Contents
- Cell select analysis
- load the file info to variable in workspace
- Load Parameters
- load file to variable in workspace
- scaling image from 0-255
- Display Scaled Images 1st and nth
- Create bw mask from scaled image
- Plot frames 1-20 of BWmaskclean
- If skipping the shift correction set an array Icellsx as the
- Perform Image shift Adjustment by Crosscorrelation
- Plot the Background Fluorescence Intensity (scaled with bar) and publish
- Select single/isolated cells of interest from an
- Track cells by using centroids of in the first frame
- Create an array of cell coordinates for printing cell names
- Correct the cell selection bwconncomp, error in selecting cells that
- Display each frame with cells of interest tagged by colors
- Get Restricted cell area intensities
- Compute Descriptive Statistics for each cell
- Make a color index to color cells
- Make a character string of numbers for all cells
- Make marker symbol lookup table
- Display and publish the 1st and last image (in original intensity)
- Plot Intensity vs Time for each cell and publish
- Plot Intensity vs Time(frame) and publish
- Plot a Curve Fit to response after treatment
- Save Variables
- Plot Radialwise Intensity and publish
- Save Variables
- Make Avi Movie of original images and Mean +/- SD Inensity of Cell Selection
Cell select analysis
back to top%--------------------------------------------------- %cd('C:\XXX')% where XXX = your file path to image data %---------------------------------------------------
load the file info to variable in workspace
back to top%--------------------------------------------------- %files: % 'B8_treatment_0,1_M_f-14_(GFP,0,2Hz,180f).tif' % 'C8 psychosinie 5,0 M_f-12_(GFP, 0.2Hz, 180f).tif' % 'D8 vehicle_f-12_(GFP, 0.2Hz, 180f).tif' % 'T3-A04_5uM psychosine_40_(GFP, 0.1Hz, 80).tif' %--------------------------------------------------- %Trial 30_05/11/12 %file = 'B2 f-12 treatment XuM H2O (conditions).tif'; %where B2=well number, %f-12=treatment frame, XuM=concentration of tretment, conditions=treatment conditions file = 'B2 f-12 treatment XuM H2O (conditions).tif'; %---------------------------------------------------
Load Parameters
back to top%--------------------------------------------------- %CellArea = 'WholeCell'; %final area may not equal initial area CellArea = 'Restricted'; %final area equals initial area Treatment = file(1:strfind(file,'.tif')-1); %remove .tif from file names if ~isempty(strfind(Treatment,'frame')) %find frame treat when frame notation used FrameTreat = Treatment(strfind(Treatment,'frame')+6:strfind(Treatment,'frame')+7); FrameTreat = str2double(FrameTreat); elseif ~isempty(strfind(Treatment,'f-')) %find frame treat when f- notation used FrameTreat = Treatment(strfind(Treatment,'f-')+2:strfind(Treatment,'f-')+3); FrameTreat = str2double(FrameTreat); elseif ~isempty(strfind(Treatment,'no treatment')) %find if no treatment FrameTreat = 0; else FrameTreat = 0; %assume not treated end FrameRate = ' (0.2 Hz)'; %0.2Hz = 1frame/5seconds %FrameRate = ' (0.1 Hz)'; SampleFreq = 0.2; %sample rate Hz TimeTreat = roundn(FrameTreat/SampleFreq/60,-1); %minute integer FramePostTreat = FrameTreat + 1; Printcolor = 'BW'; %color of plots and figures %Printcolor = 'Color'; %will print graphs in color AxisFont = 20; Itemp = imread(file); %read first frame width = size(Itemp,2); height = size(Itemp,1); clear Itemp; info = imfinfo(file); %load info of series frames = length(info); %frames = 30; %set frames to process less than all frames clear info; Well = strtok(file); %well on plate Passage = 'P-29'; %cell passage number MyDate = '2012-05-11'; %date of experiment %FLuoXunits = 'frame'; FluoXunits = 'minutes'; FluoYLim = [0 255]; %set the fluo y-axis limits FluoYTick = [0; 50; 100; 150; 200; 250]; %make standard fluo y-axis FluoYTickS = num2str(FluoYTick); %make standard fluo y-axis char FluoXt = frames/SampleFreq; %seconds FluoXt = FluoXt/60; %minutes FluoXdiv = floor(FluoXt/5); %integer space between 5 ticks min Min2Frame = FluoXdiv*60*SampleFreq; %space between 5 ticks frames FluoXLim = [1-5 frames+5]; %set the fluo x-axis limits FluoXTick = [1; 1*Min2Frame; 2*Min2Frame;... 3*Min2Frame; 4*Min2Frame; 5*Min2Frame]; %standard fluo x-axis FluoXTickS = num2str([0; 1*FluoXdiv; 2*FluoXdiv;... 3*FluoXdiv; 4*FluoXdiv; 5*FluoXdiv]); %standard fluo x-axis char %Focus = 'Foocus: 10x'; %focus on microscope %Focus = 'Focus: 20x'; Focus = 'Foxus: 40x'; if strcmp(Focus,'Focus: 10x') %pixel to distance conversion pixeldist = '3 Pixels ~ 2um'; elseif strcmp(Focus,'Focus: 20x') pixeldist = '3 Pixels ~ 1um'; else %strcmp(Focus, 'Focus: 40x') pixeldist = '3 Pixels ~ 0.5um'; end %-------------------------------------------------- %%Determine if need to do shift correction %--------------------------------------------------- shiftCorrection = 'Yes'; %shiftCorrection = 'No'; %---------------------------------------------------
load file to variable in workspace
back to top%--------------------------------------------------- I = ones(height,width,frames,'uint8'); %preallocate for k = 1:frames; I(:,:,k) = imread(file,'index',k); end clear width height %---------------------------------------------------
scaling image from 0-255
back to top%--------------------------------------------------- %(01/11/12)-need to create array for storing scaled images (double, uint8) % I1 = I(:,:,1); % I1sc = double(I1); %store to array % I1sc = I1sc - min(min(I1sc)); % I1sc = I1sc * 256/(max(max(I1sc))); % I1sc = uint8(I1sc); %store to array Isc = zeros(size(I),'uint8'); Iscd = double(Isc(:,:,1)); for k = 1:frames; Iscd(:,:,1) = double(I(:,:,k)) - min(min(double(I(:,:,k)))); Isc(:,:,k) = uint8(Iscd * 256 / (max(Iscd(:)))); end clear Iscd; %---------------------------------------------------
Display Scaled Images 1st and nth
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%--------------------------------------------------- % figure; % subplot(1,2,1), imshow(Isc(:,:,1)),title(num2str(1)); % subplot(1,2,2),imshow(Isc(:,:,frames)),title(num2str(frames)); %---------------------------------------------------
Create bw mask from scaled image
back to top%--------------------------------------------------- % level = graythresh(I1sc); % BWI1sc = im2bw(I1sc,level); % figure,imshow(BWI1sc) % %(01/11/12)-need to create array for storing BWIclean images % BWI1clean = bwareaopen(BWI1sc,280); % se = strel('disk',3); % BWI1clean2 = imclose(BWI1clean,se); %store to array BWIclean = logical(I); %preallocate array se = strel('disk',3); %create shifting filter disk for k = 1:frames; if k <=FrameTreat level = 0.75*graythresh(Isc(:,:,k)); %variable percent thresh else level = 0.75*graythresh(Isc(:,:,k)); %75 percent thresh end BWIsc = im2bw(Isc(:,:,k),level); BackgI(:,:,k) = uint8(I(:,:,k)).*uint8(~(BWIsc)); %show background of image BWIclean0 = bwareaopen(BWIsc,200); %delete small objects BWIclean(:,:,k) = imclose(BWIclean0,se); %get rid of pepper in objects end clear Isc level BWIsc BWIclean0 se ; %--------------------------------------------------
Plot frames 1-20 of BWmaskclean
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%--------------------------------------------------- % figure; % subplot(1,2,1), imshow(BWIclean(:,:,1)),title(num2str(1)); % subplot(1,2,2),imshow(BWIclean(:,:,frames)),title(num2str(frames)); % % for k = 1:20; % figure(1),imshow(BWIclean(:,:,k)) % title({['Frame Number ', num2str(k)]}); % pause(2); % end %---------------------------------------------------
If skipping the shift correction set an array Icellsx as the
back to topmask multiplied by the original images. Perform all data analysis using Icellsx, for original intensity values.
%--------------------------------------------------- if strcmp(shiftCorrection,'No') Icellsx = uint8(double(BWIclean).*double(I)); clear I BWIclean end %---------------------------------------------------
Perform Image shift Adjustment by Crosscorrelation
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select cell(s) of interest for registering shift correction Only need to do once on first(might need to adjust later) image for stack of images
%--------------------------------------------------- if strcmp(shiftCorrection,'Yes') [BWIroiI1,xi,yi] = roipoly(double(BWIclean(:,:,1))); %return subroi xi = ceil(xi); yi = ceil(yi); roiymin = max( [ (min(yi))-125, 1 ] ); roixmin = max( [ (min(xi))-125, 1 ] ); roiymax = min( [ (max(yi))+125, size(BWIroiI1,1) ] ); roixmax = min( [ (max(xi))+125, size(BWIroiI1,2) ] ); %BWroiI1b = BWI1clean2(roiymin:roiymax, roixmin:roixmax); %larger roi A = BWIclean(:,:,1); A = A(min(yi):max(yi),min(xi):max(xi)); B = BWIroiI1(min(yi):max(yi),min(xi):max(xi)); C = A.*B; BWItemplate = C; %create and store template for shift registration clear xi yi A B C BWIroiI1 close(gcf) % figure; % imshow(BWItemplate); %-------------------------------------------------- %%Compute the offset between image 1 BWtemplate and image n %-------------------------------------------------- BWIroiI = BWIclean(uint16(roiymin:roiymax), uint16(roixmin:roixmax),:); ypeak = ones(1,frames); xpeak = ypeak; corr_offset = ones(1,2,frames); offset = corr_offset; for k = 1:frames; % BWroitemp = BWIclean2(:,:,k); % BWroiI(:,:,k) = BWroitemp(roiymin:roiymax, roixmin:roixmax); %larger roi cc = normxcorr2(BWItemplate, double(BWIroiI(:,:,k))); [max_cc, imax] = max(abs(cc(:))); [ypeak(k), xpeak(k)] = ind2sub(size(cc),imax(1)); corr_offset(1,:,k) = [ypeak(k)-size(BWItemplate,1),... xpeak(k)-size(BWItemplate,2)]; %Calculate the offset with respect to first image offset(1,:,k) = corr_offset(1,:,k) - corr_offset(1,:,1); %store to array end clear BWIroiI BWItemplate xpeak ypeak corr_offset max_cc imax clear roiymin roiymax roixmin roixmax %-------------------------------------------------- %%Offset cropping for all images with respect to image one %-------------------------------------------------- upy = min(offset(1,1,:));%max shift up (-y) from 2D image shift correlation dny = max(offset(1,1,:));%get max shift down (+y) from 2D image shift correlation lftx = min(offset(1,2,:));%get max shift left (-x) from 2D image shift correlation rgtx = max(offset(1,2,:));%get max shift right (+x) from 2D image shift correlation if upy < 0 %trim the top of canvas yminC = 1 - upy; else %no trim yminC = 1; end if dny > 0 %trim the bottom of canvas ymaxC = size(I(:,:,1),1) - dny; else %no trim ymaxC = size(I(:,:,1),1); end if lftx < 0 %trim the left of canvas xminC = 1 - lftx; else %no trim xminC = 1; end if rgtx > 0 %trim the right of canvas xmaxC = size(I(:,:,1),2) - rgtx; else %no trim xmaxC = size(I(:,:,1),2); end %-------------------------------------------------- % %shift correction for image n versus image 1 %-------------------------------------------------- for k = 1:frames; Isctemp = uint8(double(BWIclean(:,:,k)).*double(I(:,:,k))); offset2 = offset(1,:,k); Icellsx(:,:,k) = Isctemp( (yminC:ymaxC)+offset2(1),... (xminC:xmaxC)+offset2(2)); end clear cc Isctemp offset offset1 offset2 dny upy lftx rgtx %need to limit to +1 and max length or width clear I BWIclean; end %---------------------------------------------------
Plot the Background Fluorescence Intensity (scaled with bar) and publish
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%--------------------------------------------------- for k = [1,frames] figure; if strcmp(shiftCorrection,'Yes') imshow(BackgI(yminC:ymaxC,xminC:xmaxC,k),'DisplayRange',... [min(min((BackgI(:,:,k)))),max(max((BackgI(:,:,k))))],... 'InitialMagnification', 25,'Border', 'loose'), axis on %do not scale 8bit map else imshow(BackgI(:,:,k), 'DisplayRange',... [min(min((BackgI(:,:,k)))),max(max((BackgI(:,:,k))))],... 'InitialMagnification', 25, 'Border', 'loose'), axis on %do not scale 8bit map end %imagesc(BackgI(yminC:ymaxC,xminC:xmaxC,k)), colormap gray %scaled 8bit %map set(gca,'FontSize',10) title({Treatment;... ['Background Fluorescence frame','-',num2str(k)]},'FontSize',AxisFont) xlabel(['pixels',' (',pixeldist,')'],'FontSize',AxisFont) ylabel(['pixels',' (',pixeldist,')'],'FontSize',AxisFont,... 'VerticalAlignment','baseline') hcfig = colorbar; %create colorbar and get handle set(hcfig,'FontSize', 10) cTYfig = 'Fluorescence Intensity'; hcTYfig = get(hcfig,'YLabel'); %get colorbar Yaxis title handle set(hcTYfig,'String',cTYfig,'Rotation',270,... 'VerticalAlignment','bottom','FontSize',AxisFont) %insert string %save figure saveas(gcf, [Treatment, ' Background', ' Frame ', num2str(k),... '.pdf'], 'pdf'); close gcf end clear yminC ymaxC xminC xmaxC BackgI %---------------------------------------------------
Select single/isolated cells of interest from an
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image mask of original image, for analysis
%--------------------------------------------------- k=1; BW2(:,:,k) = bwselect(double(Icellsx(:,:,k))); cc = bwconncomp(BW2(:,:,k)); STATS = regionprops(cc,(Icellsx(:,:,k)), 'BoundingBox',... 'Centroid', 'Extrema', 'PixelList', 'PixelValues','Image'); %%centroid = center of cell mass %%extrema = coordinates of cell corners %%pixelvalues = pixel intensities if strcmp(Printcolor,'Color') L(:,:,k) = labelmatrix(cc); RGB(:,:,:,k) = label2rgb(L(:,:,k)); end objects = cc.NumObjects; %RGB(:,:,:,k) = label2rgb(L(:,:,k),'Lines','k'); %alternate colormap close(gcf) %---------------------------------------------------
Track cells by using centroids of in the first frame
back to topFind same cells in all frames. Assuming the cells are not moving/migrating more than a half length of a cell beween sequential images
%--------------------------------------------------- try %attempt to find cell with given centroid else change centroid for k = 2:frames centroids = cat(1,STATS(:,k-1).Centroid); %use k-1 centroid to find %kth cell %centroids = cat(1,STATS(:,1).Centroid); %use 1st centroid to find kth cell r = centroids(:,2); %y c = centroids(:,1); %x BW2 = bwselect(Icellsx(:,:,k),c,r); cc = bwconncomp(BW2); STATS(:,k) = regionprops(cc, (Icellsx(:,:,k)),... 'BoundingBox', 'Centroid', 'Extrema',... 'PixelList', 'PixelValues','Image'); if strcmp(Printcolor,'Color') L(:,:,k) = labelmatrix(cc); RGB(:,:,:,k) = label2rgb(L(:,:,k)); %RGB(:,:,:,k) = label2rgb(L(:,:,k),'Lines', 'k'); end end clear BW2 centroids r c cc catch err if strcmp(err.identifier,'MATLAB:badsubscript') try for k = 2:frames %centroids = cat(1,STATS(:,k-1).Centroid); %use k-1 centroid to find %kth cell centroids = cat(1,STATS(:,1).Centroid); %use 1st centroid to find kth cell r = centroids(:,2); %y c = centroids(:,1); %x BW2 = bwselect(Icellsx(:,:,k),c,r); cc = bwconncomp(BW2); STATS(:,k) = regionprops(cc, (Icellsx(:,:,k)),... 'BoundingBox', 'Centroid', 'Extrema',... 'PixelList', 'PixelValues','Image'); if strcmp(Printcolor,'Color') L(:,:,k) = labelmatrix(cc); RGB(:,:,:,k) = label2rgb(L(:,:,k)); %RGB(:,:,:,k) = label2rgb(L(:,:,k),'Lines', 'k'); end end clear BW2 centroids r c cc catch err2 rethrow(err) end else rethrow(err); %if any other error stop end end %if error mismatch index dimension compare k-1 and k-2 %---------------------------------------------------
Correct the cell selection bwconncomp, error in selecting cells that
back to topshift between consecutive frames 07-5-12
%--------------------------------------------------- SwapObjects = zeros(objects,1); %initialize the swap object array for i=1:objects; cent1 = STATS(i,1).Centroid; %get the first frame selection centroid n=1; for k=1:frames; cent2 = STATS(i,k).Centroid; d = sqrt((cent1(1)-cent2(1))^2 + (cent1(2)-cent2(2))^2); if d > 45 %arbitrary threshold if exceeds then centroid switched SwapObjects(i,n) = k; %store error condition frames for objects n = n+1; end end end %%Swap the flagged Objects SwapObjects_1 = ceil(mean(SwapObjects,2)/frames); %make binary array of flaged objects if sum(SwapObjects_1) == 2; %case for when only two objects are swapping n = 1; for k = 1:length(SwapObjects_1) %step through binary array if SwapObjects_1(k) == 1 TwoObjects(n) = k; %get the object indices n = n+1; end end for k = SwapObjects(TwoObjects(1),:); TwoObjects_1 = STATS(TwoObjects(1),k); %temp store STATS object 1 TwoObjects_2 = STATS(TwoObjects(2),k); %temp store STATS object 2 STATS(TwoObjects(1),k) = TwoObjects_2; %switch STATS object 1 STATS(TwoObjects(2),k) = TwoObjects_1; %switch STATS object 2 end else if sum(SwapObjects_1) > 2; %case for when 2+ objects are switching for k = 1:size(SwapObjects,2) %step through the columns for i = 1:size(SwapObjects,1) %step through the rows if SwapObjects(i,k) > 0 cent1 = STATS(i,1).Centroid; %get the objects 1st centroid %d_temp(1) = sqrt( ( n=1; for j = 1:objects; cent2 = STATS(j,SwapObjects(i,k)).Centroid; %variable, flagged frame object D(n) = sqrt( (cent1(1)-cent2(1))^2 + (cent1(2)-cent2(2))^2 ); n = n+1; end [~,minObject] = min(D); %get object index Objects_1 = STATS(i,SwapObjects(i,k)); %temp stats flagged object Objects_2 = STATS(minObject,SwapObjects(i,k)); %temp stats minD object STATS(i,SwapObjects(i,k)) = Objects_2; %switch flagged object with minD object STATS(minObject,SwapObjects(i,k)) = Objects_1; %switch minD object with flagged object end end end end end %---------------------------------------------------
Create an array of cell coordinates for printing cell names
back to topnext to cells (Extrema(2,:) = [x y] of top right corner)
%--------------------------------------------------- % pos = ones(objects,2,frames); % for k = 1:frames % for i = 1:objects % pos(i,:,k) = STATS(i,k).Extrema(2,:); %pos[x y] % end % end %---------------------------------------------------
Display each frame with cells of interest tagged by colors
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%--------------------------------------------------- % for k = 1:1 % imshow(RGB(:,:,:,k),'InitialMagnification', 25,... % 'Border', 'loose'),title({['Frame ', num2str(k)];... % 'Color coded cells selected for analysis'}) % axis on % xlabel(['pixels',pixeldist]) % ylabel(['pixels',pixeldist],'VerticalAlignment','middle') % for i = 1:objects % text(floor(pos(i,1,k)), ceil(pos(i,2,k)), num2str(i),... % 'HorizontalAlignment', 'left',... % 'VerticalAlignment', 'baseline'); % end % pause(0.3) % end % close(gcf) %---------------------------------------------------
Get Restricted cell area intensities
back to top%attempt to correct for inequal cell cropping as image contrast changes %--------------------------------------------------- for i = 1:objects for k = 1:frames Areas(i,k) = length(STATS(i,k).PixelList); %store object areas at each frame end [Sarea(i) PosSa(i)] = min(Areas(i,:)); %store smallest area size and frame for each object end Template = zeros(700); for i = 1:objects Ismall = STATS(i,PosSa(i)).Image; %smallest object image uLsmall = floor(STATS(i,PosSa(i)).BoundingBox(1:2)); %smallest image uL corner for k = 1:frames uLcurrent = floor(STATS(i,k).BoundingBox(1:2)); %current uL corner Ishift = uLsmall -uLcurrent; %determine if any difference in position of uL corners Icurrent = STATS(i,k).Image; %current object image IcurrentTemplate = Template; IcurrentTemplate(300: 300 +size(Icurrent,1) -1,... 300: 300 +size(Icurrent,2) -1) = Icurrent; %place the current object image on template IsmallTemplate = Template; IsmallTemplate(300 +Ishift: 300 +(size(Ismall,1) -1 +Ishift),... 300 +Ishift: 300 + (size(Ismall,2) -1 +Ishift)) = Ismall; Ioverlay = IcurrentTemplate.*IsmallTemplate; %compute the logical operation && Overlayidx = find(Ioverlay == 1); %find linear indices of active pixels [Irow Icol] = ind2sub(size(Ioverlay),Overlayidx); %convert linear indeces to image coordinates Irow = Irow -(300 -1); %correct for the offset placement onto the template Icol = Icol -(300 -1); %correct for the offset placement onto the template Irow = Irow +uLcurrent(2); %correct for the offset of object on original image Icol = Icol +uLcurrent(1); %correct for the offset of object on original image Overlayidx = sub2ind(size(Icellsx(:,:,k)),Irow,Icol); %get linear indices of original object embedded in original image Iorig = Icellsx(:,:,k); STATS(i,k).MyPixelValues = Iorig(Overlayidx); end end %---------------------------------------------------
Compute Descriptive Statistics for each cell
back to topArea Mean Intensity Max Intensity Standard Error of the Mean
%--------------------------------------------------- for k = 1:frames PixInt = []; for i = 1:objects if strcmp(CellArea,'WholeCell') pixInt = double(STATS(i,k).PixelValues); else pixInt = double(STATS(i,k).MyPixelValues); end areaInt(i,1,k) = length(pixInt); meanInt(i,1,k) = mean(pixInt); maxInt(i,1,k) = max(pixInt); semInt(i,1,k) = std(pixInt)/sqrt(length(pixInt)); PixInt = cat(1,PixInt,pixInt); end MeanInt(k) = mean(PixInt); %compute the mean intensity for frame StdInt(k) = std(PixInt); %compute the std for frame SemInt(k) = std(PixInt)/sqrt(length(PixInt)); %compute the SEM for frame end clear pixInt PixInt; % %%Find outliers, use function % if FrameTreat == 0; ObjectOut = zeros(1,objects); %fix later. Do not find outliers else [ObjectOut Alphas Taus] = myout3(meanInt,FrameTreat); end % %%Compute new MeanInt,StdInt,SemInt % if ~isempty(find(ObjectOut,1))%compute if outliers found for k = 1:frames PixInt = []; for i = 1:objects if strcmp(CellArea,'WholeCell') %use whole cell fluorescence pixInt = double(STATS(i,k).PixelValues); else pixInt = double(STATS(i,k).MyPixelValues); %use area select fluorescence end if ~ObjectOut(i) %if object out is not true concatenate the array PixInt = cat(1,PixInt,pixInt); end end MeanIntadj(k) = mean(PixInt); StdIntadj(k) = std(PixInt); SemIntadj(k) = std(PixInt)/sqrt(length(PixInt)); end clear pixInt PixInt; else MeanIntadj = ''; StdIntadj = ''; SemIntadj = ''; end %---------------------------------------------------
Make a color index to color cells
back to top%--------------------------------------------------- figure(1) colors = colormap; close(1) colorIdx = 1:objects; for k = 1:objects colorIdx(k) = floor( (k-1)*(64-1)/(objects-1) ) +1; end colorId = colors(colorIdx,:); clear colorIdx %---------------------------------------------------
Make a character string of numbers for all cells
back to top%--------------------------------------------------- cellnumd = (1:objects)'; cellnumd = num2str(cellnumd); %---------------------------------------------------
Make marker symbol lookup table
back to topcan only use markers if #objects <= 13
%--------------------------------------------------- mymark = ['s','o','+','d','^','p','v','*','>','x','<','.','h']; %---------------------------------------------------
Display and publish the 1st and last image (in original intensity)
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display the 1st and last image selection (in original intensity) need to store the 1st and last images I for displaying
%--------------------------------------------------- cTYfig = 'Fluorescence Intensity'; %create text % Icellsxx = uint8(logical(L).*Icellsx); % figure,imshow(Icellsxx(:,:,1),'InitialMagnification', 25,... % 'Border', 'loose'), colorbar ,title('Frame 1 selection') % xlabel('pixels') % ylabel('pixels','VerticalAlignment','middle') % hcfig = colorbar; %create colorbar and get handle % hcTYfig = get(hcfig,'YLabel'); %get colorbar Yaxis title handle % set(hcTYfig,'String',cTYfig,'Rotation',270,... % 'VerticalAlignment','bottom') %insert string % axis on; % figure,imshow(Icellsxx(:,:,frames),'InitialMagnification', 25,... % 'Border', 'loose'), colorbar ,title({'Frame', num2str(frames), 'selection'}) % xlabel('pixels') % ylabel('pixels','VerticalAlignment','middle') % hcfig = colorbar; %create colorbar and get handle % hcTYfig = get(hcfig,'YLabel'); %get colorbar Yaxis title handle % set(hcTYfig,'String',cTYfig,'Rotation',270,... % 'VerticalAlignment','bottom') %insert string % axis on; figure,imshow(uint8(Icellsx(:,:,1)),'InitialMagnification', 25,... 'Border', 'loose'), colorbar, title('Frame 1','FontSize',AxisFont) set(gca,'FontSize',10) xlabel(['pixels',' (',pixeldist,')'],'FontSize',AxisFont) ylabel(['pixels',' (',pixeldist,')'],'FontSize',AxisFont,... 'VerticalAlignment','baseline') hcfig = colorbar; %create colorbar and get handle hcTYfig = get(hcfig,'YLabel'); %get colorbar Yaxis title handle set(hcTYfig,'String',cTYfig,'Rotation',270,... 'FontSize',AxisFont,'VerticalAlignment','bottom') %insert string axis on; k = 1; %set the frame index for cell number assignment Ix = size(Icellsx,2)/2; %half length of image x-axis Iy = size(Icellsx,1)/2; %half length of image y-axis for i = 1:objects rectangle('Position',STATS(i,k).BoundingBox,... 'EdgeColor',[0 1 0]) %draw box around object Objx = STATS(i,k).Centroid(1); %centroid of object Objy = STATS(i,k).Centroid(2); %centroid of object ObjExt = floor(STATS(i,k).Extrema); if Objx <= Ix && Objy <= Iy %quadrant 2 of image text(ObjExt(5,1), ObjExt(5,2), num2str(i),... 'HorizontalAlignment', 'left','VerticalAlignment',... 'top','color',[1 1 1]); elseif Objx > Ix && Objy <= Iy %quadrant 1 of image text(ObjExt(6,1), ObjExt(6,2), num2str(i),... 'HorizontalAlignment', 'right','VerticalAlignment',... 'top','color',[1 1 1]); elseif Objx <= Ix && Objy > Iy %quadrant 3 of image text(ObjExt(2,1), ObjExt(2,2), num2str(i),... 'HorizontalAlignment', 'left','VerticalAlignment',... 'bottom','color',[1 1 1]); else %Objx > Ix && Objy > Iy %quadrant 4 of image text(ObjExt(1,1), ObjExt(1,2), num2str(i),... 'HorizontalAlignment', 'right','VerticalAlignment',... 'bottom','color',[1 1 1]); end end %save file saveas(gcf, [Treatment, ' Selection', ' Frame ', num2str(1),... '.pdf'], 'pdf'); close gcf figure,imshow(uint8(Icellsx(:,:,frames)),'InitialMagnification', 25,... 'Border', 'loose'), colorbar, title({'Frame', num2str(frames)},... 'FontSize',AxisFont) set(gca,'FontSize',10) xlabel(['pixels',' (',pixeldist,')'],'FontSize',AxisFont) ylabel(['pixels',' (',pixeldist,')'],'FontSize',AxisFont,... 'VerticalAlignment','baseline') hcfig = colorbar; %create colorbar and get handle hcTYfig = get(hcfig,'YLabel'); %get colorbar Yaxis title handle set(hcTYfig,'String',cTYfig,'Rotation',270,... 'FontSize',AxisFont,'VerticalAlignment','bottom') %insert string axis on; k = frames; %set the frame index for cell number assignment Ix = size(Icellsx,2)/2; %half length of image x-axis Iy = size(Icellsx,1)/2; %half length of image y-axis for i = 1:objects rectangle('Position',STATS(i,k).BoundingBox,... 'EdgeColor',[0 1 0]) %draw box around object Objx = STATS(i,k).Centroid(1); %centroid of object Objy = STATS(i,k).Centroid(2); %centroid of object ObjExt = floor(STATS(i,k).Extrema); if Objx <= Ix && Objy <= Iy text(ObjExt(5,1), ObjExt(5,2), num2str(i),... 'HorizontalAlignment', 'left','VerticalAlignment',... 'top','color',[1 1 1]); elseif Objx > Ix && Objy <= Iy text(ObjExt(6,1), ObjExt(6,2), num2str(i),... 'HorizontalAlignment', 'right','VerticalAlignment',... 'top','color',[1 1 1]); elseif Objx <= Ix && Objy > Iy text(ObjExt(2,1), ObjExt(2,2), num2str(i),... 'HorizontalAlignment', 'left','VerticalAlignment',... 'bottom','color',[1 1 1]); else %Objx > Ix && Objy > Iy text(ObjExt(1,1), ObjExt(1,2), num2str(i),... 'HorizontalAlignment', 'right','VerticalAlignment',... 'bottom','color',[1 1 1]); end end %save file saveas(gcf, [Treatment, ' Selection', ' Frame ', num2str(frames),... '.pdf'], 'pdf'); close gcf %clear Icellsxx %---------------------------------------------------
Plot Intensity vs Time for each cell and publish
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%--------------------------------------------------- PlotTitle = {[Treatment ' ' MyDate]}; % tickx = 0:60:frames; %make ticks at intervals of 60 % tickx(tickx==0) = 1; %make all zeros into ones % tickxs = cell(length(tickx),1); %preallocate % for k = 1:length(tickx) % tickxs{k} = num2str(tickx(k)); %make a cell array of tick names % end tickx = FluoXTick; %make tick intervals for markers hfig1 = figure; %handle to figure hsub1 = subplot(1,2,1); %handle to subplot hold on; %hold figure plots, dont overwrite title('Mean Intensity','FontSize',AxisFont); ylabel('Average Fluorescence Intensity','FontSize',AxisFont) if strcmp(FluoXunits,'frame') xlabel('Frame (Rate = 0.2Hz)','FontSize',AxisFont); else xlabel('Time (minutes)','FontSize',AxisFont); end X1 = (1:frames)'; if objects <= 13 && strcmp(Printcolor,'BW') for i = 1:objects Y1 = meanInt(i,1,:); Y1 = Y1(:); if ObjectOut(i) plot(X1(tickx),Y1(tickx),mymark(i),'MarkerSize',6,... 'MarkerFaceColor',[1 1 1], 'MarkerEdgeColor','k') else plot(X1(tickx),Y1(tickx),mymark(i),'MarkerSize',6,... 'MarkerFaceColor',[0 0 0], 'MarkerEdgeColor','k') end end hlegend = legend(cellnumd, 'Location',... 'NorthEastOutside'); for i = 1:objects %make diff markers for line series Y1 = meanInt(i,1,:); Y1 = Y1(:); if ObjectOut(i) plot(X1,Y1, 'color',[.5 .5 .5], 'LineStyle','-') else plot(X1,Y1, 'k-') end end else for i = 1:objects Y1 = meanInt(i,1,:); Y1 = Y1(:); if ObjectOut(i) plot(X1,Y1, '--','color',[0.5 0.5 0.5]... ,'Marker','o','MarkerSize',3,... 'MarkerFaceColor',[0.5 0.5 0.5] ) else plot(X1,Y1, '--','color',colorId(i,:)... ,'Marker','o','MarkerSize',3,... 'MarkerFaceColor',colorId(i,:) ) end end hlegend = legend(cellnumd, 'Location',... 'NorthEastOutside'); end ylim(FluoYLim); hold off; hsub2 = subplot(1,2,2); title('Max Intensity','FontSize',AxisFont); ylabel('Fluorescence Intensity','FontSize',AxisFont) if strcmp(FluoXunits,'frame') xlabel('Frame (Rate = 0.2Hz)','FontSize',AxisFont); else xlabel('Time (minutes)','FontSize',AxisFont); end hold on; if objects <= 13 && strcmp(Printcolor,'BW') for i = 1:objects Y1 = maxInt(i,1,:); Y1 = Y1(:); if ObjectOut(i) plot(X1(tickx),Y1(tickx),mymark(i),'MarkerSize',6,... 'MarkerFaceColor',[1 1 1], 'MarkerEdgeColor','k') else plot(X1(tickx),Y1(tickx),mymark(i),'MarkerSize',6,... 'MarkerFaceColor',[0 0 0], 'MarkerEdgeColor','k') end end for i = 1:objects %make diff markers for line series Y1 = maxInt(i,1,:); Y1 = Y1(:); if ObjectOut(i) plot(X1,Y1, 'color',[0.5 0.5 0.5], 'LineStyle','-') else plot(X1,Y1, 'k-') end end else for i = 1:objects Y1 = maxInt(i,1,:); Y1 = Y1(:); if ObjectOut(i) plot(X1,Y1, '--','color',[0.5 0.5 0.5]... ,'Marker','o','MarkerSize',3,... 'MarkerFaceColor',[0.5 0.5 0.5] ) else plot(X1,Y1, '--','color',colorId(i,:)... ,'Marker','o','MarkerSize',3,... 'MarkerFaceColor',colorId(i,:) ) end end end ylim(FluoYLim); htitle = text(1, 1, PlotTitle, 'FontSize', AxisFont,... 'FontWeight', 'bold', 'HorizontalAlignment',... 'center'); hold off; titlepos = [-36 303.382 17.3205]; legpos =[0.8551, 0.1112, 0.1283, 0.7641]; %position legout = [0.86, 0.1061, 0.1390, 0.7691]; %position legPBR = [1 3.1250 1]; %aspect ratio legDAR = [1 0.3200 2]; %aspect ratio sub1pos = [0.0820, 0.10, 0.32, 0.7]; %position sub1out = [0.01, -0.109, 0.42, 1.025]; %position sub2pos = [0.5, 0.10, 0.32, 0.7]; %position sub2out = [0.4492, -0.109, 0.42, 1.025]; %position set(hfig1,'PaperOrientation','landscape'); set(htitle,'Position', titlepos); set(hlegend,'FontSize', 10); set(hlegend,'Position',legpos); set(hlegend,'OuterPosition',legout); set(hlegend,'DataAspectRatio',legDAR); set(hlegend,'PlotBoxAspectRatio',legPBR); set(hsub1,'TickDir','out'); set(hsub1,'Position',sub1pos); set(hsub1,'OuterPosition',sub1out); set(hsub1,'XLim',FluoXLim); %x-axis limits set(hsub1,'XTick', FluoXTick); set(hsub1,'XTickLabel',FluoXTickS); set(hsub1,'YTick', FluoYTick); set(hsub1,'YTickLabel',FluoYTickS); set(hsub2,'TickDir','out'); set(hsub2,'Position',sub2pos); set(hsub2,'OuterPosition',sub2out); set(hsub2,'Xlim',FluoXLim); set(hsub2,'XTick', FluoXTick); set(hsub2,'XTickLabel',FluoXTickS); set(hsub2,'YTick', FluoYTick); set(hsub2,'YTickLabel',FluoYTickS); if FrameTreat ~= 0 %mark treat time if treated hsub1at = text(FrameTreat,FluoYLim(1),... '{\fontname{arial}v}',... 'Parent',hsub1,'Color',[.1 .1 .1],'Fontsize', 10,... 'HorizontalAlignment','Center',... 'VerticalAlignment','baseline'); hsub1bt = text(FrameTreat,FluoYLim(1)+10,... ['Treat ',num2str(TimeTreat), ' min'],... 'Parent',hsub1,'Color',[.1 .1 .1],'Fontsize', 10,... 'HorizontalAlignment','Left',... 'VerticalAlignment','baseline'); hsub2at = text(FrameTreat,FluoYLim(1),... '{\fontname{arial}v}',... 'Parent',hsub2,'Color',[.1 .1 .1],'Fontsize', 10,... 'HorizontalAlignment','Center',... 'VerticalAlignment','baseline'); hsub2bt = text(FrameTreat,FluoYLim(1)+10,... ['Treat ',num2str(TimeTreat), ' min'],... 'Parent',hsub2,'Color',[.1 .1 .1],'Fontsize', 10,... 'HorizontalAlignment','Left',... 'VerticalAlignment','baseline'); hlegendt = text(0.5,1,'Cell','Parent',hlegend,'units','normalized',... 'VerticalAlignment','bottom','HorizontalAlignment','center',... 'FontSize',AxisFont); end %save figure saveas(gcf, [Treatment, ' Mean and Max Intensity Plots', '.pdf'], 'pdf'); close gcf %---------------------------------------------------
Plot Intensity vs Time(frame) and publish
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%--------------------------------------------------- Ytemp = {SemInt; StdInt; SemIntadj; StdIntadj}; Titletemp = {'Mean and SEM'; 'Mean and SD'; 'Mean and SEM (no outliers)';... 'Mean and SD (no outliers)' }; MeanIntTemp = {MeanInt; MeanInt; MeanIntadj; MeanIntadj}; for k = 1:4 if k>2 && isempty(find(ObjectOut,1)) continue %skip iteration if k>2 and if there were no outliers identified end figure; herr = errorbar(MeanIntTemp{k},Ytemp{k},'k'); %plot using errorbar function % herrleg = legend({'Mean Fluorescence Intensity'},... % 'Location','NorthEastOutside','Orientation',... % 'vertical'); title({Treatment;Titletemp{k}},'FontSize',AxisFont); %title(file) ylabel('Average Fluorescence Intensity','FontSize',AxisFont); if strcmp(FluoXunits,'frame') xlabel('Frame (Rate = 0.2Hz)','FontSize',AxisFont); else xlabel('Time (minutes)','FontSize',AxisFont); end xlim(FluoXLim) ylim(FluoYLim) haxes = gca; %get current axes handle set(haxes,'TickDir','out', 'box','off'); set(haxes,'XTick', FluoXTick); %set xticks set(haxes,'XTickLabel', FluoXTickS,'FontSize',10); %set the Xlabels set(haxes,'YTick', FluoYTick); %set yticks set(haxes,'YTickLabel', FluoYTickS,'FontSize',10); %set the Ylabels if FrameTreat ~= 0 %mark treat time if treated h3atxt = text(FrameTreat,FluoYLim(1),... '{\fontname{arial}v}',... 'Color',[.1 .1 .1],'Fontsize', 10,... 'HorizontalAlignment','Center',... 'VerticalAlignment','baseline'); h3btxt = text(FrameTreat,FluoYLim(1)+10,... ['Treat ',num2str(TimeTreat), ' min'],... 'Color',[.1 .1 .1],'Fontsize', 10,... 'HorizontalAlignment','Left',... 'VerticalAlignment','baseline'); end %save figure saveas(gcf, [Treatment, ' ', Titletemp{k}, ' Plot', '.pdf'], 'pdf'); close gcf end clear Ytemp Titletemp MeanIntTemp %-----------------------------------------------
Plot a Curve Fit to response after treatment
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%----------------------------------------------- if FrameTreat ~= 0 fitcurve = []; goodness = []; TitleTemp = 'Fit Curve (Post Treatment)'; eval('[fitcurve goodness] = myfit2(MeanInt,FrameTreat)') %get fit curve to data with outliers set(gca,'Box','off'); gcf; title({Treatment; TitleTemp},'FontSize',AxisFont); saveas(gcf,[Treatment, ' ', TitleTemp, 'Plot', '.pdf'], 'pdf'); close gcf; if ~isempty(find(ObjectOut,1)) fitcurveadj = []; goodnessadj = []; TitleTemp = 'Fit Curve (Post Treatment, no outliers)'; eval('[fitcurveadj goodnessadj] = myfit2(MeanIntadj,FrameTreat)') %get fit curve to data without outliers set(gca,'Box','off'); gcf; title({Treatment; TitleTemp},'FontSize',AxisFont); saveas(gcf,[Treatment, ' ', TitleTemp, 'Plot', '.pdf'], 'pdf'); close gcf; end end %-----------------------------------------------
Save Variables
back to top%----------------------------------------------- if FrameTreat ~= 0 && ~isempty(find(ObjectOut,1)) %save when Treated and when outliers exist save([Treatment,' variables','.mat'], 'MeanInt',... 'STATS','SemInt','StdInt','maxInt','meanInt','semInt','fitcurve',... 'goodness','fitcurveadj','goodnessadj'); elseif FrameTreat ~= 0 && isempty(find(ObjectOut,1)) %save when Treated and no outliers save([Treatment,' variables','.mat'], 'MeanInt',... 'STATS','SemInt','StdInt','maxInt','meanInt','semInt','fitcurve',... 'goodness'); elseif FrameTreat == 0 && ~isempty(find(ObjectOut,1)) %save when not treated and outliers exist save([Treatment,' variables','.mat'], 'MeanInt',... 'STATS','SemInt','StdInt','maxInt','meanInt','semInt'); else %save when not treated and no outliers save([Treatment,' variables','.mat'], 'MeanInt',... 'STATS','SemInt','StdInt','maxInt','meanInt','semInt'); end %-----------------------------------------------
Plot Radialwise Intensity and publish
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%----------------------------------------------- %%Create Rings % rings will overlap betwen adjacent radii because % of discrete shape of image pixels %%Set Constant Parameter & preallocate arrays Theta = linspace(0,2*pi,40); Ring = cell(objects,frames); %create cell to store cell r data cellboxw = 1:objects; cellboxh = cellboxw; Cellsub = cell(objects,1); %create cell array to store cropped cells %%Get Parameters one frame at a time for fr = 1:frames; %iterate through frames for i = 1:objects %iterate through objects centroid1 = floor(STATS(i,fr).Centroid); uLcorner1 = ceil(STATS(i,fr).BoundingBox(1:2)); centroid1sub = centroid1 - uLcorner1 + 1; %get sub centroid coordinates cellboxw(i) = ceil(STATS(i,fr).BoundingBox(3))-1; cellboxh(i) = ceil(STATS(i,fr).BoundingBox(4))-1; cellboxW = uLcorner1(1):uLcorner1(1)+cellboxw(i);%turn over for each object cellboxH = uLcorner1(2):uLcorner1(2)+cellboxh(i); %turn over for each object Cellsub{i,1} = Icellsx(cellboxH,cellboxW,fr); %turn over for ea frame rlim = ceil(1.415*max([cellboxw(i),cellboxh(i)])/2); %find max angular radii for object i Ring{i,fr} = cell(rlim,1); %create cell arrays in Ring to store radii data %Make circle r1 for k=1 %iterate through r1 r = k; %radius of circle in pixels Rho = ones(1,40)*r; %create array of angles [X Y] = pol2cart(Theta,Rho); %convert to cartesian X = ((X+centroid1sub(1))); %shift center of circles Y = (Y+size(Cellsub{i,1},1)-centroid1sub(2)+1); BWr = roipoly(Cellsub{i,1},X,Y); %use Cellsub vs Template %BWr = roipoly(Template,X,Y); %create bw template for r BWr = bwmorph(BWr,'spur'); %remove "thorns" [R C] = find(BWr); %find all coordinates of ring Idx = sub2ind(size(BWr),R,C); %convert coordinates to linear index Ring{i,fr}{k,1}(1,:) = Idx; %store linear index V = Cellsub{i,1}(Idx); %get intensity Ring{i,fr}{k,1}(2,:) = V; %store intensity clear C R X Y V end %Make circles 2-rlim %For loop start with ring 3 end with ring rlim %counter will be set to k, create r=k+2 and r=k rings for k = 2:rlim %iterate through r2+ r = k; %radius of circle in pixels, not inclusive Rho = ones(1,40)*r; %create array of angles [X Y] = (pol2cart(Theta,Rho)); %convert to cartesian X = ((X+centroid1sub(1))); %shift center of circles Y = (Y+size(Cellsub{i,1},1)-centroid1sub(2)+1); BWr = roipoly(Cellsub{i,1},X,Y); %create bw template for r BWr = bwmorph(BWr,'spur'); BWr = bwmorph(BWr,'remove'); clear X Y [R C] = find(BWr); %get coordinates of ring if isempty(R) %stop iterating through radii if radii rings fall off break end Idx = sub2ind(size(BWr),R,C); %convert coordinates to linear index V = Cellsub{i,1}(Idx); %get intensity if r >= round(1/1.415*(rlim)) %start checking values at orthoganol radii if sum(V) == 0 %stop writing values if equal to zero break end end Ring{i,fr}{k,1}(1,:) = Idx; %store index Ring{i,fr}{k,1}(2,:) = V; %store intensity clear C R X Y V end end end clear BWr* cellbox* Cellsub %%Go through all frames and compute the average r values for each object %%and for all cells %Robjects = cell(objects,1); Rframes = cell(1,frames); %replace second entry with frames for i = 1:frames %iterate through frames for j = 1:size(Ring(:,i),1) %iterate through objects a(j) = size(Ring{j,i},1); %get size of radii for each object in frame end a = max(a); %get max radii size for frame i Rframes{1,i} = cell(a,1); %preallocate cell size of length max radii for j = 1:objects %iterate through objects for k = 1:size(Ring{j,i},1) %iterate through radii %Store radii collapse objects, maintain frames Rframes{1,i}{k,1} = cat(2,Rframes{1,i}{k,1},... Ring{j,i}{k,1}(2,:)); end end end clear Ring %%Find the mean value for radii of each frame for i = 1:frames %iterate through frames for j = 1:size(Rframes{1,i},1) %iterate through radii Rmeanframes{1,i}(j,1) = mean(nonzeros(Rframes{1,i}{j,1})); Rstd = std(nonzeros(Rframes{1,i}{j,1})); Rstderrframes{1,i}(j,1) = Rstd/sqrt(nnz(Rframes{1,i}{j,1})); end end clear Rframes %create a matrix of radii %Yaxis Frame # %Xaxis Radial distance from centroid %Zaxis Average Radialwise Fluorescence Intensity %A = cell2mat(Rmeanframes); %create a matrix, might error if dim wrong A = []; for k = 1:frames %iterate through frames A(1:size(Rmeanframes{1,k},1),k) = Rmeanframes{1,k}(:); %(RFIxFrame) end A = fliplr(A); %flip matrix columns, mirror the frame axis (FramexRFI) hfsurf = figure; %create figure and get handle hsurf = surf(A); %create mesh plot for Rmeanframes if strcmp(Printcolor,'BW') colormap gray else colormap jet end title({Treatment;... 'Average Radialwise Fluorescence Intensity';... ['Cells n = ', num2str(objects)] },'FontSize',AxisFont) %update if strcmp(FluoXunits,'minutes') xlabel('Time (minutes)','FontSize',AxisFont) else xlabel('Frame (Rate = 0.2Hz)','FontSize',AxisFont) end ylabel('Radial Distance from Centroid (pixels)','FontSize',AxisFont) zlabel('Radialwise Average Fluorescence Intensity','FontSize',AxisFont) set(hsurf,'EdgeColor','flat') %set edge box color set(hfsurf,'Position',[50 250 550 400]); %position figure window view(90,90); %change surf orientation hcsurf = colorbar; %create colorbar and get handle % xtickdiv = frames/6; %create x-axis divisions space % xtickdiv = 0:xtickdiv:frames; %create x-axis % xtickdiv(xtickdiv==0)=1; %make 0 = 1 haxes = gca; %get current axes handle set(haxes,'XLim',[1 frames]) %set x-axis limits set(haxes,'XTick',FluoXTick) %set x-axis ticks set(haxes,'XTickLabel',flipud(FluoXTickS)) %write reverse labels % xtickL = get(haxes,'XTick'); %get x-axis labels % xtickL = fliplr(xtickL); %make the reverse of x-axis labels % set(haxes,'XTickLabel', xtickL); %write reverse the Xlabels radialLim = size(A); set(haxes,'Ylim',[1 radialLim(1)]) set(haxes,'XGrid','off','YGrid','off','ZGrid','off') set(haxes,'TickDir','out') cTLsurf = get(hcsurf,'YLim'); %get colorbar limits set(hcsurf,'YTick',cTLsurf) %create ticks at limits set(hcsurf,'YTickLabel',{'Low','High'}) %create Yticklabels cTYSsurf = {'Average'; 'Fluorescence Intensity'}; %create colorbar title string hcTYsurf = get(hcsurf,'YLabel'); %get colorbar Yaxis title handle set(hcTYsurf,'String',cTYSsurf,'Rotation',270,... 'VerticalAlignment','baseline','FontSize',AxisFont) %insert string %tightmap %eliminate/reduce white boarder ztemp = zlim; ztemp = ztemp(2); if FrameTreat ~= 0 %mark treat time if treated text(frames-FrameTreat,1,ztemp,['< Treat ',num2str(TimeTreat),' min'],... 'fontsize',10,'Color',[.1 .1 .1],... 'BackgroundColor','w') end %save file saveas(gcf, [Treatment, ' Average Radial Fluorescence', '.pdf'], 'pdf'); close gcf %-----------------------------------------------
Save Variables
back to top%----------------------------------------------- if FrameTreat ~= 0 save([Treatment,' variables','.mat'], 'Treatment','MeanInt','Rmeanframes','Rstderrframes',... 'STATS','SemInt','StdInt','maxInt','meanInt','semInt'); else save([Treatment,' variables','.mat'], 'Treatment','MeanInt','Rmeanframes','Rstderrframes',... 'STATS','SemInt','StdInt','maxInt','meanInt','semInt','fitcurve','goodness'); end %-----------------------------------------------
Make Avi Movie of original images and Mean +/- SD Inensity of Cell Selection
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%----------------------------------------------- %Open video object and append frames from Image stack aviobj = VideoWriter([Treatment,' ', MyDate,'.avi']); %make file name aviobj.FrameRate = 12; %frames per second aviobj.Quality = 100; %percent quality open(aviobj); %open an object file for k = 1:frames; fig = figure(1); h12=subplot(1,2,1); imshow(Icellsx(:,:,k),'DisplayRange',... [min(min(Icellsx(:,:,k))),max(max(Icellsx(:,:,k)))],... 'InitialMagnification', 25,'Border', 'loose'); %do not scale 8bit map %imshow(Icellsx(:,:,k)),... hcb = colorbar('location','SouthOutside'); if k < FramePostTreat; title({'Pre Treat'; []},'backgroundcolor',[0 1 0]); else title({[]; 'Post Treat'},'backgroundcolor',[1 0 0]); end htxt = text(1,1.35,{['NSC-34 ', Passage, ' ', Treatment,' ', MyDate,];... ['Frame ', num2str(k),' of ', num2str(frames),... FrameRate]}, 'Fontsize', 18,'HorizontalAlignment',... 'Center', 'Units','normalized'); h22=subplot(1,2,2); errorbar(MeanInt,StdInt,['k','-']), hold on,... %hleg = legend('mean', 'max','Location', 'SouthOutside'); plot(k,MeanInt(k),['b','*']),... %plot(k,maxall(k), ['k','*']),... title('Mean +/- SD'), ylabel('Fluorescence Intensity'),... xlabel('Frame (Rate = 0.2Hz)'), xlim([0 frames]); h2txt = text(12,ceil(0.95*(max(max(MeanInt)))),... {['{\fontname{Webdings}6} ','Treat']},... 'Fontsize', 10, 'HorizontalAlignment','Left'); hold off; axis(h12,'image'),axis(h12,'off'); set(h12,'Position',[0 0 0.6 1]); set(h12,'OuterPosition',[-0.08 -0.08 0.6 1]); axis(h22,'square'); set(h22,'OuterPosition',[0.49 -0.4 0.5 1.5]); set(h22,'Position',[0.52 -0.07 0.4 1.07]); set(h22,'YAxisLocation', 'right'); %set(hleg,'Position',[0.67 0.05 0.16 0.1]) set(hcb,'XTick',[0 max(max(Icellsx(:,:,k)))]); set(hcb,'Position',[0.005 0.1 0.5 0.0500]); xlabel(hcb,'Fluorescence Intensity'); % for i = 1:objects %original bounding box drawing % if k < FramePostTreat % rectangle('Position',STATS(i,k).BoundingBox,... % 'EdgeColor',[0 0 1],'Parent',h12) % else % rectangle('Position',STATS(i,k).BoundingBox,... % 'EdgeColor',[0 0 1],'Parent',h12) % end % end for i = 1:objects %new bounding box drawing with numbers rectangle('Position',STATS(i,k).BoundingBox,... 'EdgeColor',[0 0 1],'Parent',h12) %draw box around object Objx = STATS(i,k).Centroid(1); %centroid of object Objy = STATS(i,k).Centroid(2); %centroid of object ObjExt = floor(STATS(i,k).Extrema); if Objx <= Ix && Objy <= Iy %quadrant 2 of image text(ObjExt(5,1), ObjExt(5,2), num2str(i),... 'HorizontalAlignment', 'left','VerticalAlignment',... 'top','color',[1 1 1],'Parent',h12); elseif Objx > Ix && Objy <= Iy %quadrant 1 of image text(ObjExt(6,1), ObjExt(6,2), num2str(i),... 'HorizontalAlignment', 'right','VerticalAlignment',... 'top','color',[1 1 1],'Parent',h12); elseif Objx <= Ix && Objy > Iy %quadrant 3 of image text(ObjExt(2,1), ObjExt(2,2), num2str(i),... 'HorizontalAlignment', 'left','VerticalAlignment',... 'bottom','color',[1 1 1],'Parent',h12); else %Objx > Ix && Objy > Iy %quadrant 4 of image text(ObjExt(1,1), ObjExt(1,2), num2str(i),... 'HorizontalAlignment', 'right','VerticalAlignment',... 'bottom','color',[1 1 1],'Parent',h12); end end F = getframe(gcf); %get current figure handle writeVideo(aviobj,F); %write current frame to aviobj end close(gcf); close(aviobj);
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