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im_nucseg.m
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im_nucseg.m
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%% im_nucseg.m Multi Channel
% Alistair Boettiger Date Begund: 01/21/11
% Levine Lab, UC Berkeley Version Complete: 02/01/11
% Functionally complete Last Modified: 02/07/11
%
%
%% Attribution:
% Feel free to use modify and distribute this code provided that you
% attribute Alistair Boettiger and Jacques Bothma for development and abide
% by the provisions of the Creative Commons License 3.0, BY-NC-SA.
% http://creativecommons.org/licenses/by-nc-sa/3.0/.
%
%
%
%% Important Notes:
% This version written for Mac. To impliment in PC just change directory
% paths from using '/' to using '\'.
%
% Before running, go scroll down to function setup and save the default
% parameters to your data folder
%
%
%
%% Overview:
% This code uses DNA staining to associate cytoplasmic domains with the
% nearest nucleus. High reslolution mRNA FISH localizes transcripts
%
%
%% Required subroutines
% fxn_nuc_seg.m -- segmentation filter, identifies all nuclei
% fxn_nuc_reg.m -- expands nuclei to assign all regions of embryo to one
% nuclei or another.
% dotfinder.m -- locates dots using difference of gaussians and watershed
% DuplicateDots.m -- compares layers to ID duplicate dots
% vect2rast.m -- simple vector to raster conversion, called by DuplicateDots
%
%% Updates:
% 02/07/11 moved plotting
function varargout = im_nucseg(varargin)
% IM_NUCSEG M-file for im_nucseg.fig
% IM_NUCSEG, by itself, launches the GUI
%
% IM_NUCSEG('CALLBACK',hObject,eventData,handles,...) calls the local
% function named CALLBACK in IM_NUCSEG.M with the given input arguments.
%
% IM_NUCSEG('Property','Value',...) creates a new IM_NUCSEG or raises the
% existing singleton*. Starting from the left, property value pairs are
% applied to the GUI before im_nucseg_OpeningFcn gets called. An
% unrecognized property name or invalid value makes property application
% stop. All inputs are passed to im_nucseg_OpeningFcn via varargin.
%
% See also: GUIDE, GUIDATA, GUIHANDLES
% Edit the above text to modify the response to help im_nucseg
% Last Modified by GUIDE v2.5 10-Mar-2011 12:19:13
% Begin initialization code - DO NOT EDIT
gui_Singleton = 1;
gui_State = struct('gui_Name', mfilename, ...
'gui_Singleton', gui_Singleton, ...
'gui_OpeningFcn', @im_nucseg_OpeningFcn, ...
'gui_OutputFcn', @im_nucseg_OutputFcn, ...
'gui_LayoutFcn', [] , ...
'gui_Callback', []);
if nargin && ischar(varargin{1})
gui_State.gui_Callback = str2func(varargin{1});
end
if nargout
[varargout{1:nargout}] = gui_mainfcn(gui_State, varargin{:});
else
gui_mainfcn(gui_State, varargin{:});
end
% End initialization code - DO NOT EDIT
% --- Executes just before im_nucseg is made visible.
function im_nucseg_OpeningFcn(hObject, eventdata, handles, varargin)
% This function has no output args, see OutputFcn.
% hObject handle to figure
% eventdata reserved - to be defined in a future version of MATLAB
% handles structure with handles and user data (see GUIDATA)
% varargin command line arguments to im_nucseg (see VARARGIN)
handles.output = hObject; % Choose default command line output for im_nucdots_v5
% Some initial setup
% Folder to save .mat data files in for normal script function.
handles.fdata = '/Users/alistair/Documents/Berkeley/Levine_Lab/ImageProcessing/';
handles.step = 0; % starting step is step 0
set(handles.stepnum,'String',handles.step); % change step label in GUI
handles.output = hObject; % update handles object with new step number
guidata(hObject, handles); % update GUI data with new handles
setup(hObject, eventdata, handles); % set up labels and default values for new step
guidata(hObject, handles); % update GUI data with new labels
% Update handles structure
guidata(hObject, handles);
% UIWAIT makes im_nucseg wait for user response (see UIRESUME)
% uiwait(handles.figure1);
%=========================================================================%
% Primary Analysis Section %
%=========================================================================%
% % All of the functional processing script is in this function
function run_Callback(hObject, eventdata, handles)
step = handles.step;
% Step 0: Load Data into script
if step == 0;
disp('running...'); tic
handles.NucChn = str2double(get(handles.in1,'String')); %
handles.imsize = str2double(get(handles.in2,'String'));
% save([handles.fdata,'/','test']);
% load([handles.fdata,'/','test']);
handles.output = hObject; % update handles object with new step number
% guidata(hObject, handles); % update GUI data with new handles
handles = imload(hObject, eventdata,handles); % load new embryo
guidata(hObject, handles); % update GUI data with new handles
toc
end
% Step 1: Nuclear Threshold
% uses fxn: fxn_nuc_seg
if step == 1;
% load appropriate data
disp('running step 1...'); tic
minN = str2double(get(handles.in1,'String')); %
imblur = str2double(get(handles.in2,'String'));
sigmaE = str2double(get(handles.in3,'String'));
sigmaI = str2double(get(handles.in4,'String'));
FiltSize = str2double(get(handles.in5,'String'));
I = handles.Nucs;
% get threshold image 'bw' and nuclei centroids 'cent'
[handles.bw,handles.cent] = fxn_nuc_seg(I,minN,sigmaE,sigmaI,FiltSize,imblur);
% Save data values
% handles.output = hObject; guidata(hObject, handles);
guidata(hObject, handles); % update GUI data with new handles
toc;
end
% Step 2: Get Region for each Nuclei
% uses fxn fxn_nuc_reg
if step == 2;
tic
disp('running step 2...');
Mthink = str2double(get(handles.in1,'String')); %
Mthin = str2double(get(handles.in2,'String'));
Imnn = str2double(get(handles.in3,'String'));
[NucLabeled,Nuc_overlay,conn_map,cell_bords] = fxn_nuc_reg(handles.Nucs,handles.bw,Mthink,Mthin,Imnn);
% save([handles.fdata,'/','test']);
% load([handles.fdata,'/','test']);
[h,w] = size(NucLabeled);
Cell_bnd = false(h,w);
Cell_bnd(cell_bords) = 1;
Cell_bnd = bwareaopen( Cell_bnd,100);
int = class(handles.Nucs);
if strcmp(int,'uint16')
Nucs = handles.Nucs + uint16(2^16*Cell_bnd);
elseif strcmp(int,'uint8')
Nucs = handles.Nucs + uint8(2^8*Cell_bnd);
end
figure(21); clf; imagesc(Nucs); colormap('hot');
handles.NucLabeled = NucLabeled;
handles.Cell_bnd = Cell_bnd;
handles.conn_map = conn_map;
guidata(hObject, handles); % update GUI data with new handles
toc
end
% export data
if step == 3
tic
fout = get(handles.fout,'String');
fname = get(handles.in1,'String');
disp(['exporting data to ',fout,fname,'...']);
NucLabeled = handles.NucLabeled;
nuc_cents = handles.cent;
Nucs = handles.Nucs;
conn_map = handles.conn_map;
Cell_bnd = handles.Cell_bnd;
save([fout,fname,'_nucdata'],...
'NucLabeled','nuc_cents','conn_map','Cell_bnd','Nucs');
guidata(hObject, handles);
toc
disp('data saved');
end
%========================================================================%
% end of functional processing script
% The rest of this code is GUI manipulations
% --- Executes on button press in VarButton.
function VarButton_Callback(hObject, eventdata, handles)
% ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ %
% File managining scripts %
% ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ %
% This function sets up the new steps with the appropriate input labels and
% defalut label parameters
% handles.fdata = '/Users/alistair/Documents/Berkeley/Levine_Lab/ImageProcessing/';
function setup(hObject,eventdata,handles)
if handles.step == 0;
load([handles.fdata, 'imnucseg_pars0']);
% pars = {'3','512',' ',' ',' ',' '}; save([handles.fdata,'imnucseg_pars0'], 'pars' );
set(handles.in1label,'String','Nuclei channel');
set(handles.in1,'String', pars(1));
set(handles.in2label,'String','imsize');
set(handles.in2,'String', pars(2));
set(handles.in3label,'String',' ');
set(handles.in3,'String', pars(3));
set(handles.in4label,'String',' ');
set(handles.in4,'String', pars(4));
set(handles.in5label,'String',' ');
set(handles.in5,'String', pars(5));
set(handles.in6label,'String',' ');
set(handles.in6,'String', pars(6));
set(handles.VarButtonName,'String','');
dir = {
'Load max-projected image from folder.'} ;
set(handles.directions,'String',dir);
end
if handles.step == 1;
load([handles.fdata,'/','imnucseg_pars1']);
% pars = {'100','4','20','23','30',' '}; save([handles.fdata,'imnucseg_pars1'], 'pars' );
set(handles.in1label,'String','min Nuc size'); % number of pixels in filter (linear dimension of a square)
set(handles.in1,'String', pars{1});
set(handles.in2label,'String','Imblur'); % width of Gaussian in pixels
set(handles.in2,'String',pars{2});
set(handles.in3label,'String','Excitation Width');
set(handles.in3,'String',pars{3});
set(handles.in4label,'String','Inhibition Width');
set(handles.in4,'String', pars{4});
set(handles.in5label,'String','FilterSize');
set(handles.in5,'String', pars{5});
set(handles.in6label,'String',' ');
set(handles.in6,'String', pars{6});
dir = {
'Step 1: Find nuclei. Uses a difference of Gaussian filter with';
'a min nucleus size filter and watershed splitter'};
set(handles.directions,'String',dir);
end
if handles.step == 2; % nuclei segmentation
load([handles.fdata,'/','imnucseg_pars2']);
% pars = {'45','3','2','','','',''}; save([handles.fdata,'imnucseg_pars2'], 'pars' );
set(handles.in1label,'String','thicken nuclei');
set(handles.in1,'String', pars{1});
set(handles.in2label,'String','thin boundaries');
set(handles.in2,'String', pars{2});
set(handles.in3label,'String','erode');
set(handles.in3,'String', pars{3});
set(handles.in4label,'String',' ');
set(handles.in4,'String', pars{4});
set(handles.in5label,'String',' ');
set(handles.in5,'String', pars{5});
set(handles.in6label,'String',' ');
set(handles.in6,'String', pars{6});
dir = {'Step 2: Map nuclear region';
'nuclei expand until they collide. Borders are assigned to different nuclei'} ;
set(handles.directions,'String',dir);
end
if handles.step == 3;
load([handles.fdata,'/','imnucseg_pars3']);
% pars = {' ',' ',' ',' ',' ',' '}; save([handles.fdata,'imnucseg_pars3'], 'pars' );
froot = get(handles.froot,'String');
emb = get(handles.embin,'String');
fname = [froot,'_',emb];
set(handles.in1label,'String','Save Name');
set(handles.in1,'String', fname);
set(handles.in2label,'String',' ');
set(handles.in2,'String', pars{2});
set(handles.in3label,'String',' ');
set(handles.in3,'String', pars{3});
set(handles.in4label,'String',' ');
set(handles.in4,'String', pars{4});
set(handles.in5label,'String',' ');
set(handles.in5,'String', pars{5});
set(handles.in6label,'String',' ');
set(handles.in6,'String', pars{6});
set(handles.VarButtonName,'String','Manual Reg Select');
dir = {'Step 3: Save Data'};
set(handles.directions,'String',dir);
end
guidata(hObject, handles); % update GUI data with new labels
% --- Executes on button press in savePars.
function savePars_Callback(hObject, eventdata, handles)
% record the values of the 6 input boxes for the step now showing
p1 = get(handles.in1,'String');
p2 = get(handles.in2,'String');
p3 = get(handles.in3,'String');
p4 = get(handles.in4,'String');
p5 = get(handles.in5,'String');
p6 = get(handles.in6,'String');
try % for some reason the parameters are sometimes retrived as cells instead of strings
% we need to make sure they are strings.
pars = {p1{:}, p2{:}, p3{:}, p4{:}, p5{:}, p6{:}}; % cell array of strings
catch
pars = {p1, p2, p3, p4, p5, p6}; % cell array of strings
end
% Export parameters
stp_label = get(handles.stepnum,'String');
savelabel = ['imnucseg_pars',stp_label];
% labeled as nucdot_parsi.mat where "i" is the step number
save([handles.fdata, savelabel], 'pars'); % export values
disp([handles.fdata, savelabel]);
pars
% save([handles.fdata,'/','test']);
% load([handles.fdata,'/','test']);
guidata(hObject, handles); % update GUI data with new labels
% ----------------------STEP CONTROLS----------------------- %
% interfaces to main analysis code
% --- Executes on button press in nextstep.
function nextstep_Callback(hObject, eventdata, handles)
handles.step = handles.step + 1; % forward 1 step
set(handles.stepnum,'String', handles.step); % change step label in GUI
handles.output = hObject; % update handles object with new step number
guidata(hObject, handles); % update GUI data with new handles
setup(hObject, eventdata, handles); % set up labels and default values for new step
guidata(hObject, handles); % update GUI data with new labels
% --- Executes on button press in back.
function back_Callback(hObject, eventdata, handles)
handles.step = handles.step-1; % go back a step
set(handles.stepnum,'String',handles.step); % Change step label in GUI
handles.output = hObject; % update handles object with new step number
guidata(hObject, handles); % update GUI data with new handles
setup(hObject, eventdata, handles); % set up labels and default values for new step
guidata(hObject, handles); % update GUI data with new labels
% -------------------------------------------------------- %
% --- Executes on button press in LoadNext.
function LoadNext_Callback(hObject, eventdata, handles)
embn = handles.emb + 1; % update embryo number
if embn<10
emb = ['0',num2str(embn)];
else
emb = num2str(embn);
end
set(handles.embin,'String',emb); % update emb number field in GUI
handles.emb = emb; % update emb number in handles structure
[handles] = imload(hObject, eventdata, handles); % load new embryo
guidata(hObject, handles); % save for access by other functions
% Reset step to step 1;
handles.step = 1; % forward 1 step
set(handles.stepnum,'String', handles.step); % change step label in GUI
handles.output = hObject; % update handles object with new step number
guidata(hObject, handles); % update GUI data with new handles
setup(hObject, eventdata, handles); % set up labels and default values for new step
guidata(hObject, handles); % update GUI data with new labels
%========== change source images ================%
function [handles] = imload(hObject, eventdata,handles)
disp('loading image...');
% handles.source
handles.fin = get(handles.source,'String'); % folder
handles.fname = get(handles.froot,'String'); % embryo name
handles.embn = get(handles.embin,'String'); % embryo number
handles.emb = str2double(handles.embn);
% fname = [handles.fin,'/',handles.fname,'_',handles.embn,'_max.tif'];
fname = [handles.fin,'/','max_',handles.fname,'_',handles.embn,'.tif'];
handles.Im = imread(fname);
h = size(handles.Im,1);
Nuc = handles.Im(:,:,handles.NucChn);
scale = handles.imsize/h;
handles.Nucs = imresize(Nuc,scale);
disp(['rescaling to ',num2str(scale*100,3), ' percent']);
try
Imini = imresize(handles.Im,scale);
figure(1); clf; imshow(Imini);
catch
try % try just nuclear layer.
figure(1); clf; imagesc(Nuc); colormap gray;
catch me
disp(me.message);
end
end
handles.output = hObject;
guidata(hObject,handles);% pause(.1);
disp('image loaded');
%====================================================%
% ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ %
% Automatically return the program to step 0 if the image source directory,
% file name, or image number are changed.
function froot_Callback(hObject, eventdata, handles)
handles.step = 0; % starting step is step 0
set(handles.stepnum,'String',handles.step); % change step label in GUI
handles.output = hObject; % update handles object with new step number
guidata(hObject, handles); % update GUI data with new handles
setup(hObject, eventdata, handles); % set up labels and default values for new step
guidata(hObject, handles); % update GUI data with new labels
function embin_Callback(hObject, eventdata, handles)
handles.step = 0; % starting step is step 0
set(handles.stepnum,'String',handles.step); % change step label in GUI
handles.output = hObject; % update handles object with new step number
guidata(hObject, handles); % update GUI data with new handles
setup(hObject, eventdata, handles); % set up labels and default values for new step
guidata(hObject, handles); % update GUI data with new labels
function source_Callback(hObject, eventdata, handles)
handles.step = 0; % starting step is step 0
set(handles.stepnum,'String',handles.step); % change step label in GUI
handles.output = hObject; % update handles object with new step number
guidata(hObject, handles); % update GUI data with new handles
setup(hObject, eventdata, handles); % set up labels and default values for new step
guidata(hObject, handles); % update GUI data with new labels
% Open file browser to select source folder
function SourceBrowse_Callback(hObject, eventdata, handles)
sourcefile = uigetdir; % prompts user to select directory
set(handles.source,'String',sourcefile);
%% GUI Interface Setup
% The rest of this code just sets up the GUI interface
% --- Outputs from this function are returned to the command line.
function varargout = im_nucseg_OutputFcn(hObject, eventdata, handles)
varargout{1} = handles.output;
% --- Executes during object creation, after setting all properties.
function embin_CreateFcn(hObject, eventdata, handles)
if ispc && isequal(get(hObject,'BackgroundColor'), get(0,'defaultUicontrolBackgroundColor'))
set(hObject,'BackgroundColor','white');
end
function source_CreateFcn(hObject, eventdata, handles)
if ispc && isequal(get(hObject,'BackgroundColor'), get(0,'defaultUicontrolBackgroundColor'))
set(hObject,'BackgroundColor','white');
end
function froot_CreateFcn(hObject, eventdata, handles)
if ispc && isequal(get(hObject,'BackgroundColor'), get(0,'defaultUicontrolBackgroundColor'))
set(hObject,'BackgroundColor','white');
end
function in2_Callback(hObject, eventdata, handles)
function in2_CreateFcn(hObject, eventdata, handles)
if ispc && isequal(get(hObject,'BackgroundColor'), get(0,'defaultUicontrolBackgroundColor'))
set(hObject,'BackgroundColor','white');
end
function in3_Callback(hObject, eventdata, handles)
function in3_CreateFcn(hObject, eventdata, handles)
if ispc && isequal(get(hObject,'BackgroundColor'), get(0,'defaultUicontrolBackgroundColor'))
set(hObject,'BackgroundColor','white');
end
function in1_Callback(hObject, eventdata, handles)
function in1_CreateFcn(hObject, eventdata, handles)
if ispc && isequal(get(hObject,'BackgroundColor'), get(0,'defaultUicontrolBackgroundColor'))
set(hObject,'BackgroundColor','white');
end
function in5_Callback(hObject, eventdata, handles)
function in5_CreateFcn(hObject, eventdata, handles)
if ispc && isequal(get(hObject,'BackgroundColor'), get(0,'defaultUicontrolBackgroundColor'))
set(hObject,'BackgroundColor','white');
end
function in6_Callback(hObject, eventdata, handles)
function in6_CreateFcn(hObject, eventdata, handles)
if ispc && isequal(get(hObject,'BackgroundColor'), get(0,'defaultUicontrolBackgroundColor'))
set(hObject,'BackgroundColor','white');
end
function in4_Callback(hObject, eventdata, handles)
function in4_CreateFcn(hObject, eventdata, handles)
if ispc && isequal(get(hObject,'BackgroundColor'), get(0,'defaultUicontrolBackgroundColor'))
set(hObject,'BackgroundColor','white');
end
function fout_Callback(hObject, eventdata, handles)
% --- Executes during object creation, after setting all properties.
function fout_CreateFcn(hObject, eventdata, handles)
if ispc && isequal(get(hObject,'BackgroundColor'), get(0,'defaultUicontrolBackgroundColor'))
set(hObject,'BackgroundColor','white');
end