Read and analyze cell image data.
Pythologist 1) reads exports from InForm software or other sources into a common storage format, and 2) extracts basic analysis features from cell image data. This software is generally intended to be run from a jupyter notebook and provides hooks into the image data so that the user can have the flexability to execute analyses they design or find in the primary literature.
List of image analysis publications
Pythologist is based on IrisSpatialFeatures (C.D. Carey, ,D. Gusenleitner, M. Lipshitz, et al. Blood. 2017) https://doi.org/10.1182/blood-2017-03-770719, and is implemented in the python programming language.
Features Pythologist add are:
- An common CellProjectGeneric storage class, and classical inheritance conventions to organize the importation of different data types.
- A mutable CellDataFrame class that can be used for slicing, and combining projects.
- The ability to add binary features to cells based on cell-cell contacts or cell proximity.
- Customizable images based on the cell segmentation or heatmaps spaninng the cartesian coordinates.
- Specify cell populations through a SubsetLogic syntax for quick selection of mutually exclusive phenotypes or binary features
- A set of Quality Check functions to identify potential issues in imported data.
Read files from sources like inForm software by PerkinElmer.
Storage Object - Read the Docs
For function details please Read The Docs
Functions for working with image data in python. This set of functions are used in the construction of pythologist-reader intermediate storage format, and in the analysis of image data .. finding neighbors .. or the generation of images.
- Check assumptions for input types prior to ingestion.
- Define a universal intermediate storage format suitable for downstream analysis.
- Define input parameters suitable custom for pipeline execution of feature extraction.
This package should facilitate the validation of inputs.
This section is in development.
Primary Software
pythologistThis software package uses a CellDataFrame class, an extension of a Pandas DataFrame to modify data and execute analyses [Read the Docs] [source]pythologist-schemasThis submodule documents/defines the formats of inputs and outputs expected in this pipeline. [source]pythologist-readerThis submodule facillitates reading platform-specific data into a harmonized format. [Read the Docs] [source]pythologist-test-imagesThis submodule has some example data [source]pythologist-image-utilitiesThis submodule has helper functions to work with images [Read the Docs] [source]
Additional Analytics
good-neighborsThis package facilitates the analysis of cellular data based on their proximal "cellular neighborhoods" [Read the Docs] [source]
The docker is the fastest way to get working with the latest version of pytholgoist.
To start a jupyter lab notebook with the required software as your user in your current drectory you can use the following command
docker run -v "$(pwd)":"/work" --rm -p 8888:8888 --user $(id -u):$(id -g) vacation/pythologist:latest
This will start jupyter lab on port 8888 as your user and group.
Any of the test data examples should work fine in this environment.
git clone https://github.com/dfci/pythologist.git
The assumption here is that the exports are grouped so that sample folders contain one or more image exports, and that sample name can be inferred from the last folder name.
from pythologist_test_images import TestImages
from pythologist_reader.formats.inform.sets import CellProjectInForm
import matplotlib.pyplot as plt
# Get the path of the test dataset
path = TestImages().raw('IrisSpatialFeatures')
# Create the storage opbject where the project will be saved
cpi = CellProjectInForm('pythologist.h5',mode='w')
# Read the project data
cpi.read_path(path,require=False,verbose=True,microns_per_pixel=0.496,sample_name_index=-1)
# Display one of the cell map images
for f in cpi.frame_iter():
break
print(f.frame_name)
plt.imshow(f.cell_map_image(),origin='upper')
plt.show()MEL2_7
Another format supported for a project import is one with a custom tumor and invasive margin definition. Similar to above, the project is organized into sample folders, and each image within each sample folder has a tif file defining the tumor and invasive margin. These come in the form of a <image name prefix>_Tumor.tif and <image name prefix>_Invasive_Margin.tif for each image. The _Tumor.tif is an area filled in where the tumor is, and transparent elsewhere. The _Invasive_Margin.tif is a drawn line of a known width. steps is used to grow the margin out that many pixels in each direction to establish an invasive margin region. Here we also rename some markers during read-in to clean up the syntax of thresholding on binary features.
from pythologist_test_images import TestImages
from pythologist_reader.formats.inform.custom import CellProjectInFormLineArea
# Get the path of the test dataset
path = TestImages().raw('IrisSpatialFeatures')
# Specify where the data read-in will be stored as an h5 object
cpi = CellProjectInFormLineArea('test.h5',mode='w')
# Read in the data (gets stored on the fly into the h5 object)
cpi.read_path(path,
sample_name_index=-1,
verbose=True,
steps=76,
project_name='IrisSpatialFeatures',
microns_per_pixel=0.496)
for f in cpi.frame_iter():
break
print(f.frame_name)
print('hand drawn margin')
plt.imshow(f.get_image(f.get_data('custom_images').\
set_index('custom_label').loc['Drawn','image_id']),origin='upper')
plt.show()
print('hand drawn tumor area')
plt.imshow(f.get_image(f.get_data('custom_images').\
set_index('custom_label').loc['Area','image_id']),origin='upper')
plt.show()
print('Mutually exclusive Margin, Tumor, and Stroma')
plt.imshow(f.get_image(f.get_data('regions').\
set_index('region_label').loc['Margin','image_id']),origin='upper')
plt.show()
plt.imshow(f.get_image(f.get_data('regions').\
set_index('region_label').loc['Tumor','image_id']),origin='upper')
plt.show()
plt.imshow(f.get_image(f.get_data('regions').\
set_index('region_label').loc['Stroma','image_id']),origin='upper')
plt.show()MEL2_2
hand drawn margin
hand drawn tumor area
Mutually exclusive Margin, Tumor, and Stroma
Here we will use the mask, but not expand or subtract from it.
from pythologist_test_images import TestImages
from pythologist_reader.formats.inform.custom import CellProjectInFormCustomMask
import matplotlib.pyplot as plt
path = TestImages().raw('IrisSpatialFeatures')
cpi = CellProjectInFormCustomMask('test.h5',mode='w')
cpi.read_path(path,
microns_per_pixel=0.496,
sample_name_index=-1,
verbose=True,
custom_mask_name='Tumor',
other_mask_name='Not-Tumor')
for f in cpi.frame_iter():
rs = f.get_data('regions').set_index('region_label')
for r in rs.index:
print(r)
plt.imshow(f.get_image(rs.loc[r]['image_id']),origin='upper')
plt.show()
breakMEL2_2
Tumor
Not-Tumor
Check general status of the CellDataFrame
cdf = cpi.cdf
cdf.db = cpi
cdf.qc(verbose=True).print_results()prints the following QC metrics to stdout
==========
Check microns per pixel attribute
PASS
Microns per pixel is 0.496
==========
Check storage object is set
PASS
h5 object is set
==========
Is there a 1:1 correspondence between sample_name and sample_id?
PASS
Good concordance.
Issue count: 0/2
==========
Is there a 1:1 correspondence between frame_name and frame_id?
PASS
Good concordance.
Issue count: 0/4
==========
Is there a 1:1 correspondence between project_name and project_id?
PASS
Good concordance.
Issue count: 0/1
==========
Is the same frame name present in multiple samples?
PASS
frame_name's are all in their own samples
Issue count: 0/4
==========
Are the same phenotypes listed and following rules for mutual exclusion?
PASS
phenotype_calls and phenotype_label follows expected rules
==========
Are the same phenotypes included on all images?
PASS
Consistent phenotypes
Issue count: 0/4
==========
Are the same scored names included on all images?
PASS
Consistent scored_names
Issue count: 0/4
==========
Are the same regions represented the same with an image and across images?
PASS
Consistent regions
Issue count: 0/5
==========
Are the same regions listed matching a valid region_label
PASS
regions and region_label follows expected rules
==========
Do we have any region sizes so small they should consider being excluded?
WARNING
[
"Very small non-zero regions are included in the data['IrisSpatialFeatures', 'MEL2', 'MEL2_7', {'Margin': 495640, 'Tumor': 947369, 'Stroma': 116}]"
]
Issue count: 1/2
The cell phenotypes set prior to calling cartesian are the phenotypes available to plot.
from pythologist_test_images import TestImages
from plotnine import *
proj = TestImages().project('IrisSpatialFeatures')
cdf = TestImages().celldataframe('IrisSpatialFeatures')
cdf.db = proj
cart = cdf.cartesian(verbose=True,step_pixels=50,max_distance_pixels=75)
df,cols,rngtop = cart.rgb_dataframe(red='CD8+',green='SOX10+')
shape = cdf.iloc[0]['frame_shape']
(ggplot(df,aes(x='frame_x',y='frame_y',fill='color_str'))
+ geom_point(shape='h',size=4.5,color='#777777',stroke=0.2)
+ geom_vline(xintercept=-1,color="#555555")
+ geom_vline(xintercept=shape[1],color="#555555")
+ geom_hline(yintercept=-1,color="#555555")
+ geom_hline(yintercept=shape[0],color="#555555")
+ facet_wrap('frame_name')
+ scale_fill_manual(cols,guide=False)
+ theme_bw()
+ theme(figure_size=(8,8))
+ theme(aspect_ratio=shape[0]/shape[1])
+ scale_y_reverse()
)
from pythologist_test_images import TestImages
from plotnine import *
proj = TestImages().project('IrisSpatialFeatures')
cdf = TestImages().celldataframe('IrisSpatialFeatures')
cdf.db = proj
ch = cdf.db.qc().channel_histograms()
sub = ch.loc[(~ch['threshold_value'].isna())&(ch['channel_label']=='PDL1')]
(ggplot(sub,aes(x='bins',y='counts'))
+ geom_bar(stat='identity')
+ facet_wrap('frame_name')
+ geom_vline(aes(xintercept='threshold_value'),color='red')
+ theme_bw()
+ ggtitle('Thresholding of PDL1\ngiven image pixel intensities')
)The original component images were not available for IrisSpatialFeatures example, so pixel intensities are simulated and don't necessarily match the those which would have been used to set the original threshold values.
from pythologist_test_images import TestImages
from pythologist_reader.formats.inform.custom import CellProjectInFormCustomMask
from pythologist import SubsetLogic as SL
cpi = TestImages().project('IrisSpatialFeatures')
cdf = cpi.cdf
cdf.db = cpi
sub = cdf.loc[cdf['frame_name']=='MEL2_7'].dropna()
cont = sub.contacts().threshold('CD8+','CD8+/contact').contacts().threshold('SOX10+','SOX10+/contact')
cont = cont.threshold('CD8+','SOX10+/contact',
positive_label='CD8+ contact',
negative_label='CD8+').\
threshold('SOX10+','CD8+/contact',
positive_label='SOX10+ contact',
negative_label='SOX10+')
schema = [
{'subset_logic':SL(phenotypes=['OTHER']),
'edge_color':(50,50,50,255),
'watershed_steps':0,
'fill_color':(0,0,0,255)
},
{'subset_logic':SL(phenotypes=['SOX10+']),
'edge_color':(166,206,227,255),
'watershed_steps':0,
'fill_color':(0,0,0,0)
},
{'subset_logic':SL(phenotypes=['CD8+']),
'edge_color':(253,191,111,255),
'watershed_steps':0,
'fill_color':(0,0,0,0)
},
{'subset_logic':SL(phenotypes=['CD8+ contact']),
'edge_color':(253,191,111,255),
'watershed_steps':0,
'fill_color':(255,127,0,255)
},
{'subset_logic':SL(phenotypes=['SOX10+ contact']),
'edge_color':(166,206,227,255),
'watershed_steps':0,
'fill_color':(31,120,180,255)
}
]
sio = cont.segmentation_images().build_segmentation_image(schema,background=(0,0,0,255))
sio.write_to_path('test_edges',overwrite=True)MEL2_7
Image is zoomed-in and cropped to show the contours better.
This happens frequently because current InForm exports only permit two features to be scored per export
merged,fail = cdf1.merge_scores(cdf2,on=['sample_name','frame_name','x','y'])cdf.scored_names['PD1', 'PDL1']
cdf.phenotypes['CD8+', 'OTHER', 'SOX10+']
cdf.regions['Margin', 'Stroma', 'Tumor']
collapsed = cdf.collapse_phenotypes(['CD8+','OTHER'],'non-Tumor')
collapsed.phenotypes['SOX10+', 'non-Tumor']
Rename TUMOR to Tumor
renamed = cdf.rename_region('TUMOR','Tumor')renamed = cdf.rename_scored_calls({'PDL1 (Opal 520)':'PDL1'})Make CYTOK into CYTOK PDL1+ and CYTOK PDL1-
raw_thresh = raw.threshold('CYTOK','PDL1')CD68_CD163 = raw.threshold('CD68','CD163').\
threshold('CD68 CD163+','PDL1').\
threshold('CD68 CD163-','PDL1')generate counts and fractions of the current phenotypes and export them to a csv
cdf.counts().frame_counts().to_csv('my_frame_counts.csv')generate counts and fractions of the current phenotypes and export them to a csv
cdf.counts().sample_counts().to_csv('my_sample_counts.csv')The follow command creates a new CellDataFrame that has an additional binary feature representative of the contact with 'T cell' phenotype cells.
cdf = cdf.contacts().threshold('T cell')The follow command creates a new CellDataFrame that has an additional binary feature representative of being inside or outisde 75 microns of 'T cell' phenotype cells.
cdf = cdf.nearestneighbors().threshold('T cell','T cell/within 75um',distance_um=75)To ensure we are generating expected outs we can check against the outputs of IrisSpatialFeatures [github].
- Jupyter Notebook: Test against IrisSpatialFeatures outputs