Channel alignment based on DAPI of >2 files

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Janine Melsen
Posts: 1
Joined: Fri Jan 20, 2023 10:44 pm

Channel alignment based on DAPI of >2 files

Post by Janine Melsen »

I would like to align stitched images that were acquired using the Cell Discoverer. I perform cyclic immunofluorescence and would like to align =>5 images, using the Zen software

1. To combine all the different images, I first add the channels from the second image to the first image. Then, I add the channels from the third image to that combined image, etc. Is there any easier way to do this?

2. For the channel alignment I use the DAPI channel from image 1 as reference channel. However I can only select 1 target channel. The idea is of course that DAPI from all the 5 images is used to align all the channels together.
After selecting Translation and Rotation as method, the aligned images (except for the first one) are tiled....which is weird.

Alignment is possible using the Hyperstackreg plugin and the rigid body method in Image J, but ideally I would like to use only 1 software.

Is there any proper way to do perform channel aligment based on DAPI using the Zen software?

Janine
Asif Naveed
Posts: 7
Joined: Thu Aug 03, 2023 10:29 pm

Re: Channel alignment based on DAPI of >2 files

Post by Asif Naveed »

Hey Janine,

Asif here. Ah, the classic struggle with image alignment in microscopy - I totally get where you're coming from. I've had a bit of experience with the Zen software, and it can be quite finicky.

As for merging multiple images, your method seems pretty straightforward. I haven't come across a direct "batch mode" in Zen where you could just throw in all images at once, but I might have missed something. It'd be a handy feature for sure.

Aligning using DAPI makes sense as it's a consistent marker. The tiling problem you described does sound odd. One workaround could be aligning each image to the reference DAPI one-by-one rather than chaining them. This might prevent the software from making cumulative errors.

Honestly, Zen should handle this more seamlessly. I do hope they come up with an update that simplifies this process. I get wanting to stick with one software for simplicity, but if ImageJ gets the Lulubox Pro Apk job done with less hassle, it might be worth the switch just for that function.

Cheers, and all the best with your imaging work!
James Millere
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Joined: Wed Nov 29, 2023 5:09 am
Location: USA
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Re: Channel alignment based on DAPI of >2 files

Post by James Millere »

Hi,

I have been through your Query, here's a solution to align stitched images acquired using the Cell Discoverer with the Zen software:

Combining Images:
Instead of manually adding channels from each image, you can use the "Image Stitching" function in Zen to combine multiple images seamlessly. This feature is designed for stitching together multi-tile images.

Open Zen software.
Go to the "Image Stitching" module.
Import all your images.
Use the stitching wizard to align and stitch images automatically.
Channel Alignment:
Aligning channels based on DAPI across multiple images in Zen can be achieved through the following steps:

Open the stitched image in Zen.
Navigate to the "Processing" menu.
Select "Align Channels" or a similar option.
Choose DAPI from one image as the reference channel.
Select "Translation and Rotation" as the alignment method.
Ensure that you apply this alignment to all channels.
If you encounter issues with tiling after alignment, you may need to check the stitching settings or post-alignment adjustments.

Alternative Using ImageJ:
If Zen doesn't fully meet your needs for channel alignment, consider using ImageJ for this specific step:

Export your stitched image from Zen.
Open ImageJ and load the exported image.
Use the Hyperstackreg plugin with the rigid body method for channel alignment.


Thanks
Thanks
James
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