Flatfield correction followed by stitching
It is often the case that tiled microscopy images contain artifacts due to uneven illumination.
This is due to the shape of the illumination, the quality of the optics and the size of the field of view, but losses of up to 50% can be seen between the periphery and the center of a field of view. This loss usually follows a parabolic function, but is not necessarily centered, causing artifacts when stitching tiled acquisitions.
While many methods exist to compensate for this, the simplest consists in acquiring a 'flat field' image by taking a homogeneous sample (Chroma slides or even better, dye solutions, see reference below and our associated Wiki page) and acquiring it for each channel of the dataset
This image can then be used to divide the original one and thus help 'flatten' the illumination.
Furthermore, excellent tools already exist to perform image stitching such as the Stitch Grid/Collection Plugin
By leveraging the scriptability of that plugin, this script
- Flattens the intensities based on a user-provided Flat Field image stack (1 slice per channel)
- Saves a tiff (and possiby downsampled) version of the images
- Reads the XY Position of each tile to create a TileConfiguration file readable by the Stitch Grid/Collection Plugin
- Eventually calls said plugin
References
See the associated article
M. A. MODEL, J. L. BLANK, Concentrated dyes as a source of two-dimensional fluorescent field for characterization of a confocal microscope
Journal of Microscopy, Vol. 229, Pt 1 2008, pp. 12–16