--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/CommandSingleCellExtraction.py	Fri Mar 12 00:19:24 2021 +0000
@@ -0,0 +1,11 @@
+#Script for parsing command line arguments and running single-cell
+#data extraction functions
+#Joshua Hess
+import ParseInput
+import SingleCellDataExtraction
+
+#Parse the command line arguments
+args = ParseInput.ParseInputDataExtract()
+
+#Run the MultiExtractSingleCells function
+SingleCellDataExtraction.MultiExtractSingleCells(**args)

--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/ParseInput.py	Fri Mar 12 00:19:24 2021 +0000
@@ -0,0 +1,23 @@
+#Functions for parsing command line arguments for ome ilastik prep
+import argparse
+
+
+def ParseInputDataExtract():
+   """Function for parsing command line arguments for input to single-cell
+   data extraction"""
+
+#if __name__ == '__main__':
+   parser = argparse.ArgumentParser()
+   parser.add_argument('--masks',nargs='*')
+   parser.add_argument('--image')
+   parser.add_argument('--channel_names')
+   parser.add_argument('--output')
+   #parser.add_argument('--suffix')
+   args = parser.parse_args()
+   #Create a dictionary object to pass to the next function
+   dict = {'masks': args.masks, 'image': args.image,\
+    'channel_names': args.channel_names,'output':args.output}
+   #Print the dictionary object
+   print(dict)
+   #Return the dictionary
+   return dict

--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/SingleCellDataExtraction.py	Fri Mar 12 00:19:24 2021 +0000
@@ -0,0 +1,272 @@
+#Functions for reading in single cell imaging data
+#Joshua Hess
+
+#Import necessary modules
+import skimage.io
+import h5py
+import pandas as pd
+import numpy as np
+import os
+import skimage.measure as measure
+from pathlib import Path
+import csv
+
+import sys
+
+
+def MaskChannel(mask_loaded,image_loaded_z):
+    """Function for quantifying a single channel image
+
+    Returns a table with CellID according to the mask and the mean pixel intensity
+    for the given channel for each cell"""
+    print(f'Mask loaded: {mask_loaded.shape}', file=sys.stderr)
+    print(f'Image loaded: {image_loaded_z.shape}', file=sys.stderr)
+    dat = measure.regionprops(mask_loaded, image_loaded_z)
+    n = len(dat)
+    intensity_z = np.empty(n)
+    for i in range(n):
+        intensity_z[i] = dat[i].mean_intensity
+        # Clear reference to avoid memory leak -- see MaskIDs for explanation.
+        dat[i] = None
+    return intensity_z
+
+
+def MaskIDs(mask):
+    """This function will extract the CellIDs and the XY positions for each
+    cell based on that cells centroid
+
+    Returns a dictionary object"""
+
+    dat = measure.regionprops(mask)
+    n = len(dat)
+
+    # Pre-allocate numpy arrays for all properties we'll calculate.
+    labels = np.empty(n, int)
+    xcoords = np.empty(n)
+    ycoords = np.empty(n)
+    area = np.empty(n, int)
+    minor_axis_length = np.empty(n)
+    major_axis_length = np.empty(n)
+    eccentricity = np.empty(n)
+    solidity = np.empty(n)
+    extent = np.empty(n)
+    orientation = np.empty(n)
+
+    for i in range(n):
+        labels[i] = dat[i].label
+        xcoords[i] = dat[i].centroid[1]
+        ycoords[i] = dat[i].centroid[0]
+        area[i] = dat[i].area
+        major_axis_length[i] = dat[i].major_axis_length
+        minor_axis_length[i] = dat[i].minor_axis_length
+        eccentricity[i] = dat[i].eccentricity
+        solidity[i] = dat[i].solidity
+        extent[i] = dat[i].extent
+        orientation[i] = dat[i].orientation
+        # By clearing the reference to each RegionProperties object, we allow it
+        # and its cache to be garbage collected immediately. Otherwise memory
+        # usage creeps up needlessly while this function is executing.
+        dat[i] = None
+
+    IDs = {
+        "CellID": labels,
+        "X_centroid": xcoords,
+        "Y_centroid": ycoords,
+        "column_centroid": xcoords,
+        "row_centroid": ycoords,
+        "Area": area,
+        "MajorAxisLength": major_axis_length,
+        "MinorAxisLength": minor_axis_length,
+        "Eccentricity": eccentricity,
+        "Solidity": solidity,
+        "Extent": extent,
+        "Orientation": orientation,
+    }
+
+    return IDs
+
+
+def PrepareData(image,z):
+    """Function for preparing input for maskzstack function. Connecting function
+    to use with mc micro ilastik pipeline"""
+
+    image_path = Path(image)
+    print(f'{image_path} at {z}', file=sys.stderr)
+
+    #Check to see if image tif(f)
+    if image_path.suffix == '.tiff' or image_path.suffix == '.tif' or image_path.suffix == '.btf':
+        #Check to see if the image is ome.tif(f)
+        if  image.endswith(('.ome.tif','.ome.tiff')):
+            #Read the image
+            image_loaded_z = skimage.io.imread(image,img_num=z,plugin='tifffile')
+            #print('OME TIF(F) found')
+        else:
+            #Read the image
+            image_loaded_z = skimage.io.imread(image,img_num=z,plugin='tifffile')
+            #print('TIF(F) found')
+            # Remove extra axis
+            #image_loaded = image_loaded.reshape((image_loaded.shape[1],image_loaded.shape[3],image_loaded.shape[4]))
+
+    #Check to see if image is hdf5
+    elif image_path.suffix == '.h5' or image_path.suffix == '.hdf5':
+        #Read the image
+        f = h5py.File(image,'r+')
+        #Get the dataset name from the h5 file
+        dat_name = list(f.keys())[0]
+        ###If the hdf5 is exported from ilastik fiji plugin, the dat_name will be 'data'
+        #Get the image data
+        image_loaded = np.array(f[dat_name])
+        #Remove the first axis (ilastik convention)
+        image_loaded = image_loaded.reshape((image_loaded.shape[1],image_loaded.shape[2],image_loaded.shape[3]))
+        ###If the hdf5 is exported from ilastik fiji plugin, the order will need to be
+        ###switched as above --> z_stack = np.swapaxes(z_stack,0,2) --> z_stack = np.swapaxes(z_stack,0,1)
+
+    #Return the objects
+    return image_loaded_z
+
+
+def MaskZstack(masks_loaded,image,channel_names_loaded):
+    """This function will extract the stats for each cell mask through each channel
+    in the input image
+
+    mask_loaded: dictionary containing Tiff masks that represents the cells in your image.
+
+    z_stack: Multichannel z stack image"""
+
+    #Get the names of the keys for the masks dictionary
+    mask_names = list(masks_loaded.keys())
+    #Get the CellIDs for this dataset by using only a single mask (first mask)
+    IDs = pd.DataFrame(MaskIDs(masks_loaded[mask_names[0]]))
+    #Create empty dictionary to store channel results per mask
+    dict_of_chan = {m_name: [] for m_name in mask_names}
+    #Get the z channel and the associated channel name from list of channel names
+    print(f'channels: {channel_names_loaded}', file=sys.stderr)
+    print(f'num channels: {len(channel_names_loaded)}', file=sys.stderr)
+    for z in range(len(channel_names_loaded)):
+        #Run the data Prep function
+        image_loaded_z = PrepareData(image,z)
+
+        #Iterate through number of masks to extract single cell data
+        for nm in range(len(mask_names)):
+            #Use the above information to mask z stack
+            dict_of_chan[mask_names[nm]].append(MaskChannel(masks_loaded[mask_names[nm]],image_loaded_z))
+        #Print progress
+        print("Finished "+str(z))
+
+    #Iterate through the rest of the masks to modify names of channels and convert to data table
+    for nm in mask_names:
+        #Check if this is the first mask
+        if nm == mask_names[0]:
+            #Create channel names for this mask
+            new_names = [channel_names_loaded[i]+"_"+str(nm) for i in range(len(channel_names_loaded))]
+            #Convert the channel names list and the list of intensity values to a dictionary and combine with CellIDs and XY
+            dict_of_chan[nm] = pd.concat([IDs,pd.DataFrame(dict(zip(new_names,dict_of_chan[nm])))],axis=1)
+            #Get the name of the columns in the dataframe so we can reorder to histoCAT convention
+            cols = list(dict_of_chan[nm].columns.values)
+            #Reorder the list (Move xy position to end with spatial information)
+            cols.append(cols.pop(cols.index("X_centroid")))
+            cols.append(cols.pop(cols.index("Y_centroid")))
+            cols.append(cols.pop(cols.index("column_centroid")))
+            cols.append(cols.pop(cols.index("row_centroid")))
+            cols.append(cols.pop(cols.index("Area")))
+            cols.append(cols.pop(cols.index("MajorAxisLength")))
+            cols.append(cols.pop(cols.index("MinorAxisLength")))
+            cols.append(cols.pop(cols.index("Eccentricity")))
+            cols.append(cols.pop(cols.index("Solidity")))
+            cols.append(cols.pop(cols.index("Extent")))
+            cols.append(cols.pop(cols.index("Orientation")))
+            #Reindex the dataframe with new order
+            dict_of_chan[nm] = dict_of_chan[nm].reindex(columns=cols)
+        #Otherwise, add no spatial information
+        else:
+            #Create channel names for this mask
+            new_names = [channel_names_loaded[i]+"_"+str(nm) for i in range(len(channel_names_loaded))]
+            #Use the above information to mask z stack
+            dict_of_chan[nm] = pd.DataFrame(dict(zip(new_names,dict_of_chan[nm])))
+
+    #Concatenate all data from all masks to return
+    dat = pd.concat([dict_of_chan[nm] for nm in mask_names],axis=1)
+
+    #Return the dataframe
+    return dat
+
+
+def ExtractSingleCells(masks,image,channel_names,output):
+    """Function for extracting single cell information from input
+    path containing single-cell masks, z_stack path, and channel_names path."""
+
+    #Create pathlib object for output
+    output = Path(output)
+
+    #Check if header available
+    #sniffer = csv.Sniffer()
+    #sniffer.has_header(open(channel_names).readline())
+    #If header not available
+    #if not sniffer:
+        #If header available
+        #channel_names_loaded = pd.read_csv(channel_names)
+        #channel_names_loaded_list = list(channel_names_loaded.marker_name)
+    #else:
+        #print("negative")
+        #old one column version
+        #channel_names_loaded = pd.read_csv(channel_names,header=None)
+        #Add a column index for ease
+        #channel_names_loaded.columns = ["marker"]
+        #channel_names_loaded = list(channel_names_loaded.marker.values)
+
+    #Read csv channel names
+    channel_names_loaded = pd.read_csv(channel_names)
+    #Check for size of columns
+    if channel_names_loaded.shape[1] > 1:
+        #Get the marker_name column if more than one column (CyCIF structure)
+        channel_names_loaded_list = list(channel_names_loaded.marker_name)
+    else:
+        #old one column version -- re-read the csv file and add column name
+        channel_names_loaded = pd.read_csv(channel_names, header = None)
+        #Add a column index for ease and for standardization
+        channel_names_loaded.columns = ["marker"]
+        channel_names_loaded_list = list(channel_names_loaded.marker)
+
+    #Check for unique marker names -- create new list to store new names
+    channel_names_loaded_checked = []
+    for idx,val in enumerate(channel_names_loaded_list):
+        #Check for unique value
+        if channel_names_loaded_list.count(val) > 1:
+            #If unique count greater than one, add suffix
+            channel_names_loaded_checked.append(val + "_"+ str(channel_names_loaded_list[:idx].count(val) + 1))
+        else:
+            #Otherwise, leave channel name
+            channel_names_loaded_checked.append(val)
+
+    #Clear small memory amount by clearing old channel names
+    channel_names_loaded, channel_names_loaded_list = None, None
+
+    #Read the masks
+    masks_loaded = {}
+    #iterate through mask paths and read images to add to dictionary object
+    for m in masks:
+        m_full_name = os.path.basename(m)
+        m_name = m_full_name.split('.')[0]
+        masks_loaded.update({str(m_name):skimage.io.imread(m,plugin='tifffile')})
+
+    scdata_z = MaskZstack(masks_loaded,image,channel_names_loaded_checked)
+    #Write the singe cell data to a csv file using the image name
+
+    im_full_name = os.path.basename(image)
+    im_name = im_full_name.split('.')[0]
+    scdata_z.to_csv(str(Path(os.path.join(str(output),str(im_name+".csv")))),index=False)
+
+
+def MultiExtractSingleCells(masks,image,channel_names,output):
+    """Function for iterating over a list of z_stacks and output locations to
+    export single-cell data from image masks"""
+
+    print("Extracting single-cell data for "+str(image)+'...')
+
+    #Run the ExtractSingleCells function for this image
+    ExtractSingleCells(masks,image,channel_names,output)
+
+    #Print update
+    im_full_name = os.path.basename(image)
+    im_name = im_full_name.split('.')[0]
+    print("Finished "+str(im_name))

--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/macros.xml	Fri Mar 12 00:19:24 2021 +0000
@@ -0,0 +1,24 @@
+<?xml version="1.0"?>
+<macros>
+    <xml name="requirements">
+        <requirements>
+            <requirement type="package" version="3.6.10">python</requirement>
+            <requirement type="package" version="0.17.2">scikit-image</requirement>
+            <requirement type="package" version="2.10.0">h5py</requirement>
+            <requirement type="package" version="1.0.4">pandas</requirement>
+            <requirement type="package" version="1.18.5">numpy</requirement>
+            <requirement type="package" version="1.0.1">pathlib</requirement>
+        </requirements>
+    </xml>
+
+    <xml name="version_cmd">
+        <version_command>echo @VERSION@</version_command>
+    </xml>
+    <xml name="citations">
+        <citations>
+        </citations>
+    </xml>
+
+    <token name="@VERSION@">1.3.1</token>
+    <token name="@CMD_BEGIN@">python ${__tool_directory__}/CommandSingleCellExtraction.py</token>
+</macros>

--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/quantification.xml	Fri Mar 12 00:19:24 2021 +0000
@@ -0,0 +1,71 @@
+<tool id="quantification" name="Quantification" version="@VERSION@.5" profile="17.09">
+    <description>Single cell quantification, a module for single-cell data extraction given a segmentation mask and multi-channel image.</description>
+    <macros>
+        <import>macros.xml</import>
+    </macros>
+ 
+    <expand macro="requirements"/>
+    @VERSION_CMD@
+
+    <command detect_errors="exit_code"><![CDATA[
+    ln -s '$image' '${image.name}'.ome.tiff;
+    ln -s '$primary_mask' '${primary_mask.name}'.ome.tiff; 
+    #for $mask in $supp_masks:
+    ln -s '$mask' '${mask.name}'.ome.tiff;
+    #end for
+
+    mkdir ./tool_out;
+
+    @CMD_BEGIN@
+    
+    --masks 
+    '${primary_mask.name}'.ome.tiff
+    #if $supp_masks
+    #for $mask in $supp_masks:
+    '${mask.name}'.ome.tiff
+    #end for
+    #end if
+
+    --image '${image.name}'.ome.tiff
+    --output ./tool_out
+    --channel_names '$channel_names';
+
+    mv ./tool_out/*.csv ./tool_out/quantified.csv;
+    ]]></command>
+
+    <inputs>
+        <param name="image" type="data" format="tiff" label="Registered TIFF"/>
+        <param name="primary_mask" type="data" format="tiff" label="Primary Cell Mask"/>
+        <param name="supp_masks" type="data" multiple="true" optional="true" format="tiff" label="Additional Cell Masks"/>
+        <param name="channel_names" type="data" format="csv" label="Marker Channels"/>
+    </inputs>
+
+    <outputs>
+        <data format="csv" name="quant_out" from_work_dir="./tool_out/quantified.csv" label="${tool.name} on ${on_string}"/>
+    </outputs>
+    <help><![CDATA[
+# Single cell quantification
+Module for single-cell data extraction given a segmentation mask and multi-channel image. The CSV structure is aligned with histoCAT output.
+
+**CommandSingleCellExtraction.py**:
+
+* `--masks` Paths to where masks are stored (Ex: ./segmentation/cellMask.tif) -> If multiple masks are selected the first mask will be used for spatial feature extraction but all will be quantified
+
+* `--image` Path to image(s) for quantification.  (Ex: ./registration/*.h5) -> works with .h(df)5 or .tif(f)
+
+* `--output` Path to output directory. (Ex: ./feature_extraction)
+
+* `--channel_names` csv file containing the channel names for the z-stack (Ex: ./my_channels.csv)
+
+# Run script
+`python CommandSingleCellExtraction.py --masks ./segmentation/cellMask.tif ./segmentation/membraneMask.tif --image ./registration/Exemplar_001.h5  --output ./feature_extraction --channel_names ./my_channels.csv`
+
+# Main developer
+Denis Schapiro (https://github.com/DenisSch)
+
+Joshua Hess (https://github.com/JoshuaHess12)
+
+Jeremy Muhlich (https://github.com/jmuhlich)
+    ]]></help>
+    <expand macro="citations" />
+</tool>