spacr 0.9.1__tar.gz → 0.9.3__tar.gz

{spacr-0.9.1 → spacr-0.9.3}/LICENSE

@@ -1,6 +1,6 @@
 The MIT License (MIT)
 
-Copyright (c) <year> Adam Veldhousen
+Copyright (c) 2025 Adam Veldhousen
 
 Permission is hereby granted, free of charge, to any person obtaining a copy
 of this software and associated documentation files (the "Software"), to deal

{spacr-0.9.1 → spacr-0.9.3}/MANIFEST.in

@@ -1,8 +1,6 @@
 recursive-include spacr/resources/models/cp *
 recursive-include spacr/resources/icons *
 recursive-include spacr/resources/font *
-recursive-include spacr/resources/MEDIAR *
-recursive-include spacr/resources/MEDIAR_weights *
 
 # Include essential files
 include *.py
@@ -31,14 +29,10 @@ global-exclude docs-env/
 
 # Exclude model files from distribution
 exclude spacr/resources/models/cp/*
-prune notebooks
-prune spacr/notebooks
+prune Notebooks
+prune spacr/Notebooks
 prune spacr/datasets
 
-# Exclude model files from distribution
-#exclude spacr/resources/models/cp/*
-#prune spacr/resources/models/cp
-
 # Exclude all notebooks (including old ones)
 prune notebooks
 prune spacr/notebooks

spacr-0.9.3/PKG-INFO

@@ -0,0 +1,207 @@
+Metadata-Version: 2.1
+Name: spacr
+Version: 0.9.3
+Summary: Spatial phenotype analysis of crisp screens (SpaCr)
+Home-page: https://github.com/EinarOlafsson/spacr
+Author: Einar Birnir Olafsson
+Author-email: olafsson@med.umich.com
+Classifier: Programming Language :: Python :: 3
+Classifier: License :: OSI Approved :: MIT License
+Classifier: Operating System :: OS Independent
+Description-Content-Type: text/x-rst
+License-File: LICENSE
+Requires-Dist: numpy<2.0,>=1.26.4
+Requires-Dist: pandas<3.0,>=2.2.1
+Requires-Dist: scipy<2.0,>=1.12.0
+Requires-Dist: cellpose<4.0,>=3.0.6
+Requires-Dist: scikit-image<1.0,>=0.22.0
+Requires-Dist: scikit-learn<2.0,>=1.4.1
+Requires-Dist: scikit-posthocs<0.20,>=0.10.0
+Requires-Dist: mahotas<2.0,>=1.4.13
+Requires-Dist: btrack<1.0,>=0.6.5
+Requires-Dist: trackpy<1.0,>=0.6.2
+Requires-Dist: statsmodels<1.0,>=0.14.1
+Requires-Dist: shap<1.0,>=0.45.0
+Requires-Dist: torch<3.0,>=2.0
+Requires-Dist: torchvision<1.0,>=0.1
+Requires-Dist: torch-geometric<3.0,>=2.5
+Requires-Dist: torchcam<1.0,>=0.4.0
+Requires-Dist: transformers<5.0,>=4.45.2
+Requires-Dist: segmentation_models_pytorch>=0.3.3
+Requires-Dist: monai>=1.3.0
+Requires-Dist: captum<1.0,>=0.7.0
+Requires-Dist: seaborn<1.0,>=0.13.2
+Requires-Dist: matplotlib<4.0,>=3.8.3
+Requires-Dist: matplotlib_venn<2.0,>=1.1
+Requires-Dist: adjustText<2.0,>=1.2.0
+Requires-Dist: bottleneck<2.0,>=1.3.6
+Requires-Dist: numexpr<3.0,>=2.8.4
+Requires-Dist: opencv-python-headless<5.0,>=4.9.0.80
+Requires-Dist: pillow<11.0,>=10.2.0
+Requires-Dist: tifffile>=2023.4.12
+Requires-Dist: nd2reader<4.0,>=3.3.0
+Requires-Dist: czifile
+Requires-Dist: pylibCZIrw<6.0,>=5.0.0
+Requires-Dist: aicspylibczi
+Requires-Dist: readlif
+Requires-Dist: imageio<3.0,>=2.34.0
+Requires-Dist: pingouin<1.0,>=0.5.5
+Requires-Dist: umap-learn<1.0,>=0.5.6
+Requires-Dist: ttkthemes<4.0,>=3.2.2
+Requires-Dist: xgboost<3.0,>=2.0.3
+Requires-Dist: PyWavelets<2.0,>=1.6.0
+Requires-Dist: ttf_opensans>=2020.10.30
+Requires-Dist: customtkinter<6.0,>=5.2.2
+Requires-Dist: biopython<2.0,>=1.80
+Requires-Dist: lxml<6.0,>=5.1.0
+Requires-Dist: psutil<6.0,>=5.9.8
+Requires-Dist: gputil<2.0,>=1.4.0
+Requires-Dist: gpustat<2.0,>=1.1.1
+Requires-Dist: pyautogui<1.0,>=0.9.54
+Requires-Dist: tables<4.0,>=3.8.0
+Requires-Dist: rapidfuzz<4.0,>=3.9
+Requires-Dist: keyring<16.0,>=15.1
+Requires-Dist: screeninfo<1.0,>=0.8.1
+Requires-Dist: fastremap>=1.14.1
+Requires-Dist: pytz>=2023.3.post1
+Requires-Dist: tqdm>=4.65.0
+Requires-Dist: wandb>=0.16.2
+Requires-Dist: openai<2.0,>=1.50.2
+Requires-Dist: gdown
+Requires-Dist: IPython<9.0,>=8.18.1
+Requires-Dist: ipykernel
+Requires-Dist: ipywidgets<9.0,>=8.1.2
+Requires-Dist: brokenaxes<1.0,>=0.6.2
+Requires-Dist: huggingface-hub<0.25,>=0.24.0
+Provides-Extra: dev
+Requires-Dist: pytest<3.11,>=3.9; extra == "dev"
+Provides-Extra: headless
+Requires-Dist: opencv-python-headless; extra == "headless"
+Provides-Extra: full
+Requires-Dist: opencv-python; extra == "full"
+
+.. |Docs| image:: https://github.com/EinarOlafsson/spacr/actions/workflows/pages/pages-build-deployment/badge.svg
+   :target: https://einarolafsson.github.io/spacr/index.html
+.. |PyPI version| image:: https://badge.fury.io/py/spacr.svg
+   :target: https://badge.fury.io/py/spacr
+.. |Python version| image:: https://img.shields.io/pypi/pyversions/spacr
+   :target: https://pypistats.org/packages/spacr
+.. |Licence: MIT| image:: https://img.shields.io/github/license/EinarOlafsson/spacr
+   :target: https://github.com/EinarOlafsson/spacr/blob/main/LICENSE
+.. |repo size| image:: https://img.shields.io/github/repo-size/EinarOlafsson/spacr
+   :target: https://github.com/EinarOlafsson/spacr/
+
+.. _docs: https://einarolafsson.github.io/spacr/index.html
+
+Badges
+------
+|Docs| |PyPI version| |Python version| |Licence: MIT| |repo size|
+
+SpaCr
+=====
+
+**Spatial phenotype analysis of CRISPR-Cas9 screens (SpaCr).**
+
+The spatial organization of organelles and proteins within cells constitutes a key level of functional regulation. In the context of infectious disease, the spatial relationships between host cell structures and intracellular pathogens are critical to understanding host clearance mechanisms and how pathogens evade them. SpaCr is a Python-based software package for generating single-cell image data for deep-learning sub-cellular/cellular phenotypic classification from pooled genetic CRISPR-Cas9 screens. SpaCr provides a flexible toolset to extract single-cell images and measurements from high-content cell painting experiments, train deep-learning models to classify cellular/subcellular phenotypes, simulate, and analyze pooled CRISPR-Cas9 imaging screens.
+
+Features
+--------
+
+-  **Generate Masks:** Generate cellpose masks of cell, nuclei, and pathogen objects.
+-  **Object Measurements:** Measurements for each object including scikit-image regionprops, intensity percentiles, shannon-entropy, Pearson’s and Manders’ correlations, homogeneity, and radial distribution. Measurements are saved to a SQL database in object-level tables.
+-  **Crop Images:** Save objects (cells, nuclei, pathogen, cytoplasm) as images. Object image paths are saved in a SQL database.
+-  **Train CNNs or Transformers:** Train Torch models to classify single object images.
+-  **Manual Annotation:** Supports manual annotation of single-cell images and segmentation to refine training datasets for training CNNs/Transformers or cellpose, respectively.
+-  **Finetune Cellpose Models:** Adjust pre-existing Cellpose models to your specific dataset for improved performance.
+-  **Timelapse Data Support:** Track objects in timelapse image data.
+-  **Simulations:** Simulate spatial phenotype screens.
+-  **Sequencing:** Map FASTQ reads to barcode and gRNA barcode metadata.
+-  **Misc:** Analyze Ca oscillation, recruitment, infection rate, plaque size/count.
+
+.. image:: https://github.com/EinarOlafsson/spacr/raw/main/spacr/resources/icons/flow_chart_v3.png
+   :alt: SpaCr workflow
+   :align: center
+
+
+**Overview and data organization of spaCR.**
+
+**a.** Schematic workflow of the spaCR pipeline for pooled image-based CRISPR screens. Microscopy images (TIFF, LIF, CZI, NDI) and sequencing reads (FASTQ) are used as inputs (black). The main modules (teal) are: (1) Mask: generates object masks for cells, nuclei, pathogens, and cytoplasm; (2) Measure: extracts object-level features and crops object images, storing quantitative data in an SQL database; (3) Classify—applies machine learning (ML, e.g., XGBoost) or deep learning (DL, e.g., PyTorch) models to classify objects, summarizing results as well-level classification scores; (4) Map Barcodes: extracts and maps row, column, and gRNA barcodes from sequencing data to corresponding wells; (5) Regression: estimates gRNA effect sizes and gene scores via multiple linear regression using well-level summary statistics.
+**b.** Downstream submodules available for extended analyses at each stage.
+**c.** Output folder structure for each module, including locations for raw and processed images, masks, object-level measurements, datasets, and results.
+**d.** List of all spaCR package modules.
+
+Installation
+------------
+
+**Linux recommended.**  
+If using Windows, switch to Linux—it's free, open-source, and better.
+
+**macOS prerequisites (before install):**
+
+::
+
+   brew install libomp
+   brew install hdf5
+
+**Linux GUI requirement:**  
+SpaCr GUI requires Tkinter.  
+
+::
+
+   sudo apt-get install python3-tk
+
+**Installation:**
+
+::
+
+   pip install spacr
+
+**Run SpaCr GUI:**
+
+::
+
+   spacr
+
+Data Availability
+-----------------
+
+- **Raw sequencing data** are available from NCBI BioProject `PRJNA1261935 <https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1261935>`_ and SRA accessions: `SRR33531217 <https://www.ncbi.nlm.nih.gov/sra/SRR33531217>`_, `SRR33531218 <https://www.ncbi.nlm.nih.gov/sra/SRR33531218>`_, `SRR33531219 <https://www.ncbi.nlm.nih.gov/sra/SRR33531219>`_, `SRR33531220 <https://www.ncbi.nlm.nih.gov/sra/SRR33531220>`_
+
+- **Image data** is deposited at EBI BioStudies under accession: 
+  `S-BIAD2076 <https://www.ebi.ac.uk/biostudies/studies/S-BIAD2076>`_  
+  *(If the link redirects to the main BioStudies portal, copy and paste it directly into your browser.)*
+
+
+Example Notebooks
+-----------------
+
+The following example Jupyter notebooks illustrate common workflows using spaCR.
+
+- `Generate masks <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/1_spacr_generate_masks.ipynb>`_  
+  *Generate cell, nuclei, and pathogen segmentation masks from microscopy images using Cellpose.*
+
+- `Capture single cell images and measurements <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/2_spacr_generate_mesurments_crop_images.ipynb>`_  
+  *Extract object-level measurements and crop single-cell images for downstream analysis.*
+
+- `Machine learning based object classification <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/3a_spacr_machine_learning.ipynb>`_  
+  *Train traditional machine learning models (e.g., XGBoost) to classify cell phenotypes based on extracted features.*
+
+- `Computer vision based object classification <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/3b_spacr_computer_vision.ipynb>`_  
+  *Train and evaluate deep learning models (PyTorch CNNs/Transformers) on cropped object images.*
+
+- `Map sequencing barcodes <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/4_spacr_map_barecodes.ipynb>`_  
+  *Map sequencing reads to row, column, and gRNA barcodes for CRISPR screen genotype-phenotype mapping.*
+
+- `Finetune cellpose models <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/5_spacr_train_cellpose.ipynb>`_  
+  *Finetune Cellpose models using your own annotated training data for improved segmentation accuracy.*
+
+License
+-------
+spaCR is distributed under the terms of the MIT License.
+See the `LICENSE <https://github.com/EinarOlafsson/spacr/blob/main/LICENSE>`_ file for details.
+
+How to Cite
+-----------
+If you use spaCR in your research, please cite:  
+Olafsson EB, et al. SpaCr: Spatial phenotype analysis of CRISPR-Cas9 screens. *Manuscript in preparation*.
+

spacr-0.9.3/README.rst

@@ -0,0 +1,125 @@
+.. |Docs| image:: https://github.com/EinarOlafsson/spacr/actions/workflows/pages/pages-build-deployment/badge.svg
+   :target: https://einarolafsson.github.io/spacr/index.html
+.. |PyPI version| image:: https://badge.fury.io/py/spacr.svg
+   :target: https://badge.fury.io/py/spacr
+.. |Python version| image:: https://img.shields.io/pypi/pyversions/spacr
+   :target: https://pypistats.org/packages/spacr
+.. |Licence: MIT| image:: https://img.shields.io/github/license/EinarOlafsson/spacr
+   :target: https://github.com/EinarOlafsson/spacr/blob/main/LICENSE
+.. |repo size| image:: https://img.shields.io/github/repo-size/EinarOlafsson/spacr
+   :target: https://github.com/EinarOlafsson/spacr/
+
+.. _docs: https://einarolafsson.github.io/spacr/index.html
+
+Badges
+------
+|Docs| |PyPI version| |Python version| |Licence: MIT| |repo size|
+
+SpaCr
+=====
+
+**Spatial phenotype analysis of CRISPR-Cas9 screens (SpaCr).**
+
+The spatial organization of organelles and proteins within cells constitutes a key level of functional regulation. In the context of infectious disease, the spatial relationships between host cell structures and intracellular pathogens are critical to understanding host clearance mechanisms and how pathogens evade them. SpaCr is a Python-based software package for generating single-cell image data for deep-learning sub-cellular/cellular phenotypic classification from pooled genetic CRISPR-Cas9 screens. SpaCr provides a flexible toolset to extract single-cell images and measurements from high-content cell painting experiments, train deep-learning models to classify cellular/subcellular phenotypes, simulate, and analyze pooled CRISPR-Cas9 imaging screens.
+
+Features
+--------
+
+-  **Generate Masks:** Generate cellpose masks of cell, nuclei, and pathogen objects.
+-  **Object Measurements:** Measurements for each object including scikit-image regionprops, intensity percentiles, shannon-entropy, Pearson’s and Manders’ correlations, homogeneity, and radial distribution. Measurements are saved to a SQL database in object-level tables.
+-  **Crop Images:** Save objects (cells, nuclei, pathogen, cytoplasm) as images. Object image paths are saved in a SQL database.
+-  **Train CNNs or Transformers:** Train Torch models to classify single object images.
+-  **Manual Annotation:** Supports manual annotation of single-cell images and segmentation to refine training datasets for training CNNs/Transformers or cellpose, respectively.
+-  **Finetune Cellpose Models:** Adjust pre-existing Cellpose models to your specific dataset for improved performance.
+-  **Timelapse Data Support:** Track objects in timelapse image data.
+-  **Simulations:** Simulate spatial phenotype screens.
+-  **Sequencing:** Map FASTQ reads to barcode and gRNA barcode metadata.
+-  **Misc:** Analyze Ca oscillation, recruitment, infection rate, plaque size/count.
+
+.. image:: https://github.com/EinarOlafsson/spacr/raw/main/spacr/resources/icons/flow_chart_v3.png
+   :alt: SpaCr workflow
+   :align: center
+
+
+**Overview and data organization of spaCR.**
+
+**a.** Schematic workflow of the spaCR pipeline for pooled image-based CRISPR screens. Microscopy images (TIFF, LIF, CZI, NDI) and sequencing reads (FASTQ) are used as inputs (black). The main modules (teal) are: (1) Mask: generates object masks for cells, nuclei, pathogens, and cytoplasm; (2) Measure: extracts object-level features and crops object images, storing quantitative data in an SQL database; (3) Classify—applies machine learning (ML, e.g., XGBoost) or deep learning (DL, e.g., PyTorch) models to classify objects, summarizing results as well-level classification scores; (4) Map Barcodes: extracts and maps row, column, and gRNA barcodes from sequencing data to corresponding wells; (5) Regression: estimates gRNA effect sizes and gene scores via multiple linear regression using well-level summary statistics.
+**b.** Downstream submodules available for extended analyses at each stage.
+**c.** Output folder structure for each module, including locations for raw and processed images, masks, object-level measurements, datasets, and results.
+**d.** List of all spaCR package modules.
+
+Installation
+------------
+
+**Linux recommended.**  
+If using Windows, switch to Linux—it's free, open-source, and better.
+
+**macOS prerequisites (before install):**
+
+::
+
+   brew install libomp
+   brew install hdf5
+
+**Linux GUI requirement:**  
+SpaCr GUI requires Tkinter.  
+
+::
+
+   sudo apt-get install python3-tk
+
+**Installation:**
+
+::
+
+   pip install spacr
+
+**Run SpaCr GUI:**
+
+::
+
+   spacr
+
+Data Availability
+-----------------
+
+- **Raw sequencing data** are available from NCBI BioProject `PRJNA1261935 <https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1261935>`_ and SRA accessions: `SRR33531217 <https://www.ncbi.nlm.nih.gov/sra/SRR33531217>`_, `SRR33531218 <https://www.ncbi.nlm.nih.gov/sra/SRR33531218>`_, `SRR33531219 <https://www.ncbi.nlm.nih.gov/sra/SRR33531219>`_, `SRR33531220 <https://www.ncbi.nlm.nih.gov/sra/SRR33531220>`_
+
+- **Image data** is deposited at EBI BioStudies under accession: 
+  `S-BIAD2076 <https://www.ebi.ac.uk/biostudies/studies/S-BIAD2076>`_  
+  *(If the link redirects to the main BioStudies portal, copy and paste it directly into your browser.)*
+
+
+Example Notebooks
+-----------------
+
+The following example Jupyter notebooks illustrate common workflows using spaCR.
+
+- `Generate masks <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/1_spacr_generate_masks.ipynb>`_  
+  *Generate cell, nuclei, and pathogen segmentation masks from microscopy images using Cellpose.*
+
+- `Capture single cell images and measurements <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/2_spacr_generate_mesurments_crop_images.ipynb>`_  
+  *Extract object-level measurements and crop single-cell images for downstream analysis.*
+
+- `Machine learning based object classification <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/3a_spacr_machine_learning.ipynb>`_  
+  *Train traditional machine learning models (e.g., XGBoost) to classify cell phenotypes based on extracted features.*
+
+- `Computer vision based object classification <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/3b_spacr_computer_vision.ipynb>`_  
+  *Train and evaluate deep learning models (PyTorch CNNs/Transformers) on cropped object images.*
+
+- `Map sequencing barcodes <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/4_spacr_map_barecodes.ipynb>`_  
+  *Map sequencing reads to row, column, and gRNA barcodes for CRISPR screen genotype-phenotype mapping.*
+
+- `Finetune cellpose models <https://github.com/EinarOlafsson/spacr/blob/main/Notebooks/5_spacr_train_cellpose.ipynb>`_  
+  *Finetune Cellpose models using your own annotated training data for improved segmentation accuracy.*
+
+License
+-------
+spaCR is distributed under the terms of the MIT License.
+See the `LICENSE <https://github.com/EinarOlafsson/spacr/blob/main/LICENSE>`_ file for details.
+
+How to Cite
+-----------
+If you use spaCR in your research, please cite:  
+Olafsson EB, et al. SpaCr: Spatial phenotype analysis of CRISPR-Cas9 screens. *Manuscript in preparation*.
+

{spacr-0.9.1 → spacr-0.9.3}/setup.py

@@ -71,7 +71,7 @@ dependencies = [
     'huggingface-hub>=0.24.0,<0.25'
 ]
 
-VERSION = "0.9.1"
+VERSION = "0.9.3"
 
 setup(
     name="spacr",

{spacr-0.9.1 → spacr-0.9.3}/spacr/app_annotate.py

@@ -30,6 +30,10 @@ def initiate_annotation_app(parent_frame):
         settings['percentiles'] = list(map(convert_to_number, settings['percentiles'].split(','))) if settings['percentiles'] else [2, 98]
         settings['normalize'] = settings['normalize'].lower() == 'true'
         settings['normalize_channels'] = settings['normalize_channels'].split(',')
+        settings['outline'] = settings['outline'].split(',') if settings['outline'] else None
+        settings['outline_threshold_factor'] = float(settings['outline_threshold_factor']) if settings['outline_threshold_factor'] else 1.0
+        settings['outline_sigma'] = float(settings['outline_threshold_factor']) if settings['outline_threshold_factor'] else 1.0
+        
         try:
             settings['measurement'] = settings['measurement'].split(',') if settings['measurement'] else None
             settings['threshold'] = None if settings['threshold'].lower() == 'none' else int(settings['threshold'])

{spacr-0.9.1 → spacr-0.9.3}/spacr/gui_core.py

@@ -361,8 +361,8 @@ def setup_plot_section(vertical_container, settings_type):
     if settings_type == 'map_barcodes':
         current_dir = os.path.dirname(__file__)
         resources_path = os.path.join(current_dir, 'resources', 'icons')
-        gif_path = os.path.join(resources_path, 'dna_matrix.mp4')
-        display_media_in_plot_frame(gif_path, plot_frame)
+        #gif_path = os.path.join(resources_path, 'dna_matrix.mp4')
+        #display_media_in_plot_frame(gif_path, plot_frame)
 
         canvas = FigureCanvasTkAgg(figure, master=plot_frame)
         canvas.get_tk_widget().configure(cursor='arrow', highlightthickness=0)

{spacr-0.9.1 → spacr-0.9.3}/spacr/gui_elements.py

@@ -10,18 +10,23 @@ import numpy as np
 import pandas as pd
 from PIL import Image, ImageOps, ImageTk, ImageDraw, ImageFont, ImageEnhance
 from concurrent.futures import ThreadPoolExecutor
-from skimage.exposure import rescale_intensity
 from IPython.display import display, HTML
 import imageio.v2 as imageio
 from collections import deque
+from skimage.filters import threshold_otsu
+from skimage.exposure import rescale_intensity
 from skimage.draw import polygon, line
 from skimage.transform import resize
-from scipy.ndimage import binary_fill_holes, label
+from skimage.morphology import dilation, disk
+from skimage.segmentation import find_boundaries
+from skimage.util import img_as_ubyte
+from scipy.ndimage import binary_fill_holes, label, gaussian_filter
 from tkinter import ttk, scrolledtext
 from sklearn.model_selection import train_test_split
 from xgboost import XGBClassifier
 from sklearn.metrics import classification_report, confusion_matrix
 
+
 fig = None
 
 def restart_gui_app(root):
@@ -2209,7 +2214,7 @@ class ModifyMaskApp:
         self.update_display()
 
 class AnnotateApp:
-    def __init__(self, root, db_path, src, image_type=None, channels=None, image_size=200, annotation_column='annotate', normalize=False, percentiles=(1, 99), measurement=None, threshold=None, normalize_channels=None):
+    def __init__(self, root, db_path, src, image_type=None, channels=None, image_size=200, annotation_column='annotate', normalize=False, percentiles=(1, 99), measurement=None, threshold=None, normalize_channels=None, outline=None, outline_threshold_factor=1, outline_sigma=1):
         self.root = root
         self.db_path = db_path
         self.src = src
@@ -2237,7 +2242,10 @@ class AnnotateApp:
         self.measurement = measurement
         self.threshold = threshold
         self.normalize_channels = normalize_channels
-        print('self.normalize_channels',self.normalize_channels)
+        self.outline = outline #([s.strip().lower() for s in outline.split(',') if s.strip()]if isinstance(outline, str) and outline else None)
+        self.outline_threshold_factor = outline_threshold_factor
+        self.outline_sigma = outline_sigma
+        
         style_out = set_dark_style(ttk.Style())
         self.font_loader = style_out['font_loader']
         self.font_size = style_out['font_size']
@@ -2337,7 +2345,12 @@ class AnnotateApp:
             'percentiles': ','.join(map(str, self.percentiles)),
             'measurement': ','.join(self.measurement) if self.measurement else '',
             'threshold': str(self.threshold) if self.threshold is not None else '',
-            'normalize_channels': ','.join(self.normalize_channels) if self.normalize_channels else ''
+            'normalize_channels': ','.join(self.normalize_channels) if self.normalize_channels else '',
+            'outline': ','.join(self.outline) if self.outline else '',
+            'outline_threshold_factor': str(self.outline_threshold_factor) if hasattr(self, 'outline_threshold_factor') else '1.0',
+            'outline_sigma': str(self.outline_sigma) if hasattr(self, 'outline_sigma') else '1.0',
+            'src': self.src,
+            'db_path': self.db_path,
         }
 
         for key, data in vars_dict.items():
@@ -2354,7 +2367,10 @@ class AnnotateApp:
             settings['percentiles'] = list(map(convert_to_number, settings['percentiles'].split(','))) if settings['percentiles'] else [1, 99]
             settings['normalize'] = settings['normalize'].lower() == 'true'
             settings['normalize_channels'] = settings['normalize_channels'].split(',') if settings['normalize_channels'] else None
-
+            settings['outline'] = settings['outline'].split(',') if settings['outline'] else None
+            settings['outline_threshold_factor'] = float(settings['outline_threshold_factor'].replace(',', '.')) if settings['outline_threshold_factor'] else 1.0
+            settings['outline_sigma'] = float(settings['outline_sigma'].replace(',', '.')) if settings['outline_sigma'] else 1.0
+            
             try:
                 settings['measurement'] = settings['measurement'].split(',') if settings['measurement'] else None
                 settings['threshold'] = None if settings['threshold'].lower() == 'none' else int(settings['threshold'])
@@ -2379,7 +2395,12 @@ class AnnotateApp:
                 'percentiles': settings.get('percentiles'),
                 'measurement': settings.get('measurement'),
                 'threshold': settings.get('threshold'),
-                'normalize_channels': settings.get('normalize_channels')
+                'normalize_channels': settings.get('normalize_channels'),
+                'outline': settings.get('outline'),
+                'outline_threshold_factor': settings.get('outline_threshold_factor'),
+                'outline_sigma': settings.get('outline_sigma'),
+                'src': self.src,
+                'db_path': self.db_path
             })
 
             settings_window.destroy()
@@ -2389,22 +2410,32 @@ class AnnotateApp:
         
     def update_settings(self, **kwargs):
         allowed_attributes = {
-            'image_type', 'channels', 'image_size', 'annotation_column',
-            'normalize', 'percentiles', 'measurement', 'threshold', 'normalize_channels'
+            'image_type', 'channels', 'image_size', 'annotation_column', 'src', 'db_path',
+            'normalize', 'percentiles', 'measurement', 'threshold', 'normalize_channels', 'outline', 'outline_threshold_factor', 'outline_sigma'
         }
 
         updated = False
-
+                
         for attr, value in kwargs.items():
             if attr in allowed_attributes and value is not None:
+                if attr == 'outline':
+                    if isinstance(value, str):
+                        value = [s.strip().lower() for s in value.split(',') if s.strip()]
+                elif attr == 'outline_threshold_factor':
+                    value = float(value)
+                elif attr == 'outline_sigma':
+                    value = float(value)
                 setattr(self, attr, value)
                 updated = True
 
+
         if 'image_size' in kwargs:
             if isinstance(self.image_size, list):
                 self.image_size = (int(self.image_size[0]), int(self.image_size[0]))
             elif isinstance(self.image_size, int):
                 self.image_size = (self.image_size, self.image_size)
+            elif isinstance(self.image_size, tuple) and len(self.image_size) == 2:
+                self.image_size = tuple(map(int, self.image_size))
             else:
                 raise ValueError("Invalid image size")
 
@@ -2599,9 +2630,47 @@ class AnnotateApp:
         img = self.normalize_image(img, self.normalize, self.percentiles, self.normalize_channels)
         img = img.convert('RGB')
         img = self.filter_channels(img)
+        
+        if self.outline:
+            img = self.outline_image(img, self.outline_sigma)
+        
         img = img.resize(self.image_size)
         return img, annotation
     
+    def outline_image(self, img, edge_sigma=1, edge_thickness=1):
+        """
+        For each selected channel, compute a continuous outline from the intensity landscape
+        using Otsu threshold scaled by a correction factor. Replace only that channel.
+        """
+        arr = np.asarray(img)
+        if arr.ndim != 3 or arr.shape[2] != 3:
+            return img  # not RGB
+
+        out_img = arr.copy()
+        channel_map = {'r': 0, 'g': 1, 'b': 2}
+        factor = getattr(self, 'outline_threshold_factor', 1.0)
+
+        for ch in self.outline:
+            if ch not in channel_map:
+                continue
+            idx = channel_map[ch]
+            channel_data = arr[:, :, idx]
+
+            try:
+                channel_data = gaussian_filter(channel_data, sigma=edge_sigma) 
+                otsu_thresh = threshold_otsu(channel_data)
+                corrected_thresh = min(255, otsu_thresh * factor)
+                fg_mask = channel_data > corrected_thresh
+            except Exception:
+                continue
+            
+            edge = find_boundaries(fg_mask, mode='inner')
+            thick_edge = dilation(edge, disk(edge_thickness))
+
+            out_img[:, :, idx] = (thick_edge * 255).astype(np.uint8)
+
+        return Image.fromarray(out_img)
+
     @staticmethod
     def normalize_image(img, normalize=False, percentiles=(1, 99), normalize_channels=None):
         """

{spacr-0.9.1 → spacr-0.9.3}/spacr/gui_utils.py

@@ -252,7 +252,7 @@ def annotate(settings):
     app.load_images()
     root.mainloop()
 
-def generate_annotate_fields(frame):
+def generate_annotate_fields_v1(frame):
     from .settings import set_annotate_default_settings
     from .gui_elements import set_dark_style
 
@@ -281,6 +281,48 @@ def generate_annotate_fields(frame):
     
     return vars_dict
 
+def generate_annotate_fields(frame):
+    from .settings import set_annotate_default_settings
+    from .gui_elements import set_dark_style
+
+    style_out = set_dark_style(ttk.Style())
+    font_loader = style_out['font_loader']
+    font_size = style_out['font_size'] - 2
+
+    vars_dict = {}
+    settings = set_annotate_default_settings(settings={})
+    
+    for setting in settings:
+        vars_dict[setting] = {
+            'entry': ttk.Entry(frame),
+            'value': settings[setting]
+        }
+
+    # Arrange input fields and labels
+    for row, (name, data) in enumerate(vars_dict.items()):
+        tk.Label(
+            frame,
+            text=f"{name.replace('_', ' ').capitalize()}:",
+            bg=style_out['bg_color'],
+            fg=style_out['fg_color'],
+            font=font_loader.get_font(size=font_size)
+        ).grid(row=row, column=0)
+
+        value = data['value']
+        if isinstance(value, list):
+            string_value = ','.join(map(str, value))
+        elif isinstance(value, (int, float, bool)):
+            string_value = str(value)
+        elif value is None:
+            string_value = ''
+        else:
+            string_value = value
+
+        data['entry'].insert(0, string_value)
+        data['entry'].grid(row=row, column=1)
+
+    return vars_dict
+
 def run_annotate_app(vars_dict, parent_frame):
     settings = {key: data['entry'].get() for key, data in vars_dict.items()}
     settings['channels'] = settings['channels'].split(',')
@@ -349,7 +391,7 @@ def annotate_with_image_refs(settings, root, shutdown_callback):
     screen_height = root.winfo_screenheight()
     root.geometry(f"{screen_width}x{screen_height}")
 
-    app = AnnotateApp(root, db, src, image_type=settings['image_type'], channels=settings['channels'], image_size=settings['img_size'], annotation_column=settings['annotation_column'], normalize=settings['normalize'], percentiles=settings['percentiles'], measurement=settings['measurement'], threshold=settings['threshold'], normalize_channels=settings['normalize_channels'])
+    app = AnnotateApp(root, db, src, image_type=settings['image_type'], channels=settings['channels'], image_size=settings['img_size'], annotation_column=settings['annotation_column'], normalize=settings['normalize'], percentiles=settings['percentiles'], measurement=settings['measurement'], threshold=settings['threshold'], normalize_channels=settings['normalize_channels'], outline=settings['outline'], outline_threshold_factor=settings['outline_threshold_factor'], outline_sigma=settings['outline_sigma'])
 
     # Set the canvas background to black
     root.configure(bg='black')