By Ivan Calandra
This document explains the main steps to open CZI and TIFF files from Zeiss microscopes with Fiji/ImageJ and access their metadata.
ImageJ (ImageJ2 for the most recent version) is an open-source software for scientific imaging. It is a very powerful tool to process scientific images, like microscope images.
Fiji, which means "Fiji Is Just ImageJ", is "an image processing package—a “batteries-included” distribution of ImageJ2, bundling a lot of plugins which facilitate scientific image analysis". One such plugin is Bio-Formats.
CZI is an open format, part of the Bio-Formats, used for all Zeiss microscopes. It includes all the acquisition metadata. The plugin Bio-Formats is necessary to open the CZI files in ImageJ2/Fiji (see section Import CZI files).
Zeiss scanning electron microscopes (SEM) save images in TIFF format (in addition to CZI). The plugin IMBalENce is necessary to read the metadata from these TIFF files with ImageJ2/Fiji (see section Open SEM TIFF files).
The steps presented below are to be be run with ImageJ2/Fiji, including the plugins Bio-Formats & IMBalENce.
As a side note, Zeiss' CZI and TIFF files can also be processed with Python, but this is outside the scope of this document.
Download and install ImageJ2/Fiji from the official website and follow the installation instructions there.
There is actually no installation required. Unzip the downloaded file and move the extracted folder (ImageJ or Fiji.app) to a location where you have read and write access, for example in C:\Users\[your name]\.
If you installed Fiji, you do not need to install the plugin Bio-Formats because it already comes with Fiji.
If you installed ImageJ2, follow the instructions here to install the plugin Bio-Formats.
Follow the instructions here to install the plugin IMBalENce.
To open and run ImageJ2/Fiji, simply open the file ImageJ*.exe (Windows).
In ImageJ2/Fiji, go to Plugins > Bio-Formats > Bio-Formats Importer and select the CZI file you want to open.
Bio-Formats Importer.
A new window opens with import options. In what concerns metadata, there are two complementary options: Display metadata will open all metadata from the file in a new window as a table, while Display OME-XML metadata will display only the standardized OME metadata as XML.
If you choose the former, the most important information has the keys Information and Scaling (quite at the end of the list).
Bio-Formats import options.
When opening files like this, the image will be scaled automatically, that is, the pixel size (and therefore the image size in µm) will be extracted from the metadata. A scale bar can easily be added with Analyze > Tools > Scale Bar....
Add a scale bar.
In ImageJ2/Fiji, open the TIFF image for which you want to access the metadata.
Then, go to EM tool > SEM Zeiss metadata Scale.
Access metadata from a Zeiss SEM TIFF file.
A new window with some of the metadata opens. In order to access all metadata, go to EM tool > SEM metadata pro.
When using the function SEM Zeiss metadata Scale, the image will be scaled automatically, that is, the pixel size (and therefore the image size in µm) will be extracted from the metadata. A scale bar can easily be added with Analyze > Tools > Scale Bar... (see section Import CZI files).