function int_track

%=========================================================
%    November 4, 2015
% This code is for finding trajectories of background level from
%multiple region of interests. By looking at when the background level
%increases by a very low concentration of dye, you can identify BsSMC
%arrival time.

% This code assumes that the signal is moving in the y-direction

%   How to use it?
%(1) Open the raw movie file using ImageJ.
%(2) "Analyze" tab -> "Set Measurements" -> Select "Bounding rectangle"
%(3) Designate region of interest (ROI) by dragging the mouse over it.
%(4) Ctrl + t
%(5) Repeat until regions of all of the events were selected.
%(6) In the "ROI Manager" window of ImageJ, click "Measure". This will
%   give you x- and y-coordinates, width, height of each ROI.
%(7) Save it as "(File name).txt"
%(8) Now we need to find out background intensity level right next to 
%   each ROI. You can do it by "Ctrl + t"
%(9) Save it (only for background ROI - NOT including signal ROI) as
%   "(Another file name).txt"
%(10) Let's say we have 10 DNAs. Then, "(File name).txt" contains
%   coordinate information for 10 signals, and "(Another file name).txt"
%   contains those for 10 background levels.
%(11) Save averaged image. This can be done by "Image" tab -> "Stacks" ->
%   "Z Project". The averaged image must be saved under the name "AVG_
%   (File name).tif"
%(12) Save the raw data movie file as Image sequence. For example, if your
%  movie file consists of 100 frames, save individual frame as a seperate
%  TIFF file (= 100 files)

%(13) Now Open this "int_track.m" Matlab file, and run it.
 
% Details on the code is commented throughout this code.
%=========================================================

clear all
close all

% User Inputs
% select directory with images to be analyzed
filepath=uigetdir('','select directory that has images to be analyzed');
% change matlab "Current Folder" to the selected directory
cd(filepath)
% load only tif image files
D=dir([filepath,filesep,'*.tif']);

% get image file prefix
% Assume that my image files are named as "abc100mM-0001.tif"
% "abc100mM-0002.tif",..., and so on. If your file names are named
% differently, simply change below two lines accordingly.
fname=D(1).name(1:regexp(D(1).name,'mM-')+2);
% beginning frame
frame_min=input('starting frame = ');
% ending frame
frame_max=input(['ending frame (',num2str(sum(strncmp(fname,{D.name},length(fname)))),...
    ' images available) = ']);
% frame increment
step=input('frame increment = ');
% ccd integration time in secs
int_time=input('time per frame (seconds) = ');
% select ROI text file to be analyzed
roi_file=uigetfile('*.txt','select DNA ROI.txt');
% select DNA background ROI text file
bgr_roi_file=uigetfile('*.txt','select DNA background ROI.txt (or click ''cancel'')');
% directory to save analyzed data and figures
outdir=uigetdir('','select directory to save output');
outfname=input('name output (e.g. ''kymo1''): ');

% read ROI data
ROIs=dlmread(roi_file,'\t',1,0);
if bgr_roi_file~=0
    bgrROI=dlmread(bgr_roi_file,'\t',1,0);
    % Change index of ROIs that marked in ImageJ to matlab format
    bgrxmin=bgrROI(2)+1; %minimum x pixel number
    bgrymin=bgrROI(3)+1; %minimum y pixel number
    bgrwidth=bgrROI(4); %change in x (pixels)
    bgrheight=bgrROI(5); %change in y (pixels)
    bgrxmax=bgrxmin+bgrwidth-1; %maximum x pixel number
    bgrymax=bgrymin+bgrheight-1; %maximum y pixel number
end

for ii=1:size(ROIs,1)
    % Change index of ROIs that marked in ImageJ to matlab format
    xmin=ROIs(ii,2)+1; %minimum x pixel number
    ymin=ROIs(ii,3)+1; %minimum y pixel number
    width=ROIs(ii,4); %change in x (pixels)
    height=ROIs(ii,5); %change in y (pixels)
    xmax=xmin+width-1; %maximum x pixel number
    ymax=ymin+height-1; %maximum y pixel number
    
    for i=frame_min:step:frame_max
        % 2012-07-27 LS: modified name for HK's images
        %Constructs file name --> meta vue default
        if i<10
            name=[fname '000' int2str(i)];
        else if i>9 & i<100
                name=[fname '00' int2str(i)];
            else if i> 99 & i<1000
                    name=[fname '0' int2str(i)];
                else
                    name=[fname int2str(i)];
                end
            end
        end
        % loads image
        data=imread(name,'tiff');
        % clips image using user defined rectangle: x=col, y=row
        data_crop=data(ymin:ymax,xmin:xmax);
        % calculates integrated intensity
        int((i - frame_min)/step+1)=sum(sum(data_crop));
        % calculates dark background using the given ROI
        if bgr_roi_file~=0
            back=mean(mean(data(bgrymin:bgrymax,bgrxmin:bgrxmax)));
            % subtracts background
            data2=imsubtract(data,back);
            data2_crop=data2(ymin:ymax,xmin:xmax);
            int_noback((i - frame_min)/step+1)=sum(sum(data2_crop));
        end
    end
    % constructs time axis
    t=int_time*(frame_min-1:step:frame_max-1);
    % save intensity trajectory and time data
    %save([outdir '\' outfname '_int_traj_ROI-' int2str(ii) '.txt'],'int','-ascii')
    %save([outdir '\' outfname '_time_traj_ROI-' int2str(ii) '.txt'],'t','-ascii')
    save([outdir '\' outfname '_int-' int2str(ii) '.txt'],'int','-ascii')
    save([outdir '\' outfname '_time-' int2str(ii) '.txt'],'t','-ascii')
    if bgr_roi_file~=0
        save([outdir '\' outfname '_int_noback_traj-' int2str(ii) '.txt'],'int_noback','-ascii')
    end
    
    % plot intensity trajectory over time
    figure(ii),plot(t,int,'b.','MarkerSize',16)
    if bgr_roi_file~=0
        hold on
        % plot background subtracted intensity trajectory over time
        plot(t,int_noback,'r.','MarkerSize',16)
        legend('Raw','NoBack','Location','NorthWest')
    end
    xlabel('Time [s]')
    ylabel('Integrated Intensity [A.U.]')
    % save intensity trajectory plot
    %saveas(gca, [outdir '\' outfname '_int_traj_ROI-' int2str(ii) '_fig.tif'], 'tif')
    %saveas(gca, [outdir '\' outfname '_int_traj_ROI-' int2str(ii) '_fig.fig'], 'fig')
    saveas(gca, [outdir '\' outfname '_int-' int2str(ii) '_fig.tif'], 'tif')
    saveas(gca, [outdir '\' outfname '_int-' int2str(ii) '_fig.fig'], 'fig')
    
    t_HJ=t';
    int_HJ=int';
    Background_Intensity_Traj(:, 1) = t_HJ;
    Background_Intensity_Traj(:, 2) = int_HJ;
    
    % save time versus x trajectory
    %save([outdir '\' outfname '_Background_time_versus_intensity-' int2str(ii) '.txt'],'Background_Intensity_Traj','-ascii')
    save([outdir '\' outfname '_BG_t_vs_I-' int2str(ii) '.txt'],'Background_Intensity_Traj','-ascii')
         
    close all
end