My code doesn't work the second time

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Ayesha ch 2016-4-12

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https://ww2.mathworks.cn/matlabcentral/answers/278531-my-code-doesn-t-work-the-second-time

编辑： Stephen23 2016-4-12

I'm using the following code for nucleus subtraction and then median filtering. My code works fine the first time but the second time I run it, the subtraction doesn't work. Could anyone please tell me what I'm doing wrong?

close all;
he=imread('2.jpg');
% imshow(he), title('original');
% filter
h=[1 1 1; 1 1 1; 1 1 1]/9;
b=imfilter(he,h);
% figure, imshow(b), title('filtered');
% segmentation
cform = makecform('srgb2lab');
lab_he = applycform(he,cform);
ab = double(lab_he(:,:,2:3));
nrows = size(ab,1);
ncols = size(ab,2);
ab = reshape(ab,nrows*ncols,2);
nColors = 3;
% repeat the clustering 3 times to avoid local minima
[cluster_idx, cluster_center] = kmeans(ab,nColors,'distance','sqEuclidean', ...
                                      'Replicates',3);
                                  pixel_labels = reshape(cluster_idx,nrows,ncols);
% imshow(pixel_labels,[]), title('image labeled by cluster index');
segmented_images = cell(1,3);
rgb_label = repmat(pixel_labels,[1 1 3]);
for k = 1:nColors
    color = he;
    color(rgb_label ~= k) = 0;
    segmented_images{k} = color;
end
% imshow(segmented_images{1}), title('objects in cluster 1');
% imshow(segmented_images{2}), title('objects in cluster 2');
% figure, imshow(segmented_images{3}), title('objects in cluster 3');
mean_cluster_value = mean(cluster_center,2);
[tmp, idx] = sort(mean_cluster_value);
blue_cluster_num = idx(1);
L = lab_he(:,:,1);
blue_idx = find(pixel_labels == blue_cluster_num);
L_blue = L(blue_idx);
is_light_blue = im2bw(L_blue,graythresh(L_blue));
nuclei_labels = repmat(uint8(0),[nrows ncols]);
nuclei_labels(blue_idx(is_light_blue==false)) = 1;
nuclei_labels = repmat(nuclei_labels,[1 1 3]);
blue_nuclei = he;
blue_nuclei(nuclei_labels ~= 1) = 0;
% figure, imshow(blue_nuclei), title('blue nuclei');
% segmentation ends here
% erosion
se = strel('ball',9,9);
e=imerode(blue_nuclei,se);
% figure, imshow(e), title('erosion');
% reconstruction erosion as marker
IM = imreconstruct(e,segmented_images{2});
% figure, imshow(IM), title('reconstructed');
% In as Mask on original image
In=b.*IM;
% figure, imshow(In), title('masked');
% Subtraction 
I=imsubtract(b,In);
figure, imshow(I), title('subtracted');
figure, imshow(he);
% median filter
g=rgb2gray(I);
J = imnoise(g,'salt & pepper',0.02);
K = medfilt2(g, [3 3]);
figure, imshow(J), figure, imshow(K);