Source

As part of our work on bacterial biofilms, we have been developing a new program for quantification of confocal microscopy image stacks. We have undertaken this for faster quantification of a large number of images and to obtain more qualitative and quantitative information from our image stacks than is currently available. Toward this goal, the following scripts have been written in Matlab, tested, and published.

The scripts are published in the Journal of Microbiological Methods. We request that you acknowledge the use of these scripts in any publications or other distributions of the source code. The proper references are:

S. Sommerfeld Ross, J.M. Reinhardt, J. Fiegel. Enhanced analysis of bacteria susceptibility in connected biofilms. J. Microbiol. Methods 2012. 90(1): 9-14.

- colocalization adjustment

- connected-biofilm and unconnected bacteria quantification and image generation

S. Sommerfeld Ross, M.L. Falsetta Wood, M.A. Apicella, J.M. Reinhardt, J. Fiegel. Quantification of confocal images of biofilms grown on irregular surfaces. J. Microbiol. Methods. 2014; 100: 111-120.

- modified connected volume filtration (MCVF)

- modified substratum coverage

- percent association (quantification of bacteria colocalized with stained component)

Text files containing the scripts are here or you can copy them below:
Connected and Unconnected Biofilm Bacteria

MCVF and Irregular Surface Scripts

The colocalization adjustment identifies the colocalized pixels and removes the green fluorescent signal from these pixels, retaining only the red fluorescent signal. This algorithm is given by:

for i=1:Lastimageslice;

for Rowpixel=1:Numberpixelsinrow;

for Colpixel=1:Numberpixelsincolumn;

if double(ChannelRedThresholdLogic(Rowpixel,Colpixel,i))==1 && double(ChannelGreenThresholdLogic(Rowpixel,Colpixel,i))==1 || double(ChannelRedThresholdLogic(Rowpixel,Colpixel,i))==1;

AllRedPixels(Rowpixel, Colpixel,i)=1;

AllGreenPixels(Rowpixel,Colpixel, i)=0;

elseif double(ChannelGreenThresholdLogic(Rowpixel,Colpixel,i))==1 && double(ChannelRedThresholdLogic(Rowpixel,Colpixel,i))==0;

AllRedPixels(Rowpixel,Colpixel,i)=0;

AllGreenPixels(Rowpixel,Colpixel, i)=1;

elseif double(ChannelRedThresholdLogic(Rowpixel,Colpixel,i))==0 || double(ChannelGreenThresholdLogic(Rowpixel,Colpixel,i))==0;

AllRedPixels(Rowpixel,Colpixel,i)=0;

AllGreenPixels(Rowpixel,Colpixel, i)=0;

end;

end;

end;

end;

where the Lastimageslice, Numberpixelsinrow, and Numberpixelsincolumn values are obtained from the “.info” file. ChannelRedThresholdLogic is the binary image obtained after thresholding for the red fluorescent channel. ChannelGreenThresholdLogic is the binary image obtained after thresholding for the green fluorescent channel.

To maintain both the connected-biofilm bacteria pixels and the unconnected bacteria pixels, we use the following algorithm:

for i=1:Lastimageslice;

for Rowpixel=1:Numberpixelsinrow;

for Colpixel=1:Numberpixelsincolumn;


if AllRedPixels(Rowpixel,Colpixel,i) ==1 && filt_images(Rowpixel,Colpixel,i) ==1;

ConnectedBiofilmRedPixel(Rowpixel,Colpixel,i)=1;

UnconnectedBacteriaRedPixel(Rowpixel,Colpixel,i)=0;

elseif AllRedPixels(Rowpixel,Colpixel,i) ==1 && filt_images(Rowpixel,Colpixel,i) ==0;

ConnectedBiofilmRedPixel(Rowpixel,Colpixel,i)=0;

UnconnectedBacteriaRedPixel(Rowpixel,Colpixel,i)=1;

elseif AllRedPixels(Rowpixel,Colpixel,i) ==0 && filt_images(Rowpixel,Colpixel,i) ==1 || AllRedPixels(Rowpixel,Colpixel,i) ==0 && filt_images(Rowpixel,Colpixel,i) ==0;

ConnectedBiofilmRedPixel(Rowpixel,Colpixel,i)=0;

UnconnectedBacteriaRedPixel(Rowpixel,Colpixel,i)=0;

end;


if AllGreenPixels(Rowpixel,Colpixel,i)==1 && filt_images(Rowpixel,Colpixel,i) ==1;

ConnectedBiofilmGreenPixel(Rowpixel,Colpixel,i)=1;

UnconnectedBacteriaGreenPixel(Rowpixel,Colpixel,i)=0;

elseif AllGreenPixels(Rowpixel,Colpixel,i)==1 && filt_images(Rowpixel,Colpixel,i) ==0;

ConnectedBiofilmGreenPixel(Rowpixel,Colpixel,i)=0;

UnconnectedBacteriaGreenPixel(Rowpixel,Colpixel,i)=1;

elseif AllGreenPixels(Rowpixel,Colpixel,i)==0 && filt_images(Rowpixel,Colpixel,i) ==0 || AllGreenPixels(Rowpixel,Colpixel,i)==0 && filt_images(Rowpixel,Colpixel,i) ==1;

ConnectedBiofilmGreenPixel(Rowpixel,Colpixel,i)=0;

UnconnectedBacteriaGreenPixel(Rowpixel,Colpixel,i)=0;

end;

end;

end;

end;

where the Lastimageslice, Numberpixelsinrow, and Numberpixelsincolumn values are obtained from the “.info” file. AllRedPixels and AllGreenPixels were defined in the colocalization adjustment algorithm. The filt_images is the output from the connected volume filtration. This algorithm compares each pixel in the image sequence to the obtained color channel pixels of interest (AllRedPixels or AllGreenPixels) and the filt-images output (1=connected-biofilm bacteria, 0=unconnected bacteria or background noise).

The image matrices can be saved as separate “.tif” image sequences. These “.tif” image sequences are created via:

folder='Sample Name';

parentfolderConnectedBiofilm=strcat(folder,'\ConnectedBiofilmBacteria\');

parentfolderUnconnectedBacteria=strcat( folder,'\UnconnectedBacteria\');

mkdir(parentfolderConnectedBiofilm);

mkdir(parentfolderUnconnectedBacteria);


for i=1:Lastimageslice;

FinalConnectedBiofilmBacteriaRed(:,:,i)=ConnectedBiofilmRedPixel(:,:,i).*double(OriginalBacteria(:,:,i));

name = ['ConnectedBiofilmBacteriaRed',int2str(i),'.tif'];

filename= strcat(parentfolderConnectedBiofilm, name);

imwrite(uint8(FinalConnectedBiofilmBacteriaRed(:,:,i)),filename,'tiff');

FinalConnectedBiofilmBacteriaGreen(:,:,i)=ConnectedBiofilmGreenPixel(:,:,i).*double(OriginalBacteria(:,:,i));

name = ['ConnectedBiofilmBacteriaGreen',int2str(i),'.tif'];

filename= strcat(parentfolderConnectedBiofilm, name);

imwrite(uint8(FinalConnectedBiofilmBacteriaGreen(:,:,i)),filename,'tiff');

FinalUnconnectedBacteriaRed(:,:,i)=UnconnectedBacteriaRedPixel(:,:,i).*double(OriginalBacteria(:,:,i));

name = ['UnconnectedBacteriaRed',int2str(i),'.tif'];

filename= strcat(parentfolderUnconnectedBacteria, name);

imwrite(uint8(FinalUnconnectedBacteriaRed(:,:,i)),filename,'tiff');

FinalUnconnectedBacteriaGreen(:,:,i)=UnconnectedBacteriaGreenPixel(:,:,i).*double(OriginalBacteria(:,:,i));

name = ['UnconnectedBacteriaGreen',int2str(i),'.tif'];

filename= strcat(parentfolderUnconnectedBacteria, name);

imwrite(uint8(FinalUnconnectedBacteriaGreen(:,:,i)),filename,'tiff');

end;

where folders are made to organize the produced images and then the images are developed by multiplying the binary image files (ConnectedBiofilmRedPixel, ConnectedBiofilmGreenPixel, UnconnectedBacteriaRedPixel, and UnconnectedBacteriaGreenPixel) by the original bacteria image (OriginalBacteria) with gray scale values between 1 and 256 for 8-bit images. The “.tif” image sequences can be visualized in 3D rendering software, such as Volocity (PerkinElmer, Waltham, MA).

The percent dead (red) and live (green) bacterial populations can be quantified by the following scripts:

PercentOverallConnectedBiofilmRed=sum(sum(sum(ConnectedBiofilmRedPixel(:,:,:))))./(sum(sum(sum(ConnectedBiofilmRedPixel(:,:,:))))+sum(sum(sum(ConnectedBiofilmGreenPixel(:,:,:)))))*100;

PercentOverallConnectedBiofilmGreen=sum(sum(sum(ConnectedBiofilmGreenPixel(:,:,:))))./(sum(sum(sum(ConnectedBiofilmRedPixel(:,:,:))))+sum(sum(sum(ConnectedBiofilmGreenPixel(:,:,:)))))*100;

PercentOverallUnconnectedBacteriaRed=sum(sum(sum(UnconnectedBacteriaRedPixel(:,:,:))))./(sum(sum(sum(UnconnectedBacteriaRedPixel(:,:,:))))+sum(sum(sum(UnconnectedBacteriaGreenPixel(:,:,:)))))*100;

PercentOverallUnconnectedBacteriaGreen=sum(sum(sum(UnconnectedBacteriaGreenPixel(:,:,:))))./(sum(sum(sum(UnconnectedBacteriaRedPixel(:,:,:))))+sum(sum(sum(UnconnectedBacteriaGreenPixel(:,:,:)))))*100;

for i=1:Lastimageslice;

PercentRedConnectedBiofilmBySlice(i)=sum(sum(ConnectedBiofilmRedPixel(:,:,i)))./(sum(sum(ConnectedBiofilmRedPixel(:,:,i)))+sum(sum(ConnectedBiofilmGreenPixel(:,:,i))))*100;

PercentGreenConnectedBiofilmBySlice(i)=sum(sum(ConnectedBiofilmGreenPixel(:,:,i)))./(sum(sum(ConnectedBiofilmRedPixel(:,:,i)))+sum(sum(ConnectedBiofilmGreenPixel(:,:,i))))*100;

PercentRedUnconnectedBacteriaBySlice(i)=sum(sum(UnconnectedBacteriaRedPixel(:,:,i)))./(sum(sum(UnconnectedBacteriaRedPixel(:,:,i)))+sum(sum(UnconnectedBacteriaGreenPixel(:,:,i))))*100;

PercentGreenUnconnectedBacteriaBySlice(i)=sum(sum(UnconnectedBacteriaGreenPixel(:,:,i)))./(sum(sum(UnconnectedBacteriaRedPixel(:,:,i)))+sum(sum(UnconnectedBacteriaGreenPixel(:,:,i))))*100;

end;

The MCVF and irregular surface scripts are here:

global lsm
%Test Image 1
%Original Image
OriginalSurface(:,:,1)=[15,15,15,180,180,150,150,150,150,150;
                  20,20,20,10,190,190,190,190,190,175;
                  20,20,20,10,180,180,190,185,180,185;
                  20,20,20,10,10,185,175,180,180,180];
             
OriginalSurface(:,:,2)=[10,185,10,20,20,20,10,20,20,20;
                  10,180,180,190,20,20,20,10,10,10;
                  10,180,185,185,20,190,190,185,20,20;
                  10,190,175,175,15,180,185,190,175,185];
             
OriginalSurface(:,:,3)=[20,20,10,10,10,20,20,20,20,20;
                  10,20,20,20,20,10,10,20,20,20;
                  10,185,20,20,20,20,20,20,30,30;
                  10,170,30,30,20,20,20,10,10,10];
             
OriginalSurface(:,:,4)=[10,10,10,20,40,20,20,20,30,40;
                  60,30,30,30,30,20,30,20,20,20;
                  30,30,50,50,50,30,30,30,30,30;
                  20,20,30,30,30,20,20,20,20,30];
             
             
OriginalBacteria(:,:,1)=[50,50,30,30,30,50,40,40,30,30;
                  30,40,20,20,20,30,50,50,30,70;
                  30,70,70,50,50,50,30,30,30,30;
                  30,40,40,30,20,20,20,60,50,50];
             
OriginalBacteria(:,:,2)=[40,40,40,185,30,30,30,190,40,40;
                  40,40,20,20,168,185,179,20,40,40;
                  40,20,10,20,190,10,10,10,145,155;
                  15,10,10,30,30,20,20,20,30,50];
         
OriginalBacteria(:,:,3)=[30,30,20,150,185,20,50,50,50,20;
                  180,130,20,145,20,30,30,30,50,50;
                  40,20,40,20,129,180,185,175,190,40;
                  10,40,40,20,175,20,40,175,20,20];
             
OriginalBacteria(:,:,4)=[40,30,40,180,175,50,40,40,30,40;
                  15,20,20,160,165,40,180,30,30,40;
                  15,40,20,185,190,40,190,160,40,40;
                  30,30,30,30,175,175,180,185,30,30];
             
             
%Image After Thresholding (Separate Biofilm Components From Background
%Noise)
Surface(:,:,1)=[0,0,0,1,1,1,1,1,1,1;
                  0,0,0,0,1,1,1,1,1,1;
                  0,0,0,0,1,1,1,1,1,1;
                  0,0,0,0,0,1,1,1,1,1];
             
Surface(:,:,2)=[0,1,0,0,0,0,0,0,0,0;
                  0,1,1,1,0,0,0,0,0,0;
                  0,1,1,1,0,1,1,1,0,0;
                  0,1,1,1,0,1,1,1,1,1];
             
Surface(:,:,3)=[0,0,0,0,0,0,0,0,0,0;
                  0,0,0,0,0,0,0,0,0,0;
                  0,1,0,0,0,0,0,0,0,0;
                  0,1,0,0,0,0,0,0,0,0];
             
Surface(:,:,4)=[0,0,0,0,0,0,0,0,0,0;
                  0,0,0,0,0,0,0,0,0,0;
                  0,0,0,0,0,0,0,0,0,0;
                  0,0,0,0,0,0,0,0,0,0];
         
            
Bacteria(:,:,1)=[0,0,0,0,0,0,0,0,0,0;
                  0,0,0,0,0,0,0,0,0,0;
                  0,0,0,0,0,0,0,0,0,0;
                  0,0,0,0,0,0,0,0,0,0];
             
Bacteria(:,:,2)=[0,0,0,1,0,0,0,1,0,0;
                  0,0,0,0,1,1,1,0,0,0;
                  0,0,0,0,1,0,0,0,1,1;
                  0,0,0,0,0,0,0,0,0,0];
         
Bacteria(:,:,3)=[0,0,0,1,1,0,0,0,0,0;
                  1,1,0,1,0,0,0,0,0,0;
                  0,0,0,0,1,1,1,1,1,0;
                  0,0,0,0,1,0,0,1,0,0];
             
Bacteria(:,:,4)=[0,0,0,1,1,0,0,0,0,0;
                  0,0,0,1,1,0,1,0,0,0;
                  0,0,0,1,1,0,1,1,0,0;
                  0,0,0,0,1,1,1,1,0,0];
             
             
             
pixelCol=10;
pixelRow=4;
totalpixel=pixelCol.*pixelRow;  
%calibration to microns
micronx=0.5;
microny=0.5;
micronz=1;
%Number of image slices
yy=4;
lsm.yy=yy;
             
             
% Connected Volume Filtration (CVF) of the Bacteria and Surface
% Connected volume filtration code was provided open source by Dr. Arne Heydorn in his
% software program COMSTAT.  Permission was obtained on March 7, 2012, by Dr. Heydorn to
% use his CVF algorithm.  Source code can be obtained by Dr. Heydorn at www.imageanalysis.dk.
%
% License for CVF
% Redistribution and use in source and binary forms, with or without
% modification, are permitted provided that the following conditions are met:
%
% 1. If the program is modified, redistributions must include a notice
% indicating that the redistributed program is not identical to the software
% distributed by the Department of Microbiology, Technical University of Denmark.
% Redistributions must also include a notice indicating that the redistributed
% program includes software developed by the Department of Microbiology,
% Technical University of Denmark.
%
% 2. All advertising materials mentioning features or use of this software must
% display the following acknowledgment: This product includes software developed
% by the Department of Microbiology, Technical University of Denmark.
%
% We also request that use of this software be cited in publications as
%
% Heydorn, A., Nielsen, A.T., Hentzer, M., Sternberg, C., Givskov, M., Ersbøll,
% B.K., Molin, S. (2000) Quantification of biofilm structures by the novel computer
% program COMSTAT. Microbiology 146 (10) 2395-2407
%
% The software is provided "AS-IS" and without warranty of any kind, express,
% implied or otherwise, including without limitation, any warranty of merchantability
% or fitness for a particular purpose. In no event shall the Technical University
% of Denmark or the authors be liable for any special, incidental, indirect or
% consequential damages of any kind, or any damages whatsoever resulting from loss
% of use, data or profits, whether or not advised of the possibility of damage, and
% on any theory of liability, arising out of or in connection with the use or
% performance of this software. This code was written using MATLAB 3.1 (MathWorks,
% www.mathworks.com) and may be subject to certain additional restrictions
% as a result.
 
 
%Modification of the CVF code 
%April 2012:  Stacy Sommerfeld Ross modified the CVF from Dr. Heydorn to fit her application
%and variables (see below).  This modified CVF (MCVF) is used to complete connected
%volume filtration on bacteria grown on uneven surfaces, in this case Surface.
%This code is provided "as is" without warranty.  
 
 
%Connected Volume Filtration for Bacteria and Surface
BiofilmConnectedtoSurface=zeros(pixelRow, pixelCol, yy);
 
%Classifying Starting Point for Connected Volume Filtration
BiofilmConnectedtoSurface(:,:,1)=Bacteria(:,:,1); %This allows for any biofilm growing directly on the substratum
HorizontalGrowthForBacteriaAttachedtoBStart(:,:,1)=Bacteria(:,:,1);
   
for i = 2:yy;
 commonarea(:,:,i-1)=uint8(Surface(:,:,i-1).*double((Bacteria(:,:,i)>0))); % what pixels are common between the two layers
 [vectorx,vectory]=find(commonarea(:,:,i-1)); % vectors containing the nonzero elements of commonarea
 BiofilmConnectedtoSurface(:,:,i)=commonarea(:,:,i-1); %  expand the area enherited from the layer below
 HorizontalGrowthForBacteriaAttachedtoBStart(:,:,i)=double(bwselect(Bacteria(:,:,i),vectory,vectorx,8));
 clear vectorx
 clear vectory
end;
 
BiofilmConnectedtoSurfaceforSubstratum=BiofilmConnectedtoSurface;
for i=1:yy
    for Rowpixel=1:pixelRow;
            for Colpixel=1:pixelCol;
                if double(BiofilmConnectedtoSurface(Rowpixel,Colpixel,i))==1  && double(Surface(Rowpixel,Colpixel,i))==1
                    BiofilmConnectedtoSurfaceforSubstratum(Rowpixel,Colpixel,i)=0;
                elseif double(BiofilmConnectedtoSurface(Rowpixel,Colpixel,i))==1  && double(Surface(Rowpixel,Colpixel,i))==0
                    BiofilmConnectedtoSurfaceforSubstratum(Rowpixel,Colpixel,i)=1;
                elseif double(BiofilmConnectedtoSurface(Rowpixel,Colpixel,i))==0  && double(Surface(Rowpixel,Colpixel,i))==0
                    BiofilmConnectedtoSurfaceforSubstratum(Rowpixel,Colpixel,i)=0;
                elseif double(BiofilmConnectedtoSurface(Rowpixel,Colpixel,i))==0  && double(Surface(Rowpixel,Colpixel,i))==1
                    BiofilmConnectedtoSurfaceforSubstratum(Rowpixel,Colpixel,i)=0;   
                end;
            end;
    end;
end;
 
%Modified Substratum Coverage
Substratum_Modified=sum(sum(sum(BiofilmConnectedtoSurfaceforSubstratum(:,:,:))))/(pixelCol*pixelRow)*100;
 
for i=1:yy
    for Rowpixel=1:pixelRow;
            for Colpixel=1:pixelCol;
                if double(Bacteria(Rowpixel,Colpixel,i))==1  && double(Surface(Rowpixel,Colpixel,i))==1
                    BacteriaColocalized(Rowpixel,Colpixel,i)=1;
                elseif double(Bacteria(Rowpixel,Colpixel,i))==1  && double(Surface(Rowpixel,Colpixel,i))==0
                    BacteriaColocalized(Rowpixel,Colpixel,i)=0;
                elseif double(Bacteria(Rowpixel,Colpixel,i))==0  && double(Surface(Rowpixel,Colpixel,i))==0
                    BacteriaColocalized(Rowpixel,Colpixel,i)=0;
                elseif double(Bacteria(Rowpixel,Colpixel,i))==0  && double(Surface(Rowpixel,Colpixel,i))==1
                    BacteriaColocalized(Rowpixel,Colpixel,i)=0;   
                end;
            end;
    end;
end;
 
for i=1:yy
    PercentBacteriaAssociatedwithSurface(i)=sum(sum(BacteriaColocalized(:,:,i)))/sum(sum(Bacteria(:,:,i)))*100;
    NumberofBacteriaandSurfaceSamePixel(i)=sum(sum(BacteriaColocalized(:,:,i)));
    VolumeofBacteriaandSurfaceSamePixel(i)=NumberofBacteriaandSurfaceSamePixel(i)*(micronx*microny*micronz);
end;
PercentBacteriaAssociatedwithSurface=PercentBacteriaAssociatedwithSurface';
NumberofBacteriaandSurfaceSamePixel=NumberofBacteriaandSurfaceSamePixel';
VolumeofBacteriaandSurfaceSamePixel=VolumeofBacteriaandSurfaceSamePixel';
TotalBacteriaAssociatedwithSurface=sum(sum(sum(BacteriaColocalized)))/sum(sum(sum(Bacteria)))*100;
TotalNumberofBacteriaandSurfaceSamePixel=sum(sum(sum(BacteriaColocalized)));
TotalVolumeofBacteriaandSurfaceSamePixel=TotalNumberofBacteriaandSurfaceSamePixel*(micronx*microny*micronz);
%Any "NAN - Not a real number" will be made 0. 
PercentBacteriaAssociatedwithSurface(isnan(PercentBacteriaAssociatedwithSurface))=0;
 
 
 
 
for i=1:yy;
    for Rowpixel=1:pixelRow;
            for Colpixel=1:pixelCol;
                if double(BiofilmConnectedtoSurface(Rowpixel,Colpixel,i))==1  || double(HorizontalGrowthForBacteriaAttachedtoBStart(Rowpixel,Colpixel,i))==1
                    BasisforSubstratum(Rowpixel,Colpixel,i)=1;
                   
                else
                    BasisforSubstratum(Rowpixel,Colpixel,i)=0;
                end;
            end;
    end;
end;
 
 %Determine the start points where the Biofilm Attaches to the Surface or
 %Substratum
  k=1;
  for i = 1:yy;
          if sum(sum(BiofilmConnectedtoSurface(:,:,i)))>=1
          StartIndex(k)=i;
          k=k+1;
          end;       
   end;
 
   %Connect biofilm upward from each starting slice  
Storage=zeros(pixelRow, pixelCol, yy);  
NN=size(StartIndex);
for q=1:NN(2);
    SliceIndex=StartIndex(q);
    areainthislayer(:,:,1)=BasisforSubstratum(:,:,SliceIndex);
 
 for k = SliceIndex:yy-1; 
        commonarea3=uint8(areainthislayer(:,:,k-SliceIndex+1).*double((Bacteria(:,:,k+1)>0))); % what pixels are common between the two layers
        [vectorx,vectory]=find(commonarea3); % vectors containing the nonzero elements of commonarea
        %8 neighborhood connection for above slice
        %allows for horizontal growth of biofilm
        areainthislayer(:,:,k-SliceIndex+2)=double(bwselect(Bacteria(:,:,k+1),vectory,vectorx,8));
        BiofilmAttachedtoBiofilm(:,:,k-SliceIndex+1)=areainthislayer(:,:,k-SliceIndex+2);
       
        %Keep track of where biofilm pixels grew
        for Rowpixel=1:pixelRow;
            for Colpixel=1:pixelCol;
                if BiofilmAttachedtoBiofilm(Rowpixel,Colpixel,k-SliceIndex+1)==1
                    Storage(Rowpixel, Colpixel, k+1)=1;
                end;
            end;
        end;
      
 end;
 
 
end;
 
 
%Put together the starting point BiofilmConnectedtoSurface and the BiofilmAttachedtoBiofilm in Storage   
filt_images=zeros(pixelRow,pixelCol,yy);
for i = 1:yy;
        for Rowpixel=1:pixelRow;
            for Colpixel=1:pixelCol;
           
                    if double(BiofilmConnectedtoSurface(Rowpixel,Colpixel,i)) ||  double(BasisforSubstratum(Rowpixel,Colpixel,i)) ==1 ||  double(Storage(Rowpixel,Colpixel,i)) ==1
                        filt_images(Rowpixel,Colpixel,i)=1;
                                           
                    end;
             end;
        end;
end;  
 
 
%Quantification
UnconnectedBacteriaPixel=zeros(max(max(pixelRow)), max(max(pixelCol)), max(max(yy)));
ConnectedBiofilmBacteriaPixel=zeros(max(max(pixelRow)), max(max(pixelCol)), max(max(yy)));
AllSurfacePixels=double(Surface);
AllBacteriaPixels=double(Bacteria);
 
        for i=1:yy;
            for Rowpixel=1:pixelRow;
                for Colpixel=1:pixelCol;
                                    
                    if AllBacteriaPixels(Rowpixel, Colpixel,i)==1 && filt_images(Rowpixel, Colpixel,i) ==1;
                        ConnectedBiofilmBacteriaPixel(Rowpixel, Colpixel,i)=1;
                        UnconnectedBacteriaPixel(Rowpixel, Colpixel,i)=0;
                    elseif AllBacteriaPixels(Rowpixel, Colpixel,i)==1 && filt_images(Rowpixel, Colpixel,i) ==0;
                        ConnectedBiofilmBacteriaPixel(Rowpixel, Colpixel,i)=0;
                        UnconnectedBacteriaPixel(Rowpixel, Colpixel,i)=1;
                    elseif AllBacteriaPixels(Rowpixel, Colpixel,i)==0 && filt_images(Rowpixel, Colpixel,i) ==0 || AllBacteriaPixels(Rowpixel, Colpixel,i)==0 && filt_images(Rowpixel, Colpixel,i) ==1;
                        ConnectedBiofilmBacteriaPixel(Rowpixel, Colpixel,i)=0;
                        UnconnectedBacteriaPixel(Rowpixel, Colpixel,i)=0;
                       
                    end;
                               
                end;
            end;
        end;
       
     
%Preallocate space
AreaConnectedBiofilmBacteriaBySlice=zeros( max(max(yy)), 1);
AreaUnconnectedBacteriaBySlice=zeros( max(max(yy)), 1);
AreaSurfacebySlice=zeros(max(max(yy)), 1);
AreaPSurfacebySlice=zeros(max(max(yy)), 1);
TotSurfaceBySlice=zeros(max(max(yy)), 1);
TotConnectedBiofilmBacteriaBySlice=zeros(max(max(yy)), 1);
TotUnconnectedBacteriaBySlice=zeros(max(max(yy)), 1);
AreaPercentConnectedBiofilmBacteriaBySlice=zeros(max(max(yy)), 1);
AreaPercentUnconnectedBacteriBySlice=zeros(max(max(yy)), 1);
TotalinMicrons=micronx*microny*pixelCol*pixelRow;
 
    %Surface
    AreaSurfaceOverall=sum(sum(sum(Surface(:,:,:))))*micronx*microny;
    AreaPSurfaceOverall=AreaSurfaceOverall/(TotalinMicrons*yy)*100;
    TotSurface=sum(sum(sum(Surface(:,:,:))));
   
              
    %Bacteria
    AreaConnectedBiofilmBacteria=sum(sum(sum(ConnectedBiofilmBacteriaPixel(:,:,:))))*micronx*microny;
    AreaUnconnectedBacteria=sum(sum(sum(UnconnectedBacteriaPixel(:,:,:))))*micronx*microny;
    AreaPercentConnectedBiofilmBacteria=AreaConnectedBiofilmBacteria/(TotalinMicrons*yy)*100;
    AreaPercentUnconnectedBacteria=AreaUnconnectedBacteria/(TotalinMicrons*yy)*100;
    TotConnectedBiofilmBacteria=sum(sum(sum(ConnectedBiofilmBacteriaPixel(:,:,:))));
    TotUnconnectedBacteria=sum(sum(sum(UnconnectedBacteriaPixel(:,:,:))));
          
    for i=1:yy;
    %Surface
    AreaSurfacebySlice(i)=sum(sum(Surface(:,:,i)))*micronx*microny;
    AreaPSurfacebySlice(i)=AreaSurfacebySlice(i)/TotalinMicrons*100;
    TotSurfaceBySlice(i)=sum(sum(sum(Surface(:,:,i))));
   
      
    %Bacteria
    AreaConnectedBiofilmBacteriaBySlice(i)=sum(sum(ConnectedBiofilmBacteriaPixel(:,:,i)))*micronx*microny;
    AreaUnconnectedBacteriaBySlice(i)=sum(sum(UnconnectedBacteriaPixel(:,:,i)))*micronx*microny;
    AreaPercentConnectedBiofilmBacteriaBySlice(i)=AreaConnectedBiofilmBacteriaBySlice(i)/TotalinMicrons*100;
    AreaPercentUnconnectedBacteriBySlice(i)=AreaUnconnectedBacteriaBySlice(i)/TotalinMicrons*100;
    TotConnectedBiofilmBacteriaBySlice(i)=sum(sum(sum(ConnectedBiofilmBacteriaPixel(:,:,i))));
    TotUnconnectedBacteriaBySlice(i)=sum(sum(sum(UnconnectedBacteriaPixel(:,:,i))));
   
    end;
   
 
%Saving the Data
StackIndex=1;
lsm.slice=yy;
header={'Publication'};
filename=horzcat(num2str(StackIndex), '.xlsx');
colnames={'', 'Number of Connected Biofilm Pixels', 'Area Connected Biofilm, um^2', 'Area Connected Biofilm, %',  'Number of Unconnected Bacteria Pixels', 'Area Unconnected Bacteria, um^2', 'Area Unconnected Bacteria, %', 'Number of Surface Pixels','Area Surface, um^2', 'Area Surface, %', 'Number Bacteria and Surface Pixels the Same', 'Volume Bacteria and Surface Pixels the Same', 'Pecent Bacteria Associated with Surface, %', 'Modified Substratum Coverage, %'};
 
 
%Biofilm Associated Bacteria
GA=vertcat(AreaConnectedBiofilmBacteria, AreaConnectedBiofilmBacteriaBySlice(:));
SubstratumSave=vertcat(Substratum_Modified,0);
TotalConnectedBiofilmBacteria=vertcat(TotConnectedBiofilmBacteria,TotConnectedBiofilmBacteriaBySlice(:));
AreaPercentBio=vertcat(AreaPercentConnectedBiofilmBacteria, AreaPercentConnectedBiofilmBacteriaBySlice(:));
AssociatedBacteria=vertcat(TotalBacteriaAssociatedwithSurface, PercentBacteriaAssociatedwithSurface(:));
NumberAssociated=vertcat(TotalNumberofBacteriaandSurfaceSamePixel,NumberofBacteriaandSurfaceSamePixel(:));
VolumeAssociated=vertcat(TotalVolumeofBacteriaandSurfaceSamePixel,VolumeofBacteriaandSurfaceSamePixel(:));
 
%Non-Biofilm Associated Bacteria
FGA=vertcat(AreaUnconnectedBacteria,AreaUnconnectedBacteriaBySlice(:));
TotalUnconnectedBacteria=vertcat(TotUnconnectedBacteria, TotUnconnectedBacteriaBySlice(:));
AreaPercentUnconnectedBacteriaCombined=vertcat(AreaPercentUnconnectedBacteria, AreaPercentUnconnectedBacteriBySlice(:));
 
%Surface
TA=vertcat(AreaSurfaceOverall, AreaSurfacebySlice(:));
TotalSurface=vertcat(TotSurface, TotSurfaceBySlice(:));
SurfacePercent=vertcat( AreaPSurfaceOverall, AreaPSurfacebySlice(:));
 
 
%Thank you to Scott Hirsch (MathWorks, Natick, MA) for providing permission
% to use his xlswrite code in this application. (http://www.mathworks.com/matlabcentral/fileexchange/2855-xlswrite) 
%See xlswrite_BMT_Example.m for license information.
xlswrite_Image(StackIndex,TotalConnectedBiofilmBacteria, GA, AreaPercentBio,TotalUnconnectedBacteria, FGA, AreaPercentUnconnectedBacteriaCombined, TotalSurface, TA, SurfacePercent, NumberAssociated, VolumeAssociated, AssociatedBacteria, SubstratumSave, header, colnames, filename);
 
 
 
%Generating ".tif" Sequences of Components
FinalConnectedBiofilmBacteria=zeros(max(max(pixelRow)), max(max(pixelCol)), max(max(yy)));
FinalUnconnectedBacteria=zeros(max(max(pixelRow)), max(max(pixelCol)), max(max(yy)));
 
folder=['Sample', int2str(StackIndex)];
parentfolderConnectedBiofilm=strcat(folder,'\ConnectedBiofilmBacteriaImages\');
parentfolderUnconnectedBacteria=strcat( folder,'\UnconnectedBacteriaImages\');
parentfolderOverall=strcat( folder,'\OverallBacteriaImages\');
parentfolderSurface=strcat( folder,'\SurfaceImages\');
parentfolderBacteriaSurface=strcat( folder,'\BacteriaAssociatedWithSurfaceImages\');
parentfolderBacteriaStars=strcat( folder,'\ModifiedSubstratumCoverageBacteria\');
mkdir(parentfolderConnectedBiofilm);
mkdir(parentfolderUnconnectedBacteria);
mkdir(parentfolderOverall);
mkdir(parentfolderSurface);
mkdir(parentfolderBacteriaSurface);
mkdir(parentfolderBacteriaStars);
 
 
       for i=1:yy;
            FinalConnectedBiofilmBacteria(:,:,i)=ConnectedBiofilmBacteriaPixel(:,:,i).*double(OriginalBacteria(:,:,i));
            name = ['ConnectedBiofilmBacteria',int2str(i),'.tif'];
            filename= strcat(parentfolderConnectedBiofilm, name);
                imwrite(uint8(FinalConnectedBiofilmBacteria(:,:,i)),filename,'tiff');
            FinalUnconnectedBacteria(:,:,i)=UnconnectedBacteriaPixel(:,:,i).*double(OriginalBacteria(:,:,i));
            name = ['UnconnectedBacteria',int2str(i),'.tif'];
            filename= strcat(parentfolderUnconnectedBacteria, name);
                imwrite(uint8(FinalUnconnectedBacteria(:,:,i)),filename,'tiff');   
                     
        end;
 
  FinalOverallBacteria=zeros(max(max(pixelRow)), max(max(pixelCol)), max(max(yy)));
  FinalSurface=zeros(max(max(pixelRow)), max(max(pixelCol)), max(max(yy)));
  FinalBacteriaAssociatedWithSurface=zeros(max(max(pixelRow)), max(max(pixelCol)), max(max(yy)));
 
       for i=1:yy;
            FinalOverallBacteria(:,:,i)=AllBacteriaPixels(:,:,i).*double(OriginalBacteria(:,:,i));
            name = ['OverallBacteria',int2str(i),'.tif'];
            filename= strcat(parentfolderOverall, name);
                imwrite(uint8(FinalOverallBacteria(:,:,i)),filename,'tiff');
            FinalSurface(:,:,i)=double(Surface(:,:,i)).*double(OriginalSurface(:,:,i));
            name = ['Surface',int2str(i),'.tif'];
            filename= strcat(parentfolderSurface, name);
                imwrite(uint8(FinalSurface(:,:,i)),filename,'tiff');
            FinalBacteriaAssociatedWithSurface(:,:,i)=double(BacteriaColocalized(:,:,i)).*double(OriginalBacteria(:,:,i));
            name = ['BacteriaAssociatedWithSurface',int2str(i),'.tif'];
            filename= strcat(parentfolderBacteriaSurface, name);
                imwrite(uint8(FinalBacteriaAssociatedWithSurface(:,:,i)),filename,'tiff');  
            FinalBacteriaStars(:,:,i)=double(BiofilmConnectedtoSurface(:,:,i)).*double(OriginalBacteria(:,:,i));
            name = ['ModifiedSubstratumCoverageBacteria',int2str(i),'.tif'];
            filename= strcat(parentfolderBacteriaStars, name);
                imwrite(uint8(FinalBacteriaStars(:,:,i)),filename,'tiff');  
               
       end;