[Solved] CZI to Tiff64 s z c splitted

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Lug Maxwell
Posts: 6
Joined: Tue Sep 01, 2020 2:45 pm

[Solved] CZI to Tiff64 s z c splitted

Post by Lug Maxwell »

Hello,

Under Windows 10, I use ZEN blue 3.1 to convert and split a CZI file, 4 scenes, z=5, 3 fluorescent channels.
Here the parameters I use to export this file:
File type : Tiff format (64bit)
BigTiff checked
Convert to 8bit checked
Compression lossless
Resize 100%
Original data, Shift pixel, Apply display settings, Burn in graphics, Individual channels image are checked
Zoom : 100%
Define subset checked :
Channels : All
Z-position : All
Scene : All
Region :
Rectangle region
A rectangle is drawn on the view
Tiles:
Re-Tile : default values
Inside the Rectangle region a Drawn Spline Contour is manually put for external purpose (preserved thanks to Burn in graphics).

Since I checked Individual channels image, I expect to get each scene, z-stack, channel in a separate tiff file. Indeed I get all s z c combinations reported in the file names but when I open one of the tiff file, I see 3 sub images. What does it means and what are these sub images?
It seems to be difficult to simply have simple image files matching each s z c values.
Advise is welcome here.

Thank you.
Last edited by Lug Maxwell on Tue Nov 02, 2021 4:34 pm, edited 1 time in total.
CarlZeissMicroscopy3
Posts: 180
Joined: Wed May 20, 2020 10:10 am

Re: CZI to Tiff64 s z c splitted

Post by CarlZeissMicroscopy3 »

Hello Lug Maxwell,

just to get some further information:

Your CZI file consists of 4 scenes with z=5 and 3 fluorescent channels.

When we consider just one plane of a scene (e.g. scene 1, z-stack-plane 1, channel 1) is this a tiled image or a single image?
Lug Maxwell
Posts: 6
Joined: Tue Sep 01, 2020 2:45 pm

Re: CZI to Tiff64 s z c splitted

Post by Lug Maxwell »

Hello support,

This is a tiled CZI image (comes from Axio Scan.Z1 instrument).

Thank you.
CarlZeissMicroscopy3
Posts: 180
Joined: Wed May 20, 2020 10:10 am

Re: CZI to Tiff64 s z c splitted

Post by CarlZeissMicroscopy3 »

Hello Lug Maxwell,

I organized some similar images but when trying your export settings individual images for each s z c combination appeared, just as reported. Opening these files just show images ‘as expected’ …

But there are two questions on my side:

Which hotfix for Zen 3.1 did you install. Hotfix 12 is the latest!
Please have a look at Help/About Zen…/Show Zen Information/Service Packs

What program do you use to view the BigTiff images? I used IrfanView.
Lug Maxwell
Posts: 6
Joined: Tue Sep 01, 2020 2:45 pm

Re: CZI to Tiff64 s z c splitted

Post by Lug Maxwell »

3.1.81.0010 is the version I use. I do not know how to get Hotfix 12.
I use ImageJ to open tif files for visualization.
I just tried Irfanview. For s=1, z=1 and c=1, for example, Irfanview shows a blue colored 24 bit image. ImageJ shows 3 sub images (each being 8 bit) that now I understand are the RGB separated color channels. This is a strange way to code virtual color (normally should come from a lookup table in order to get the smallest possible image weight).
Anyway, in both cases, this is not expected since i checked "Convert to 8bit" before the conversion. Obtained images are not 8 bit.

Then I tried to zoom under Irfanview : details are blurred but they are sharp on the CZI original file. This is not expected since I set Resize to 100% before the conversion.

Did you try to draw a contour and use Burn-in option? Did you apply a rectangle for cropping ? Did you try to zoom the result ?

Thank you for your help.
CarlZeissMicroscopy3
Posts: 180
Joined: Wed May 20, 2020 10:10 am

Re: CZI to Tiff64 s z c splitted

Post by CarlZeissMicroscopy3 »

3.1.81.0010 is the version I use. I do not know how to get Hotfix 12.
Please try
https://portal.zeiss.com/download-center/softwares/mic
After registration the ‘Download Center’ opens and you can see a search field. Enter ‘3.1’ and click the search button. You will get a list where you search for ‘3.1’ and click ‘show all files’. Scrolling a bit down shows ‘ZEN blue 3.1 - Hotfix 12’.

"I use ImageJ to open tif files for visualization."
Ok, in this case you get a window with a “C” at the left side of the bottom line saying that these images are cannels. In addition the image name etc. is shown at the top of the window in blue, red or green depending on the selection of the three channels (RGB).
It is possible to split it into separate images by Image/Color/Split Channels.
This is what you also concluded. But I’m not so sure if that is in every case what you need…

“this is not expected since i checked "Convert to 8bit" before the conversion”
I played a bit with the combination of “Merge Channels Image” and “Individual Channels Image” and got different output results, even the channels individually with and without the ‘burned in graphics’.
From my point of view it is worth playing with these two settings (and maybe others) as the results are sometimes 'not as expected' ;-)

"Did you try to zoom the result ?"
I looked at the data of individual exported channels in ImageJ and compared it at 100% and 400% zoom to the original. In my case it seemed to be ok. (except that Zen has a ‘display mechanism’ to present the image and ImageJ ‘just shows’ the image)

"Did you try to draw a contour and use Burn-in option? Did you apply a rectangle for cropping ?"
I can answer all these points with yes.
CarlZeissMicroscopy3
Posts: 180
Joined: Wed May 20, 2020 10:10 am

Re: CZI to Tiff64 s z c splitted

Post by CarlZeissMicroscopy3 »

Just an additional information:
There exists an 'OME-TIFF Export' which also has features you expect.

Maybe you give it a try ...
Lug Maxwell
Posts: 6
Joined: Tue Sep 01, 2020 2:45 pm

Re: CZI to Tiff64 s z c splitted

Post by Lug Maxwell »

Thank you to give time to try deeper. Appreciated.

I installed Hotfix 12.

Under ImageJ, I can split RGB channels : I need to merge them to recover total pixel intensities for some fluorescent channels. That becomes messy when I have to analyse hundred czi files.
This drawback, indeed, does not appear with the OEM-TIFF converter. Unfortunately, this format also produces RGB images and is limited to 2 GB in RAM that I overcome in most cases. Now I understand that "Individual Channels Image" refers to RGB channels and not to fluorescent channels whereas "Merge Channels Image" really merge fluorescent channels (a bit confusing). The 2 GB limitation could be less restrictive with TIF+burn-in graphics straight in 8bit mode. That's why I am stuck with BigTiff that only produces RGB sub images which I cannot analyse before merging them.

Finally I realise that I do not need Individual/Merge Channels Image since I use Define Subset option to get separate fluorescent images. But it is not possible to uncheck Individual and Merge Channels Image at the same time! Why ?

Thank you for your help.
CarlZeissMicroscopy3
Posts: 180
Joined: Wed May 20, 2020 10:10 am

Re: CZI to Tiff64 s z c splitted

Post by CarlZeissMicroscopy3 »

Hello Lug Maxwell,

the initial idea was to get individual channels or merged channels or both, otherwise ‘no image’ would be the result.

As the software and image types evolve over time permutations increase and as an outcome you have to try a bit with the combinations to get the results needed and skip ‘side effects’.

Please be aware that this is a forum for OAD macro programming and not for ‘how to use the features of Zen’ …
Lug Maxwell
Posts: 6
Joined: Tue Sep 01, 2020 2:45 pm

Re: CZI to Tiff64 s z c splitted

Post by Lug Maxwell »

Hello,
You helped me a lot to fully understand what can be done for my purpose.

Thank you.
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