Last channel (3rd) right most fields (of 3) in .czi file displayed as zeros

Hello,

I have a number of .czi files from Ziess Axioscan Z1 which are not fully accessible in QuPath (same problem occurs in Definiens). When the images are loaded into QuPath, one of the high power regions (right most section captured) has pixel values of zero for the 3rd channel (of 3). This is only evident visually when you zoom in to resolution greater than x5, where the 3rd channel disappears.

Is this an issue that others have come across, and if so, is there a fix either prior to scanning, or that enables full access to the affected image file?

Thanks for any help.

Matt

  1. The region shows up in Zen then at all resolutions? It might be that the pyramid needs to be rebuilt, not sure.
  2. If you draw an annotation in that region and Calculate Intensity Features, are they calculated accurately? As in, is it only a visual error, but the data is actually there.
  3. I suspect this is a BioFormats thing, so if you can provide them with a sample image and a description of how the image was taken/processed (in terms of compression), that might help.

I just wanted to mention that I’ve seen something similar (where you zoom in and suddenly some of the data is blank) but never figured out what it was from. It only happened sporadically, so I never saw a pattern.

1 Like
  1. The pyramid appears as normal in Zen at all resolutions.
  2. In Zen using the show values tool the values appear as normal. In QuPath, if I select a rectangle and calculate intensity features I get normal results for all 3 channels if I select pixel size of 1um or greater (actually 0.97um), otherwise it gives 0 for the 3rd channel (ch 0 and 1 still work).
  3. If you could provide a means of transferring the image to you I will ask try to get the full details of how the image was captured and exported.

Thanks!

Thanks for the responses. I am not associated with OME, and while normally I might ask for the file just to test some things out and submit it myself, I am traveling for a week after tomorrow, and wouldn’t want to drop the process part-way through.
The submission system for BioFormats is here: https://docs.openmicroscopy.org/bio-formats/5.7.3/about/bug-reporting.html

Alternatively, in the past, I have opened topics on their GitHub page and uploaded the files I was having trouble with to my own Google Drive (free) to share them that way. Either one of those should be an option.

Hi @mjellis, you can upload a sample file for the Bio-Formats team to test at https://www.openmicroscopy.org/qa2/qa/upload/

If you try to open the CZI via BioFormats, do all the pyramid levels show up as different series with different number of pixels? (like in the image below)

image

They do all appear different:

image

I have opened all pyramid layers and it is only the top (largest) image from this high res area that has the zero’s issue.

Hi @dgault,

I have uploaded an example image, it is the image in series 9 that has the issue (see post to @sebi06).

Thanks for any help!

1 Like

Thanks @mjellis, we received the uploaded file and I was able to start testing it today. Using the latest Bio-Formats in FIJI I wasn’t able to reproduce the issue. Is it the entire plane for the 3rd channel which has zero values? For all 3 channels there is a section on the bottom left which is showing as zero as well as a small strip on the far right of the image.

Hi @dgault, thank you for having a look. You are correct, there are regions with zero values in all channels in each corner of the image; this is just unscanned area.

I have attached a word document ( series 9and10.zip (2.2 MB) ) containing what I see when I load series #9 (problematic high res image) and #10 (one res step below #9) into FIJI (bio-formats v6.0.1). You can see that when displaying channel 3/3 in series #9 there is just a black screen due to the zero values.

The errors recorded in the console when loading series #9 are also in the attached file.

If there is a newer version of bio-formats that you have access to I guess I’ll just have to wait for it to become available.

Ok, originally I was looking at the first series. I can reproduce the issue the same as shown in your doc with series 9. For some reason that seems to be the only one affected, and series 1 and 5 are ok. I will further debug the issue on Monday to see if I can find more details on the cause.

Hi @dgault

Have you been able to investigate this problem any further?

Hi @dscaglioni, I was able to some further investigation but have not got a fix for the issue as of yet. I have documented it in a GitHub issue at https://github.com/openmicroscopy/bioformats/issues/3401