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 (Fiji is just ImageJ: http://pacific.mpi-cbg.de/). Contact: tomancak@mpi-cbg.de
A key challenge in neuroscience is the expeditious reconstruction of neuronal circuits. For model systems such as Drosophila and C. elegans, the limiting step is no longer the acquisition of imagery ...but the extraction of the circuit from images. For this purpose, we designed a software application, TrakEM2, that addresses the systematic reconstruction of neuronal circuits from large electron microscopical and optical image volumes. We address the challenges of image volume composition from individual, deformed images; of the reconstruction of neuronal arbors and annotation of synapses with fast manual and semi-automatic methods; and the management of large collections of both images and annotations. The output is a neural circuit of 3d arbors and synapses, encoded in NeuroML and other formats, ready for analysis.
Light-sheet imaging of cleared and expanded samples creates terabyte-sized datasets that consist of many unaligned three-dimensional image tiles, which must be reconstructed before analysis. We ...developed the BigStitcher software to address this challenge. BigStitcher enables interactive visualization, fast and precise alignment, spatially resolved quality estimation, real-time fusion and deconvolution of dual-illumination, multitile, multiview datasets. The software also compensates for optical effects, thereby improving accuracy and enabling subsequent biological analysis.
During development, coordinated cell behaviors orchestrate tissue and organ morphogenesis. Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the ...mechanisms of morphogenesis. To study the cellular basis of limb development, we imaged transgenic fluorescently-labeled embryos from the crustacean
with multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis. The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker (MaMuT). In silico clonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb-bud formation is associated with spatial modulation of cell proliferation, while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis. Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic.
Light-sheet fluorescence microscopy is able to image large specimens with high resolution by capturing the samples from multiple angles. Multiview deconvolution can substantially improve the ...resolution and contrast of the images, but its application has been limited owing to the large size of the data sets. Here we present a Bayesian-based derivation of multiview deconvolution that drastically improves the convergence time, and we provide a fast implementation using graphics hardware.
The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial ...transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.
ImgLib2—generic image processing in Java PIETZSCH, Tobias; PREIBISCH, Stephan; TOMANCAK, Pavel ...
Bioinformatics,
11/2012, Letnik:
28, Številka:
22
Journal Article
Recenzirano
Odprti dostop
ImgLib2 is an open-source Java library for n-dimensional data representation and manipulation with focus on image processing. It aims at minimizing code duplication by cleanly separating ...pixel-algebra, data access and data representation in memory. Algorithms can be implemented for classes of pixel types and generic access patterns by which they become independent of the specific dimensionality, pixel type and data representation. ImgLib2 illustrates that an elegant high-level programming interface can be achieved without sacrificing performance. It provides efficient implementations of common data types, storage layouts and algorithms. It is the data model underlying ImageJ2, the KNIME Image Processing toolbox and an increasing number of Fiji-Plugins.
ImgLib2 is licensed under BSD. Documentation and source code are available at http://imglib2.net and in a public repository at https://github.com/imagej/imglib.
Supplementary data are available at Bioinformatics Online.
saalfeld@mpi-cbg.de
Pre-mRNA splicing is catalyzed by the spliceosome, which can assemble on pre-mRNA cotranscriptionally. However, whether splicing generally occurs during transcription has not been addressed. Indeed, ...splicing catalysis is expected to occur posttranscriptionally in yeast, where the shortness of terminal exons should leave insufficient time for splicing. Here, we isolate endogenous S. cerevisiae nascent RNA and determine gene-specific splicing efficiencies and transcription profiles, using high-density tiling microarrays. Surprisingly, we find that splicing occurs cotranscriptionally for the majority of intron-containing genes. Analysis of transcription profiles reveals Pol II pausing within the terminal exons of these genes. Intronless and inefficiently spliced genes lack this pause. In silico simulations of transcription and splicing kinetics confirm that this pausing event provides sufficient time for splicing before termination. The discovery of terminal exon pausing demonstrates functional coupling of transcription and splicing near gene ends.
► Endogenous S. cerevisiae nascent RNA was isolated by chromatin fractionation ► A majority of genes are cotranscriptionally spliced ► RNA Pol II pauses at terminal exons ► In silico simulations validate cotranscriptional splicing
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