Counting impacts on a target (Traces of heavy ions beam) with the help of kmeans clustering.

When studying effect of ionizing radiation (heavy ions with high LET) on cells, the fluence (particules/cm²) of the particles beam must be known. Detectors in the GANIL (Caen, France) particles accelerator can estimate the fluence. However, when irradiating cells, radiobiologists (CEA CIRIL) need to localize the impacts of the ions and to count the number of impact per cell.

The application TRACES (scripts for the aphelion application) was developped: It was capable of recording and saving images from a camera, segmenting and counting traces: 

The application was designed to analyse images as the following:

raw image of traces

Counting impacts  in a python notebook:

Contrary to the TRACES application, the following jupyter notebook can't capture images (It should be possible to do it in a python script with the cv2 module) but it can perform some automatic classification.

Distinguishing traces resulting from one or several impacts is a classification issue which can be explored with unsupervised classifier such k-means clustering:

chromosome classification:workflow

The work-flow from raw DAPI image to two features scatter plot can be summarised as follow:

Yellow:to be written

Combining five metaphases, with two features (the area of the segmented particles and the ratio between the particle area and the area of convex hull of the particle) yields the following scatter plot where three clusters can be distinguished:

• blue cluster: nuclei
• red cluster: overlapping chromosomes
• green cluster:single chromosomes
• pink cluster:particles resulting from segmentation artefacts, some corresponds to nuclei touching the image border and should have been classified as nuclei.

As seen in the previous post the green and red clusters partially overlap. The graphic was build as follow with pandas (with some help):

import KarIO
import os,csv
import pylab
import pandas
#make a configurator object
config=KarIO.ClassifConf()
#build the the name feature file
##list all the feature files
##répertoire courant : os.listdir(os.getcwd())
featurespath=os.path.join(os.getcwd(),"Results","features")
labelpath=os.path.join(os.getcwd(),"Results","labels")
#print featurespath
##open features csv files
featuresFilesList=os.listdir(featurespath)
labelsFilesList=os.listdir(labelpath)
##
metalist=[0,1,2,3,4]
#metaphase=2
def makeDataShape(meta):
    featfile=config.user+'-'+config.slide+'-'+config.metaphases_list[meta]+'-'+config.counterstain+'.csv'
    labelfile='shapeCateg-'+featfile
    fea=pandas.read_csv(os.path.join(featurespath,featfile),header=None,index_col=None,names=['particle','ratio','area'])
    lab=pandas.read_csv(os.path.join(labelpath,labelfile),header=None,index_col=None,names=['particle','type'])
    del lab['particle']    
    #merge columns:features and label
    data=fea.join(lab)
    data.insert(0,'meta',config.metaphases_list[meta])
    #print data
    return data
bigdata=makeDataShape(0)
for meta in metalist:
    bigdata=bigdata.append(makeDataShape(meta),ignore_index=True)
single=bigdata[bigdata['type']=='single']
touching=bigdata[bigdata['type']=='touching']
nuclei=bigdata[bigdata['type']=='nuclei']
dusts=bigdata[bigdata['type']=='dusts']
##ploting with pylab
#different colors according to the category
fig=pylab.figure()
ax = fig.add_subplot(111)
ax.scatter(single['ratio'],single['area'],c='green',marker='o')
ax.scatter(touching['ratio'],touching['area'],c='red',marker='o')
ax.scatter(nuclei['ratio'],nuclei['area'],c='blue',marker='o')
ax.scatter(dusts['ratio'],dusts['area'],c='pink',marker='o')
pylab.show()

chromosomes, overlapping chromosomes and nuclei

In an attempt to find features to distinguish chromosomes from overlapping chromosomes or from nuclei or small image segmentation artifacts ("dusts"), the area and the convex-hull area of isolated particles were computed after segmentation of a metaphasic chromosome image.

Image segmentation and convex-hull area computation were performed with scripts written previously. The features were saved in csv files.

miniKar produced an other csv file where the particle category (label) was saved.

This is an opportunity to handle the data with the pandas library and to see from this limited data set if the two features may be good candidates to classify at least particles in one of the three categories (single, overlapping, nuclei). A features file and the labels file were loaded, merged in one data frame. The features were filtered according to the label (the particle category) and then were displayed in a scatter plot:

green:single chromosomes; red overlapping chromosomes; blue:nuclei

The normalized area is the 1000*area of a particle divided by the image size (1024x1536). From this scatter plot, it's clear that at least an other feature will be necessary to separate the single chromosomes from the overlapping chromosomes. This may be due to the chromosomes bending.

A surprising thing, the ratio should be such: area/convexhull <1 and it is not true for all the particles.

import KarIO
import os,csv
import pylab
import pandas
#make a configurator object
config=KarIO.ClassifConf()
#build the the name feature file
##list all the feature files
##répertoire courant : os.listdir(os.getcwd())
featurespath=os.path.join(os.getcwd(),"Results","features")
labelpath=os.path.join(os.getcwd(),"Results","labels")
#print featurespath
##open features csv files
featuresFilesList=os.listdir(featurespath)
labelsFilesList=os.listdir(labelpath)
##
#let's find a pair of feature file/label file
#open with pandas
#two files match if they come from the same metaphase

featfile=config.user+'-'+config.slide+'-'+config.metaphases_list[2]+'-'+config.counterstain+'.csv'
labelfile='shapeCateg-'+featfile

fea=pandas.read_csv(os.path.join(featurespath,featfile),header=None,names=['particle','ratio','area'])
lab=pandas.read_csv(os.path.join(labelpath,labelfile),header=None,names=['particle','type'])
#merge columns:features and label
data=fea.join(lab)
single=data[data['type']=='single']
touching=data[data['type']=='touching']
nuclei=data[data['type']=='nuclei']
dusts=data[data['type']=='dusts']

##ploting with pylab
#different colors according to the category

fig=pylab.figure()
ax = fig.add_subplot(111)
ax.scatter(single['ratio'],single['area'],c='green',marker='o')
ax.scatter(touching['ratio'],touching['area'],c='red',marker='o')
ax.scatter(nuclei['ratio'],nuclei['area'],c='blue',marker='o')
pylab.show()