Globally optimal stitching of tiled 3D microscopic image acquisitions

TLDR

High‑resolution 3D imaging of large specimens requires tiled scans because confocal microscopy’s field of view is too small and the microscope stage’s coordinates are insufficiently precise for direct stitching. The authors aim to develop a globally optimal stitching method for large collections of 3D confocal images. Their method applies the Fourier Shift Theorem to compute all pairwise translations, selects the best overlap by cross‑correlation, and then determines the globally optimal configuration of the entire image set. The approach eliminates error propagation, runs quickly, works on both 2D and 3D data, and for small sets does not require prior knowledge of tile arrangement. The implementation is available as an ImageJ plugin in Fiji (FijiisjustImageJ: http://pacific.mpi‑cbg.de/) and can be contacted at tomancak@mpi‑cbg.de.

Abstract

Abstract Motivation: Modern anatomical and developmental studies often require high-resolution imaging of large specimens in three dimensions (3D). Confocal microscopy produces high-resolution 3D images, but is limited by a relatively small field of view compared with the size of large biological specimens. Therefore, motorized stages that move the sample are used to create a tiled scan of the whole specimen. The physical coordinates provided by the microscope stage are not precise enough to allow direct reconstruction (Stitching) of the whole image from individual image stacks. Results: To optimally stitch a large collection of 3D confocal images, we developed a method that, based on the Fourier Shift Theorem, computes all possible translations between pairs of 3D images, yielding the best overlap in terms of the cross-correlation measure and subsequently finds the globally optimal configuration of the whole group of 3D images. This method avoids the propagation of errors by consecutive registration steps. Additionally, to compensate the brightness differences between tiles, we apply a smooth, non-linear intensity transition between the overlapping images. Our stitching approach is fast, works on 2D and 3D images, and for small image sets does not require prior knowledge about the tile configuration. Availability: The implementation of this method is available as an ImageJ plugin distributed as a part of the Fiji project (FijiisjustImageJ: http://pacific.mpi-cbg.de/). Contact: [e-mail]