Image file formats

Prerequisites
Before starting this lesson, you should be familiar with:

Basic properties of images and pixels

Data types

Image calibration

Learning Objectives
After completing this lesson, learners should be able to:

Open and save various image files formats

Understand the difference between image voxel data and metadata

Understand that converting between image file formats likely leads to loss of information

Motivation

There are numerous ways how to save image data on disk. Virtually every microscope vendor has their own file format. It is thus very important to understand how to open those files and inspect their content. Moreover, some software will open only specific image file formats and thus it is sometime necessary to re-save the data. During such image file format conversions information can be lost; it is important to be aware of this and avoid such information loss as much as possible.

Concept map

graph TD F("TIFF, JPEG, XML/HDF5, CZI, LIF, ...") F --> PD("Pixel data") PD --> Values PD --> Dimensions F --> MD("Metadata") MD --> IC("Image calibration") MD --> MS("Microscope settings") MD --> DS("Display settings") MD --> NA("...")

Figure

Activities

Explore various image file formats

Open different image file formats and inspect their pixel data and also their metadata
Appreciate that image file formats
- include more or less metadata
- may contain multiple images
- may consist of multiple files, e.g. splitting metadata and pixel data across two files

Example image data

Show activity for:

ImageJ GUI

Open the files mentioned in the activity:

[Plugins > Bio-Formats > Bio-Format Importer].

Display metadata

Display OME-XML Metadata

Should be the same information as above but in XML (sometimes it is more correct than the above)

For ICS/IDS and XML/HDF5:

The ICS and XML file are the entry points that should be opened (the respective other file will be read automatically).

Also inspect the ICS and XML files in a simple text editor.

Saving 8 bit single channel image as TIFF:

Open xy_8bit__nuclei_PLK1_control.tif

[Image > Adjust > Brightness/Contrast] such that cells appear saturated

[File > Save As > TIFF…]

Open with Fiji

LUT metadata has changed, but pixel values and calibration metadata are preserved

Open with a web browser

It may not open at all

Saving 8 bit single channel image as JPEG:

Open xy_8bit__nuclei_PLK1_control.tif

[Image > Adjust > Brightness/Contrast] such that cells appear saturated

[File > Save As > JPEG…]

Open with Fiji

Pixel values have changed

Calibration metadata is gone

Open with a web browser

It should look the same as when you saved it

Saving 16 bit two channel movie as JPEG: xyzct_16bit__mitosis.tif

Select a timepoint in the middle of the movie

[File > Save As > JPEG…]

Open JPEG with Fiji

Image dimensions, data type, pixel values, and metadata have changed

Saving 8 bit single channel movie as GIF: xyt_8bit__mitocheck_incenp.tif

[Image > Adjust > Brightness/Contrast] such that cells appear saturated

[File > Save As > GIF…]

Open with Fiji

Pixel values have changed

Open with a web browser

Movie plays and looks as when you saved it

Resave images in various file formats

Resaving images in different file formats very often leads to a loss of metadata or distortion of the pixel values. It is critical to be aware of this!

Checks to be done after each resaving

Open the resaved image in all relevant applications and check whether the pixels values and/or metadata are different from the original image
- This is critical for the scientific integrity of the resaving
Open the image in a web-browser and observe how the image is rendered
- This can be useful to share previews with collaborators

Resave 8 bit single channel image as TIFF

Open xy_8bit__nuclei_PLK1_control.tif
Change the contrast limits
Save as TIFF
Do the above checks

Resave 8 bit single channel image as JPEG

Open xy_8bit__nuclei_PLK1_control.tif
Change contrast such that the cells appear saturated
Save as JPEG
Do the above checks

Resave 16 bit two channel movie as JPEG

Open xyzct_16bit__mitosis.tif
Select a timepoint in the middle of the movie
Save as JPEG
Do the above checks

Resave 8 bit single channel movie as GIF

Open xyt_8bit__mitocheck_incenp.tif
Change contrast such that cells appear saturated
Save as GIF
Do the above checks

Show activity for:

ImageJ GUI

Saving 8 bit single channel image as TIFF:

Open xy_8bit__nuclei_PLK1_control.tif

[Image > Adjust > Brightness/Contrast] such that cells appear saturated

[File > Save As > TIFF…]

Open with Fiji

LUT metadata has changed, but pixel values and calibration metadata are preserved

Open with a web browser

It may not open at all

Saving 8 bit single channel image as JPEG:

Open xy_8bit__nuclei_PLK1_control.tif

[Image > Adjust > Brightness/Contrast] such that cells appear saturated

[File > Save As > JPEG…]

Open with Fiji

Pixel values have changed

Calibration metadata is gone

Open with a web browser

It should look the same as when you saved it

Saving 16 bit two channel movie as JPEG: xyzct_16bit__mitosis.tif

Select a timepoint in the middle of the movie

[File > Save As > JPEG…]

Open JPEG with Fiji

Image dimensions, data type, pixel values, and metadata have changed

Saving 8 bit single channel movie as GIF: xyt_8bit__mitocheck_incenp.tif

[Image > Adjust > Brightness/Contrast] such that cells appear saturated

[File > Save As > GIF…]

Open with Fiji

Pixel values have changed

Open with a web browser

Movie plays and looks as when you saved it

Assessment

True or false

One could use Excel’s XLSX file format for saving image data.

Solution

One could use Excel’s XLSX file format for saving image data. True, the matrix of each sheet could represent one image plane and one could use the first sheet to store metadata and the mapping of each sheet (image plane) to the zct coordinates, e.g. sheet 12 c 2 z 3 t 1.

Discuss

What are the pros and cons of converting an image into another format?
What are the pros and cons of splitting metadata and image pixel data into separate files?
Do you know any good file formats for image metadata?

Solution

(A) Sometimes it is necessary to convert to another format to be able to open the image in a specific software. (B) Converting an image to another format typically loose information, e.g. because the file format that you are saving to cannot represent all the metadata of the original image file. Thus, it is in general recommened to keep to original image file. (C) Converting to a file format with good compression may save you considerable disk space.

(A) Metadata typically is much smaller than the pixel data. Thus, it can be a good idea to keep metadata in a separate file that can be readily inspected (inspecting the potentially TB sized pixel data files can be tricky). (B) The best file formats for metadata and pixel data can be very different due to the nature of the data, thus splitting can make sense. (C) Having separate files always bares the risk that you loose one of them, e.g. you may forget to copy both to a new folder.

TXT, XML, and JSON are good formats for image metadata, because they are human readable standard formats that can be openend with any text editor.

Explanations

Follow-up material

Recommended follow-up modules:

Learn more:

Bio-Formats

OME-TIFF

OME-NGFF