Stitching images with two channels

Hi community,
I am new around here and also working with Fiji.
I have tried to search within the forum for the kind of image I wanted to create but I was not able to find anything fitting my needs. The thing is that I have produced several pictures belonging to several fields from a 96 well plate. Each field has produced two pictures each one from two separated channel. Now I would like to use FIJI to create a single mosaic image containing all the fields altoghether for the two channels. I was able to produce such an image for each channel but the resulting images were different in size and then I cannot use them for further cell segmentation and analysis.
Somebody could help me with this?
I am using FIJI ImageJ 2.1.0/1.53c Java 1.8.0-172 (64bits) and I have tried these plugings for stitching;
-Pairwise stitching
-Grid/Collection stitching
-All the plugings within Stitching>deprecated folder.

Thanks a lot in advance.
PS: images are named as follows;
A10_f00_d0.tif, A10_f00_d1.tif, A10_f01_d0.tif, A10_f01_d1.tif, …

Why do you want to stitch the images of a 96 well plate if “Each field has produced two pictures each one from two separated channel.” ?

(=> one position per well, correct?)

Have you tried to open all images of one channel as a stack and then used to function Image>Stacks>Make Montage … to create your mosaic?
You can repeat this for both channels and combined the channel mosaics since they have exactly the same size and all image pairs are in the correct position.


  • two channel images for each position
  • no shift between the the positions of the channel pairs
1 Like

Hi, thanks for helping. I just noticed my explanation was not very accurate. I have a 96 well plate and the microscope has produced 18 images from each well. Those correspond to 9 fields by two channels (Blue and Green). I need to combine all the pictures from each well to get a 3x3 picture with two channels. Then I will analyze some spots within the cells.
I am trying to stitch the 18 images but I failed so far, with all the tools mentioned. I have also tried to rename the files to this pattern; img_row(rr)_col(cc)_time(tt).tif.
According to MIST F&Q it is posible to stitch two channels by stitching the first channel and then using the Global Positions File from the first one with the second channel. I am triying this one but still no success.
I will also try your suggestion

Hi @BioIRH,

have you tried Grid/Collection stitching? If so , what failed? As far as I remember it can handle multiple channels. It averages the channels for computing the optimal shifts. (you need to provide multichannel composite tiles as input).
Alternatively you can do the actual registration on a single channel. And then reuse the “Tileconfiguration.registered.txt” for the other channel. For the second channel it is important to then select TileConfiguration from file, and to uncheck “compute overlap” option.

See also this post here for a similar question.

Hi @noreenw,
Thanks for your suggestion. Finally I was able to stitch all the images. I just came up with the idea of using the original file type generated by the instrument ThermoFisher CellInsight CX7 (.C01) instead of using .TIF converted files. It just worked fine with Grid/Collection stitching plugin. I have also tried with 3x3 images and it also worked.
Thanks for your help.