Globally Optimal Stitching of Tiled 3D Microscopic Image Acquisitions

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
Bioinformatics (Impact Factor: 4.98). 05/2009; 25(11):1463-5. DOI: 10.1093/bioinformatics/btp184
Source: PubMed


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.
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.
The implementation of this method is available as an ImageJ plugin distributed as a part of the Fiji project (Fiji is just ImageJ:

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Available from: Stephan Preibisch
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    • "For tetraploid germ cells, " late pachytene " nuclei were defined as being within the last third of the meiotic zone; for triploid germ cells, late pachytene corresponded to the last 10th of the meiotic zone. Multiple overlapping fields covering the whole length of the gonad were acquired for each specimen, and gonads were assembled by iterative use of the " pairwise stitching " plugin (Preibisch et al. 2009) on Fiji to allow identification of nuclei within these defined positions. For analysis of volume-rendered synapsis configurations, nuclei were scored only when staining, resolution and the arrangement of chromosomes within the nucleus permitted unambiguous tracing of SC in 3D rotations; nuclei from at least two different gonads were analyzed for each genotype. "

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    • "In instances where longer working distances were necessary, fluorescently stained specimens were imaged with an upright Olympus fluorescent single photon confocal equipped with a 20X water-dipping objective (NA = 0.95). For 12 s and 16 s stage fluorescent RNA in situ, ImageJ was used for pairwise stitching of original optical stacks taken with 20X air objective (NA = 0.7), using the method previously described [37]. "
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