MATLAB: How to use matlab to count these particles

image analysisImage Processing Toolboxneurosphereparticle counting

So I have some neurospheres in a dish.
After I take these images, I run them through imagej. I sharpen the picture, "despeckle" the image, add a guassian blur, and then find the edges of the image. The end result of that process looks like this:
After this process, I take the image and run it through matlab's thresholding. The image then looks like:
Now I'd like to use some surface mapping and create peaks and valleys and count my neurospheres. The process I want to do is similar to the process found here.
I'd like to be able to count my neurospheres, come up with an area for each one, and then also have a diameter, and be able to discriminate each sphere by a minimum size diameter.
Would anyone be able to help?
Thanks,
Tushar The Ohio State University

Best Answer

Hi Tushar
this is John BG <mailto:jb2012@sky.com jb2012@sky.com>
1.- Acquiring petri image, using green layer only, manually binarising the whole image.
Play with threshold th1
clear all;clc;close all
A=imread('blood_cells_count.jpg');
% A2=A(:,:,1)+A(:,:,2)+A(:,:,3);    % a bit more contrast
A2=A(:,:,2);  % since green marker used no need for red blue layers
th1=190;  
A2(A2>th1)=255;
A2(A2<th1)=0;
figure(1);imshow(A);
figure(2);imshow(A2);
.
2.- Filling up holes in binarised cells
A20=~A2;
area_threshold=6;
A21 = bwareaopen(A20,area_threshold);
figure(10);imshow(A21);
A22=imfill(A21, 'holes');
figure(11); imshow(A22);
.
3.- It may not be really needed, but just in case, filling up and plotting cell contours
[B,L] = bwboundaries(A22,'noholes');
map=zeros(length(B),3);cmap=colormap(map);
figure(12);imshow(label2rgb(L,cmap, [.5 .5 .5]))
hold on
for k = 1:length(B)
   boundary = B{k};
   plot(boundary(:,2), boundary(:,1), 'r', 'LineWidth', 1)
end
.
.
4.- The asked cells count is:
length(B)
   ans =
      398
.
5.- Add a halo around each binarised blood cell, neurons are hairy aren't they?
PSF = fspecial('gaussian',5,5);
A3 = deconvlucy(uint8(255*(~A21)),PSF,5);
figure(3);imshow(A3);
.
Comment:
using area threshold may remove a few small cells. Sample arrowed small specks are removed but the circled small cell is also removed.
Perhaps next step would be to consider removing small AND no green marker (dark only) specks.
.
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thanks in advance
John BG
<mailto:jgb2012@sky.com jgb2012@sky.com>
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