function qdot_track

%=========================================================
%    November 4, 2015
% This code is for finding trajectories of fluorescent dye/quantum dot from
%multiple region of interests.

% This code assumes that the signal (Qdot or Cy3) 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 "qdot_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);
fnamename=D(1).name(1:regexp(D(1).name,'mM-')+1);
% 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 Qdot ROI.txt');
% select Qdot background ROI text file
% uniform background has only one roi in the file
% non-uniform background has multiple rois in the file
bgr_roi_file=uigetfile('*.txt','select Qdot background ROI.txt');
% enter a threshold for fitting a particular projection
thres=input('threshold (multiplication factor above background) = ');
% 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);
bgrROI=dlmread(bgr_roi_file,'\t',1,0);
% for uniform background, load roi information before going through all of
% the images
if size(bgrROI,1)==1
    % 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 non-uniform background, load bgrroi information for every ROI
    % assume length of ROIs and bgrROI are the same
    if size(bgrROI,1)>1
        % Change index of ROIs that marked in ImageJ to matlab format
        bgrxmin=bgrROI(ii,2)+1; %minimum x pixel number
        bgrymin=bgrROI(ii,3)+1; %minimum y pixel number
        bgrwidth=bgrROI(ii,4); %change in x (pixels)
        bgrheight=bgrROI(ii,5); %change in y (pixels)
        bgrxmax=bgrxmin+bgrwidth-1; %maximum x pixel number
        bgrymax=bgrymin+bgrheight-1; %maximum y pixel number
    end
    
    % create a new ROI of nonspecific bound QDot using z-projection image
    % with prefix "AVG" created in ImageJ
    % load z-projection image
    % 2012-07-27 LS: modified for HK's images
    img=imread(['AVG_' fnamename],'tiff');
    % clips image using user defined rectangle: x=col, y=row
    img_crop=img(ymin:ymax,xmin:xmax);
    % manually select a new ROI of the nonspecific bound QDot
    imshow(img_crop,[],'InitialMagnification',500)
    imcontrast(gca)
    % set dimension constraints to the new ROI
    fcn=makeConstrainToRectFcn('imrect',[1,width],[1,height]);
    ROI2crop=cell(1,[]);
    % select multiple new ROIs to be cropped out in a loop
    ii;
    
    % Sometimes, nonspecifically-bound Qdots will bother you and you would
    %like to remove it. Below are written for this purpose.
    counter=input('select ROI to be cropped? (yes=1, no=0) = ');
    %counter=0;
    while counter==1
        h=imrect(gca,'PositionConstraintFcn',fcn);
        ROI2=wait(h);
        ROI2crop{end+1}=createMask(h);
        counter=input('select more ROI? (yes=1, no=0) = ');
    end
    close all
    
    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');
        % calculates dark background using the given ROI
        back=mean(mean(data(bgrymin:bgrymax,bgrxmin:bgrxmax)));
        % subtracts background
        data2=imsubtract(data,back);
        % clips image using user defined rectangle: x=col, y=row
        data2_crop=data2(ymin:ymax,xmin:xmax);
        % crop out any nonspecific bound QDot in ROI using frame_min
        data3=data2_crop;
        if ~isempty(ROI2crop)
            for k=1:size(ROI2crop,2)
                data3(ROI2crop{k}==1)=0;
            end
        end
        
    
        % calculates projection to y-axis
        j=(i - frame_min)/step+1;
        proj(:,j)=sum(data3,2);
        
        % Fits a gaussian to projection data
        % Creates axis for fitting
        X=1:1:height;
        X=X';
        % Determine initial parameters
        % peak center - y_o
        [C,I]=max(proj(:,j));
        % if multiple maximum is found, return the first one's index
        y_o=I(1,1);
        % width along y-axis
        w=5;
        % offset
        off=mean(proj(:,j));
        % creates array of initial guesses
        p0=[y_o w C off];
        
        if C >=thres*((mean(proj(1:y_o-w,j))+mean(proj(y_o+w:end,j))/2))
            % fits single Gaussian to projection
            p1=lsqcurvefit('gauss',p0,X,proj(:,j),[],[]);
            % creates array of fit parameters
            x_traj(j)=p1(1);
        else
            x_traj(j)=0;
        end
    end
    
    % constructs time axis
    Xi=frame_min-1:step:frame_max-1;
    Ti=int_time.*Xi;
    % removes "zeros" from x_traj by linear interpolation
    [Xo,col,v]=find(x_traj);
    x_traj_int=interp1(step*(col-1)+frame_min,v,Xi,'linear');
    
    % Time (column #1) and x trajectory (column #2) data
    Ti_HJ=Ti';
    x_traj_HJ=x_traj';
    Time_traj(:, 1) = Ti_HJ;
    Time_traj(:, 2) = x_traj_HJ;
    
    % save projection
    %save([outdir '\' outfname '_proj_ROI-' int2str(ii) '.txt'],'proj','-ascii')
    save([outdir '\' outfname '_proj-' int2str(ii) '.txt'],'proj','-ascii')
    % save x trajectory
    %save([outdir '\' outfname '_x_traj_ROI-' int2str(ii) '.txt'],'x_traj','-ascii')
    save([outdir '\' outfname '_x_traj-' int2str(ii) '.txt'],'x_traj','-ascii')
    % save interpolated x trajectory
    %save([outdir '\' outfname '_x_traj_int_ROI-' int2str(ii) '.txt'],'x_traj_int','-ascii')
    save([outdir '\' outfname '_x_traj_int-' int2str(ii) '.txt'],'x_traj_int','-ascii')
    % save time axis
    %save([outdir '\' outfname '_qdot_time_ROI-' int2str(ii) '.txt'],'Ti','-ascii')
    save([outdir '\' outfname '_qdot_t-' int2str(ii) '.txt'],'Ti','-ascii')
    % save time versus x trajectory: HJ added on 20120811
    %save([outdir '\' outfname '_qdot_time_versus_trajectory-' int2str(ii) '.txt'],'Time_traj','-ascii')
    save([outdir '\' outfname '_qdot_t_vs_traj-' int2str(ii) '.txt'],'Time_traj','-ascii')
    
    % plot x trajectory over time
    figure,plot(Ti,x_traj,'b.','MarkerSize',8)
    xlabel('Time [s]')
    ylabel('Position Tracjectory [pixel]')
    % save x trajectory plot
    saveas(gca, [outdir '\' outfname '_qdot_traj-' int2str(ii) '_fig.tif'], 'tif')
    saveas(gca, [outdir '\' outfname '_qdot_traj-' int2str(ii) '_fig.fig'], 'fig')
    
    % plot interpolated x trajectory over time
    figure,plot(Ti,x_traj_int,'r.','MarkerSize',8)
    xlabel('Time [s]')
    ylabel('Position Tracjectory [pixel]')
    % save interpolated x trajectory plot
    saveas(gca, [outdir '\' outfname '_qdot_traj_int-' int2str(ii) '_fig.tif'], 'tif')
    saveas(gca, [outdir '\' outfname '_qdot_traj_int-' int2str(ii) '_fig.fig'], 'fig')
    
    clear proj
    clear x_traj
    clear x_traj_int
    
    close all
end