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Importing Image Files

Cookbook Topics
Introduction and Installation
Annotating Images
Color Image Processing
Image Intensity Processing
Importing Image Files
Particle Analysis
Saving and Exporting
Stack-slice Manipulations

The File / Import submenu

Most of the commands listed, particularly Fiji extensions, are also available (via ImageJ's HandleExtraFileTypes feature) simply by using the File / Open... menu item.

Built-in formats

Fiji primarily uses TIFF as the image file format. The menu command File  › Save will save in TIFF format. The menu command File  › Open will open TIFF files and import a number of other common file formats (e.g. JPEG, GIF, BMP, PGM, PNG) as well as those detailed below. Files can be also dragged and dropped to the toolbar to open them.


Many more file formats can be imported via Fiji plugins. One plugin is called Bio-Formats. This will import and export a wide number of file formats along with their important metadata. They can be accessed directly via the menu command File  › Import  › Bio-Formats or indirectly via the File  › Open menu command which will use the Bio-Formats plugin if needed. See the list of supported file formats on the Bio-Formats website for the complete list (135 as of this writing).

If Bio-Formats is used, you will be prompted with a big dialog and your preferences remembered.

Drag and dropping an image file to the Fiji toolbar will effectively open the file the same as the File  › Open command.

Some image file formats are more akin to databases rather than images (e.g. Leica LIF files). In these cases a second dialog will list the images in the 'database' prompting you to select which to open.

There are several different Bio-Formats commands for importing data into Fiji:

  • Bio-Formats Remote Importer. For this selection, the image doesn’t have to be saved onto your computer or a disk. But it is a limited feature; it is more robust to download the file and then use the regular Bio-Formats instead.
  • Bio-Formats Windowless Importer. With this option, Fiji uses the settings that were previously used for Bio-Formats, so it skips all dialog boxes at the beginning.
  • Bio-Formats Importer. Select this command if you need to change the settings for importing an image.

Zeiss LSM panel

The LSM panel plugin duplicates the Zeiss LSM file import function of the LOCI Bio-Format Importer, but will also extensively catalogue the LSM metadata. The LSM Toolbox is activated by the menu command File  › Import  › LSM. Once you select LSM the panel will appear.

Images are opened as 8-bit color images with the “no-palette” pseudo color from the LSM acquisition software. Each channel is imported as a separate image/stack. Lambda stacks are therefore imported as multiple images, not a single stack. They can be converted to a stack with the menu command: Image  › Stacks  › Images to stack.

Once opened, the file information can be accessed and the information can be irreversibly stamped into the images or exported.

Multiple files from a folder

Each time point of an experiment acquired with software such as Perkin Elmer’s UltraVIEW or Scion Image’s time lapse macro is saved by the acquisition software as a single TIF. The experimental sequence can be imported to Fiji via the menu command File  › Import  › Image Sequence.

Locate the directory, click on the first image in the sequence and OK all dialogs. (You may get a couple of error messages while Fiji tries to open any non-image files in the experimental directory.) The stack will “interleave” the multiple channels you recorded, and can be de-interleaved via Plugins  › Stacks - Shuffling  › Deinterleave

Selected images that are not the same size can be imported as individual images windows using File/Import and select each individual image you want imported into Fiji. Then you can put those images into a stack by Image  › Stacks  › Images to stack Unlike the File  › Import  › Image Sequence function, the images don’t need to be the same dimensions. If memory is limited, stacks can be opened as Virtual-Stacks with most of the stack remaining on the disk until it is required.

Multi-RAW sequence from a folder

To form an image, Fiji needs to know the image dimensions, bit-depth, slice number per file and any extraneous information in the file format (offset and header size). All you really need to tell it is the image dimension in x and y. These values should be obtainable from the software in which the images were acquired. Armed with this information follow these steps:

  1. File/Import/Raw…
  2. Select experimental directory.
  3. Typical values for the dialog box are:
    • Image type = 16-bit unsigned (or 8 bit typically)
    • Width and height as determined earlier
    • Offset = 0, number of image = 1, gap = 0, ‘white’ is zero = off
    • ‘Little-endian byte order’ = on, ‘open all files in folder’ = on to open all files in folder.

Non-images will also be opened and may appear as blank images and need deleting: Image  › Stacks  › Delete slice. The stack will "interleave” the multiple channels you recorded, and can be de-interleaved via Plugins  › Stacks - Shuffling  › DeInterleave.

AVI and MOV files

There are two plugins which can open uncompressed AVIs and some types of MOV file. AVIs can be opened via File  › Import  › AVI

Other Import functions

Animated GIF- This plugin opens animated GIF files as an RGB stack. Also opens single GIF images.

Fiji Extensions

  • Amira...: Open as a stack both Amira (.am) label and image stack files.
  • Biorad...: Open a Biorad (.pic) confocal stack.
  • Psychomorph .tem Reader
  • Show Amira Surface: Open in a 3D Window Amira .surf surface files (3D meshes).
  • DF3...: Open a POV-Ray density file.
  • MRC Leginon ...: Open an .mrc image (8, 16 and 32-bit) generated by Leginon (software for automated imaging in transmission electron microscopy).
  • PDF ...: Open a .pdf file. Optionally specify which page (or all) and at which scale (from 100% to 1000%). Note that when opening from "File / Open", "File / Import / URL" or from a method call, this plugin will by default open all pages at 100% magnification.
  • Extract Images From PDF...: Extract all images from a PDF file, in their original resolution.
  • DAT EMMENU ...: Open a .dat image file from EMMENU software for imaging with transmission electron microscopes.
  • DM3 Reader...: Open a .dm3 image file from Gatan cameras, with all its metadata (transmission electron microscopy).
  • TorstenRaw GZ Reader...: Open the files generated by the registration tools from
  • IPLab Reader...
  • Animated Gif...: Open an animated .gif file as a stack.
  • Amira as TrakEM2...: Open an amira .am image stack file as a TrakEM2 project. The labels file will be asked for. All calibrations and parameters are set to streamline the process.
  • Analyze...
  • Show LSM Toolbox: Open the LSM Toolbox window, from which LSM files can be opened, viewed in multi dimensions, montaged, their metadata directly inspected, time and z stamped and more.