Data processing & analysis

Working with recorded images

Loading 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

Converting 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 FlamingoConverter macro is designed for the latest iteration of Flamingo microscopes. It is compatible with both raw and tif output. The older Flamingo_RAWtoTIF macro can be found here.

Working with large datasets


Image files recorded in the tif format are compatible with most scientific image processing software, but datasets 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 BigDataViewer environment in Fiji allows to save, load, visualize and process HDF5 and N5.

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

Open Plugins – BigStitcher – Batch Processing – Define dataset… and select the Automatic Loader.

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.

Select HDF5 or N5 as output format and pick a target folder. In the next dialog, set format-specific parameters, such as compression.

Open HDF5/N5 in BigDataViewer

Open Fiji and select Plugins – BigDataViewer – Open XML/HDF5.