Data processing & analysis

work with recorded images

Load individual RAW Files

If ScopeControl is set to raw, the Flamingo saves recorded image data as raw files. Each stack is saved as a single file. Individual raw files can be imported into Fiji via File > Import > Raw… or by drag-and-drop. Adjust the following settings during import:

  • Image type: 16-bit Unsigned
  • Width & Height: 2048 (unless cropped)
  • Number of images: ≥ number of planes
  • Little-endian byte order

Convert a large number of files

Once you have a large number of image files in a given experiment folder, an automated conversion and pre-processing is desirable. These two Fiji tool converts image files recorded with a Flamingo microscope into tif files. Options for max projection, small preview, cropping and combining two illumination sides are present. Files are saved to subfolders.

The Flamingo Converter macro is designed for the latest iteration of Flamingo microscopes. It is compatible with both raw and tif output.

Test different lookup tables

The PreviewLUT macro automatically applies all lookup tables on a given Fiji installation to a single image. A given grayscale image is scaled down and the lookup table and its RGB histogram are presented as stack and gallery.

Multi-view Registration & Fusion

Import image data in BigStitcher

Open Fiji, head to Help – Update and activate the BigStitcher update site. Apply changes and and restart Fiji.

Open Plugins – BigStitcher – BigStitcher.

Browse your storage and select the first tif file of a recorded dataset. If you have a series of images (>1 file) set wildcards in the file name. For example, change t000000 to t* for a time-lapse. Check if the resulting list of selected files is correct. In the next dialog, assign the wildcards to dataset parameters. Correct the pixel size.

Detect Interest points

Fiji, BigStitcher & pyramidal file formats

Image files recorded in the tif format are compatible with most scientific image processing software, but data sets quickly get too large to be loaded into a computers main memory all at once. Pyramidal file formats like HDF5 and N5 – in combination with software supporting them – are a great way to navigate large datasets on everyday computers. The BigStitcher environment in Fiji allows to save, load, visualize and process HDF5 and N5.


BigStitcher: reconstructing high-resolution image datasets of cleared and expanded samples | Nature Methods

BigStitcher (

GitHub – PreibischLab/BigStitcher: ImgLib2/BDV implementation of Stitching for large datasets