Quantification of spectra in Zeiss Zen software running on 880 is relatively simple and can be exported as a spreadsheet. However, the software only operates on the microscope station and it is not clear that Zen Blue running on other computers can manage this quantification. Also, ImageJ has benefits of ease of ROI management, can sun on any computer, and many other measurement functions.

Therefore, we have macros for quantification of fluorescent spectra for ImageJ.

These macros could be more robust by automatically setting the spectral ranges by pulling from the metadata of czi files. However, this implementation settles for setting the range manually by reading the metadata. The macro maybe changed based on the spectra collected.

Individual cells must be traced. The tracings do not need to be perfect. If all cells were isolated, automated segmentation would work, but many cells touch. Alternatively, for whole field measurement, the entire field (select all) may be used.

Instructions for use:

A spectal image is collected using the Zeiss 800. This image is opened (imported with BioFormats) as a hyperstack with many channels.

Original format is Zeiss CZI. When Zeiss files are opened into ImageJ, the path, where they are located on disk, is lost. Therefore, save the file as TIF.

Cells are traced using any polygon tool (including thresholding and magic wand) and put into the ROI Manager using t.

When ROI manager is populated, click on the image (image must be active) and run the macro Save ROI Manager [F7].
This will save the ROI Manager in the same directory as the image with the same root name so they will be grouped.

You may add to or subtract from the ROI Manager at any time and resave.

If working on a new image, be sure to start a new ROI Manager. There are a few ways to do this. Perhaps the easiest is to close it.

Later, the ROI Manager and the image can be reopened together for analysis.

Running the "measure ratios from spectal image [q]" macro creates a table with the spectra of each ROI. This is saved in the same directory as the image file and may be opened in favorite spreadsheet program later.

Macros and example:

Zeiss880_spectral_macros_v102.ijm or Zeiss880_spectral_macros_v102.txt

[Need to insert here tif file and metadata and xml metadata]