot image intensity histogram
+    
+    masked_data = corrected_image * masks[np.newaxis, np.newaxis, :, :, np.newaxis].astype(float)
+    for i in range(len(channels_spots)): 
+        #plot_name_histogram = results_folder.joinpath('pixel_histogram_in_cell.png')
+        plot_name_histogram = results_folder.joinpath('pixel_histogram_in_cell_'+str(channels_spots[i])+'.png')
+        mi.Plots().plot_image_pixel_intensity_distribution(
+            image=np.mean(masked_data, axis=(0, 1)),
+            figsize=(8, 2),
+            bins=100,
+            remove_outliers=True,
+            remove_zeros=True,
+            save_plots=True,
+            plot_name=plot_name_histogram,
+            single_color=None,
+            list_colors=channel_names,
+            tracking_channel=channels_spots[0],
+            threshold_tracking=starting_threshold[i]
+        )
+    # Plot original image and tracks
+    suptitle = f'Image: {data_folder_path.stem[:16]} - {list_names[selected_image]} - Cell_ID: {selected_image}'
+    plot_name_original_image_and_tracks = results_folder.joinpath('original_image_tracking.png')
+    mi.Plots().plot_images(
+        image_ZYXC=corrected_image[0],
+        df=df_tracking,
+        masks=masks,
+        show_trajectories=True,
+        suptitle=suptitle,
+        figsize=(12, 3),
+        show_plot=True,
+        selected_time=0,
+        use_maximum_projection=True,
+        use_gaussian_filter=True,
+        cmap='binary',
+        min_max_percentile=[0.05, 99.95],
+        show_gird=False,
+        save_plots=True,
+        plot_name=plot_name_original_image_and_tracks
+    )
+    
+    # Combine the original image and the image with tracks
+    plot_name_complete_image = results_folder.joinpath('complete_image_tracking.png')
+    mi.Utilities().combine_images_vertically([plot_name_original, plot_name_original_image_and_tracks], plot_name_complete_image, delete_originals=True)
+
+    # Save the DataFrame
+    df_tracking.to_csv(results_folder.joinpath('tracking_results.csv'), index=False)
+    PLOT_FILTERED_IMAGES = True
+    normalize_each_particle = True 
+    crop_size = yx_spot_size_in_px + 5  # 3 pixels for the border
+    # add 5 pixels to crop_size, check if the crop_size is odd, if not, add 1
+    if crop_size % 2 == 0:
+        crop_size += 1
+    selected_time_point = None
+    #if PLOT_FILTERED_IMAGES:
+    filtered_image = mi.Utilities().gaussian_laplace_filter_image(corrected_image, list_spot_size_px, list_voxels)
+    croparray_filtered, mean_crop_filtered, first_appearance, crop_size = mi.CropArray(image=filtered_image, df_crops=df_tracking, crop_size=crop_size, remove_outliers=False, max_percentile=99.95,selected_time_point=selected_time_point,normalize_each_particle=normalize_each_particle).run()
+    #else:
+    #    croparray_filtered, mean_crop_filtered, first_appearance, crop_size = mi.CropArray(image=tested_image, df_crops=df_tracking, crop_size=crop_size, remove_outliers=False, max_percentile=99.9,selected_time_point=selected_time_point,normalize_each_particle=normalize_each_particle).run()
+    # Plot all crops  
+    if save_croparray:  
+        path_crop_array = results_folder.joinpath('crop_array.png')
+        mi.Plots().plot_croparray(croparray_filtered, crop_size, save_plots=True,plot_name= path_crop_array,suptitle=None,show_particle_labels=True, cmap='binary_r',max_percentile = 99) # flag_vector=flag_vector
+    # plot pair of crops
+    plot_name_crops_filter = results_folder.joinpath('crops.png')
+    mi.Plots().plot_matrix_pair_crops (mean_crop_filtered, crop_size,save_plots=True,plot_name=plot_name_crops_filter) # flag_vector=flag_vector
+    # Calculate the Mean Squared Displacement
+    plot_name_MSD = results_folder.joinpath('MSD_plot.png')
+    
+    #max_lag_for_MSD = 30
+    if image_time_interval is None:
+        image_time_interval = step_size_in_sec
+        print(f'Warning: The image_time_interval was not provided. Using the step_size_in_sec as the image_time_interval: {step_size_in_sec} seconds.')
+    else:
+        image_time_interval = float(image_time_interval)
+        # print a warning message indicating that we are using the step_size_in_sec as the image_time_interval
+    
+    if calculate_MSD:
+        diffusion_coefficient, em, time_range, model_fit, trackpy_df = mi.ParticleMotion(df_tracking,
+                                                                                         microns_per_pixel=pixel_xy_um,
+                                                                                         step_size_in_sec=image_time_interval,
+                                                                                         max_lagtime=max_lag_for_MSD,
+                                                                                         show_plot=True,
+                                                                                         remove_drift=False,
+                                                                                         plot_name=plot_name_MSD).calculate_msd()
+    else:
+        diffusion_coefficient = None
+
+    
+    if calculate_correlations:
+        # calculate and plot the autocorrelation
+        array_ch0= mi.Utilities().df_trajectories_to_array(dataframe=df_tracking, selected_field='spot_int_ch_0', fill_value='nans')
+        
+        if 'spot_int_ch_1' in df_tracking.columns:
+            array_ch1= mi.Utilities().df_trajectories_to_array(dataframe=df_tracking, selected_field='spot_int_ch_1', fill_value='nans') 
+            intensity_array_ch0_short, intensity_array_ch1_short = mi.Utilities().shift_trajectories(array_ch0, array_ch1,max_percentage_empty_data_in_trajectory=max_percentage_empty_data_in_trajectory)
+        else:
+            array_ch1 = None
+            intensity_array_ch0_short = mi.Utilities().shift_trajectories(array_ch0,max_percentage_empty_data_in_trajectory=max_percentage_empty_data_in_trajectory)
+            intensity_array_ch1_short = None
+
+        plot_name_intensity_matrix = results_folder.joinpath('intensity_matrix.png')
+        mi.Plots().plot_matrix_sample_time(intensity_array_ch0_short, intensity_array_ch1_short,plot_name=plot_name_intensity_matrix)
+
+        plot_name_AC_ch0 = results_folder.joinpath('AC_plot_ch0.png')
+        mean_correlation_ch0, std_correlation_ch0, lags_ch0, correlations_array_ch0,dwell_time_ch0 = mi.Correlation(primary_data=intensity_array_ch0_short, max_lag=None, 
+                                                                                                                nan_handling='ignore',shift_data=True,return_full=False,
+                                                                                                                time_interval_between_frames_in_seconds=image_time_interval,
+                                                                                                                show_plot=True,start_lag=1,fit_type='exponential',
+                                                                                                                use_linear_projection_for_lag_0=True,save_plots=True,plot_name=plot_name_AC_ch0).run()
+        if array_ch1 is not None:
+            plot_name_AC_ch1 = results_folder.joinpath('AC_plot_ch1.png')
+            mean_correlation_ch1, std_correlation_ch1, lags_ch1, correlations_array_ch1,dwell_time_ch1 = mi.Correlation(primary_data=intensity_array_ch1_short, max_lag=None, 
+                                                                                                                    nan_handling='ignore',shift_data=True,return_full=False,
+                                                                                                                    time_interval_between_frames_in_seconds=image_time_interval,
+                                                                                                                    show_plot=True,start_lag=1,fit_type='exponential',
+                                                                                                                    use_linear_projection_for_lag_0=True,save_plots=True,plot_name=plot_name_AC_ch1).run()
+            
+            # Plot cross-correlation
+            plot_name_cross_correlation = results_folder.joinpath('cross_correlation.png')
+            mean_cross_correlation, std_cross_correlation, lags_cross_correlation, cross_correlations_array, max_lag = mi.Correlation(primary_data=intensity_array_ch0_short, secondary_data=intensity_array_ch1_short, 
+                                                                                                                            max_lag=None, nan_handling='ignore', shift_data=False, return_full=True,
+                                                                                                                            time_interval_between_frames_in_seconds=image_time_interval,show_plot=True,
+                                                                                                                            save_plots=True,plot_name=plot_name_cross_correlation).run()
+        
+
+    # plot napari visualizer
+    if save_3d_visualization:
+        mask_expanded = masks[np.newaxis, np.newaxis, :, :, np.newaxis]
+        masked_image_TZYXC = filtered_image * mask_expanded
+        # Apply Gaussian filter to reduce background noise
+        #from scipy.ndimage import gaussian_filter
+        masked_image_TZYXC = gaussian_filter(masked_image_TZYXC, sigma=1)
+        # Remove extreme values from the image
+        masked_image_TZYXC = mi.RemoveExtrema(masked_image_TZYXC, min_percentile=0.001, max_percentile=99.995).remove_outliers()
+        plot_name_3d_visualizer = str(results_folder.joinpath('image_3d.gif'))
+        mi.Plots().Napari_Visualizer(masked_image_TZYXC, df_tracking, z_correction=7, channels_spots=0, plot_name=plot_name_3d_visualizer)
+    
+    # print the process has finished for the selected image
+    print(f'Image {list_names[selected_image]} has been processed.')
+    
+    return df_tracking, masks, original_tested_image, diffusion_coefficient