How to make a 3D Volume out of a 2D Tiff stack?

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I have a data set of 242 planes with x,y pixel resolution. It was taken from a microscope, I know the depth between image planes, and am looking to turn this Tiff Stack (split into individual files due to low RAM, named Flydata0000 - Flydata0242) into a 3D volume which I can eventually get a gui to play about with the image.
I have been trying Imshow to show an image array from reading the full file (containing only the fly data).
I will include what I have done already below (note I have not tried to make it 3D yet, would like corrections on what Ive done (if any) and an idea of how to go about making the stack 3D).
%Setting up path
fileFolder = 'E:\MyFile\Data\Low_Res\Sequence';
filePattern = fullfile(fileFolder, '*.tif');
TiffFiles = dir(filePattern);
% not using this now (fileNames =;)
numFrames = numel(TiffFiles);
for k=1:242
fileNames =;
fullfilename=fullfile(fileFolder, fileNames);
fprintf(1, 'Now reading %s\n', fullfilename);
imageArray = imread(fullfilename);
% Stack=imread(['VeryLowResFly0' num2str(k, '%03.f') '.tif']);
  1 Comment
Michael Nothem
Michael Nothem on 6 Aug 2018
Did you get it figured out? I am trying to do the same thing!

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Answers (2)

Rafael S.T. Vieira
Rafael S.T. Vieira on 1 Jun 2020
Edited: Rafael S.T. Vieira on 6 Aug 2020
We need to create a 3D array WxHxD for storing all images, such as stack = zeros(W,H,D). Then, in the for-loop, we should read each image: stack(:,:,k) = imread(TiffFiles(k).name). For instance:
fileFolder = 'E:\MyFile\Data\Low_Res\Sequence';
filePattern = fullfile(fileFolder, '*.tif');
all_tiff = dir(filePattern);
first_image = imread(all_tiff(1).name);
[W,H] = size(first_image);
D = numel(all_tiff);
stack = zeros(W,H,D);
stack(:,:,1) = first_image;
for i = 2:D
stack(:,:,i) = imread(all_tiff(i).name);
% uncomment next line for seeing the reading progress
% disp(string(i*100.0/D) + "%");
% The app volumeViewer will handle the visualization
I have tried the previous code on my files, and it works. However, the app volumeViewer won't work if any dimension is a singleton.
PS: I'm assuming all tiff images have the same dimensions (WxH) and are Grayscale or BW.
  1 Comment
r r
r r on 20 Nov 2021
How do I save this stack
Thank you for the excellent method, but how do I save it to 3D "format stack"

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yanqi liu
yanqi liu on 22 Nov 2021
sir,may be use smooth3、isosurface to generate 3D data,such as

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