How to threshold and obtain RGB and reflactance values (max, min, mean) of MULTIPLE images?

Hi! I have several '.tif' photos of seeds that I would like to threshold to obtain RGB values (max, min, mean) and reflectance values (max, min, mean) of individual images and, if it's possible, individual seeds. I used to do this on ImageJ but I need to learn it on MatLab.

I manually set up the channels thresholds with the Color Thresholder app and generated a function, but honestly I have not had success using this function in a loop. I would eventually like to have a .csv file with columns: "image, seed, red_max, red_min, red_mean, green_max, green_min, green_mean, blue_max, blue_min, blue_mean..." and the same for reflectance.

This is what my images look like.

I'd really appreciate any help you guys could provide me!

Cheers!

Joan.

Answer 1

[Image Analyst]

Try this:

clc;    % Clear the command window.
close all;  % Close all figures (except those of imtool.)
clear;  % Erase all existing variables. Or clearvars if you want.
workspace;  % Make sure the workspace panel is showing.
format long g;
format compact;
fontSize = 25;

%===============================================================================
% Get the name of the image the user wants to use.
baseFileName = 'Seeds.jpg';
% Get the full filename, with path prepended.
folder = pwd
fullFileName = fullfile(folder, baseFileName);

% Check if file exists.
if ~exist(fullFileName, 'file')
  % The file doesn't exist -- didn't find it there in that folder.
  % Check the entire search path (other folders) for the file by stripping off the folder.
  fullFileNameOnSearchPath = baseFileName; % No path this time.
  if ~exist(fullFileNameOnSearchPath, 'file')
    % Still didn't find it.  Alert user.
    errorMessage = sprintf('Error: %s does not exist in the search path folders.', fullFileName);
    uiwait(warndlg(errorMessage));
    return;
  end
end

%===============================================================================
% Read in demo image.
rgbImage = imread(fullFileName);
% Get the dimensions of the image.
[rows, columns, numberOfColorChannels] = size(rgbImage);
% Display the original image.
subplot(1, 2, 1);
imshow(rgbImage, []);
axis on;
caption = sprintf('Original Color Image, %s', baseFileName);
title(caption, 'FontSize', fontSize, 'Interpreter', 'None');
hp = impixelinfo();

% Set up figure properties:
% Enlarge figure to full screen.
set(gcf, 'Units', 'Normalized', 'OuterPosition', [0 0.05 1 0.95]);
% Get rid of tool bar and pulldown menus that are along top of figure.
% set(gcf, 'Toolbar', 'none', 'Menu', 'none');
% Give a name to the title bar.
set(gcf, 'Name', 'Demo by ImageAnalyst', 'NumberTitle', 'Off')

drawnow;
hp = impixelinfo(); % Set up status line to see values when you mouse over the image.

% Extract the individual red, green, and blue color channels.
redChannel = rgbImage(:, :, 1);
greenChannel = rgbImage(:, :, 2);
blueChannel = rgbImage(:, :, 3);

% Threshold to create a mask.
binaryImage = redChannel > 90;
% Get rid of small noise blobs.
binaryImage = bwareafilt(binaryImage, [100, inf]); 
% Erode them a bit to separate touching ones and get away from light shadows.
se = strel('disk', 7, 0);
binaryImage = imerode(binaryImage, se);
% Display the mask image.
subplot(1, 2, 2);
imshow(binaryImage, []);
axis on;
caption = sprintf('Seed Mask');
title(caption, 'FontSize', fontSize, 'Interpreter', 'None');
hp = impixelinfo();

[labeledImage, numSeeds] = bwlabel(binaryImage);
propsR = regionprops(labeledImage, redChannel, 'MeanIntensity', 'PixelValues');
propsG = regionprops(labeledImage, greenChannel, 'MeanIntensity', 'PixelValues');
propsB = regionprops(labeledImage, blueChannel, 'MeanIntensity', 'PixelValues');
allMeansR = [propsR.MeanIntensity];
allMeansG = [propsG.MeanIntensity];
allMeansB = [propsB.MeanIntensity];
overallSeedMeanR = mean(allMeansR);
overallSeedMeanG = mean(allMeansG);
overallSeedMeanB = mean(allMeansB);
% Get min and max of each seed for each color channel
for k = 1 : numSeeds
    allMinsR(k) = min(propsR(k).PixelValues);
    allMinsG(k) = min(propsG(k).PixelValues);
    allMinsB(k) = min(propsB(k).PixelValues);
    allMaxsR(k) = max(propsR(k).PixelValues);
    allMaxsG(k) = max(propsG(k).PixelValues);
    allMaxsB(k) = max(propsB(k).PixelValues);
end
% Prepare data for csvwrite
% "image, seed, red_max, red_min, red_mean, green_max, green_min, green_mean, blue_max, blue_min, blue_mean..." 
data = [(1:numSeeds)', allMaxsR', allMinsR', allMeansR', allMaxsG', allMinsG', allMeansG', allMaxsB', allMinsB', allMeansB']

% csvwrite can't handle strings, only numbers.  If you want the filenames on each line, use xlswrite().
% Get the name of the file that the user wants to save.
% Note, if you're saving an image you can use imsave() instead of uiputfile().
startingFolder = userpath; % Or "pwd" or wherever you want.
defaultFileName = fullfile(startingFolder, '*.csv');
[baseFileName, folder] = uiputfile(defaultFileName, 'Specify a file');
if baseFileName == 0
  % User clicked the Cancel button.
  return;
end
fullFileName = fullfile(folder, baseFileName)
csvwrite(filename, data);

[JoanManBar]

Thank you so much for your answer and code, it is AMAZING! I hope it's not too much to ask but could you help me a little to understand how to do this for multiple photos? After several hours trying, I gave up. I guess I'm just not good at making loops -any recommendation to learn is always welcome.

This is my poor attempt:

%%Clean and format
clc;    % Clear the command window.
close all;  % Close all figures (except those of imtool.)
clear;  % Erase all existing variables. Or clearvars if you want.
workspace;  % Make sure the workspace panel is showing.
format long g;
format compact;
fontSize = 25;

%%Define variables

imagefiles = dir('*.tif');
filenames = {imagefiles.name};
nfiles = length(imagefiles);

%%===============================================================================
% % Get the name of the image the user wants to use.
% baseFileName = 'c_sample001.tif';
% % Get the full filename, with path prepended.
% folder = pwd
% fullFileName = fullfile(folder, baseFileName);

%%Check if file exists.
% if ~exist(fullFileName, 'file')
%   % The file doesn't exist -- didn't find it there in that folder.
%   % Check the entire search path (other folders) for the file by stripping off the folder.
%   fullFileNameOnSearchPath = baseFileName; % No path this time.
%   if ~exist(fullFileNameOnSearchPath, 'file')
%     % Still didn't find it.  Alert user.
%     errorMessage = sprintf('Error: %s does not exist in the search path folders.', fullFileName);
%     uiwait(warndlg(errorMessage));
%     return;
%   end
% end

%%Functions

for ii = 1:nfiles
   currentfilename = imagefiles(ii).name;
   currentimage = imread(currentfilename);
   images = currentimage;

% Read in demo image.
% rgbImage = imread(fullFileName);
% Get the dimensions of the image.
[rows, columns, numberOfColorChannels] = size(images);
% Display the original image.
subplot(1, 2, 1);
imshow(images, []);
axis on;
caption = sprintf('Original Color Image, %s', currentfilename);
title(caption, 'FontSize', fontSize, 'Interpreter', 'None');
hp = impixelinfo();

% --- 
% Set up figure properties:
% Enlarge figure to full screen.
set(gcf, 'Units', 'Normalized', 'OuterPosition', [0 0.05 1 0.95]);
% Get rid of tool bar and pulldown menus that are along top of figure.
% set(gcf, 'Toolbar', 'none', 'Menu', 'none');
% Give a name to the title bar.
set(gcf, 'Name', 'Demo by ImageAnalyst', 'NumberTitle', 'Off')

drawnow;
hp = impixelinfo(); % Set up status line to see values when you mouse over the image.

for i = 1:length(images)
% Extract the individual red, green, and blue color channels.
redChannel  = images(:, :, 1);
greenChannel = images(:, :, 2);
blueChannel = images(:, :, 3);

end
% Threshold to create a mask.
binaryImages = redChannel(ii) > 90;
% Get rid of small noise blobs.
binaryImages = bwareafilt(binaryImages, [100, inf]); 
% Erode them a bit to separate touching ones and get away from light shadows.
se = strel('disk', 7, 0);
binaryImages = imerode(binaryImages, se);
% Display the mask image.
subplot(1, 2, 2);
imshow(binaryImages, []);
axis on;
caption = sprintf('Seed Mask');
title(caption, 'FontSize', fontSize, 'Interpreter', 'None');
hp = impixelinfo();

% -
[labeledImages, numSeeds] = bwlabel(binaryImages);
propsR = regionprops(labeledImages, redChannel, 'MeanIntensity', 'PixelValues');
propsG = regionprops(labeledImages, greenChannel, 'MeanIntensity', 'PixelValues');
propsB = regionprops(labeledImages, blueChannel, 'MeanIntensity', 'PixelValues');
allMeansR = [propsR.MeanIntensity];
allMeansG = [propsG.MeanIntensity];
allMeansB = [propsB.MeanIntensity];
overallSeedMeanR = mean(allMeansR);
overallSeedMeanG = mean(allMeansG);
overallSeedMeanB = mean(allMeansB);

Get min and max of each seed for each color channel

for k = 1 : numSeeds
    allMinsR(k) = min(propsR(k).PixelValues);
    allMinsG(k) = min(propsG(k).PixelValues);
    allMinsB(k) = min(propsB(k).PixelValues);
    allMaxsR(k) = max(propsR(k).PixelValues);
    allMaxsG(k) = max(propsG(k).PixelValues);
    allMaxsB(k) = max(propsB(k).PixelValues);
end
% Prepare data for csvwrite
% "image, seed, red_max, red_min, red_mean, green_max, green_min, green_mean, blue_max, blue_min, blue_mean..." 
data  = [(1:numSeeds)', allMaxsR', allMinsR', allMeansR', allMaxsG', allMinsG', allMeansG', allMaxsB', allMinsB', allMeansB']

% csvwrite can't handle strings, only numbers.  If you want the filenames on each line, use xlswrite().
% Get the name of the file that the user wants to save.
% Note, if you're saving an image you can use imsave() instead of uiputfile().
startingFolder = pwd; % Or "pwd" or wherever you want.
defaultFileName = fullfile(startingFolder, '*.csv');
[images, folder] = uiputfile(defaultFileName, 'Specify a file');
if images == 0
  % User clicked the Cancel button.
  return;

end
fullFileName = fullfile(folder, images)
csvwrite(filename, data);

end