Benjamin Schmid

Fiji: an open-source platform for biological-image analysis

Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.

BoneJ: Free and extensible bone image analysis in ImageJ

Bone geometry is commonly measured on computed tomographic (CT) and X-ray microtomographic (μCT) images. We obtained hundreds of CT, μCT and synchrotron μCT images of bones from diverse species that needed to be analysed remote from scanning hardware, but found that available software solutions were expensive, inflexible or methodologically opaque. We implemented standard bone measurements in a novel ImageJ plugin, BoneJ, with which we analysed trabecular bone, whole bones and osteocyte lacunae. BoneJ is open source and free for anyone to download, use, modify and distribute.

A high-level 3D visualization API for Java and ImageJ

BackgroundCurrent imaging methods such as Magnetic Resonance Imaging (MRI), Confocal microscopy, Electron Microscopy (EM) or Selective Plane Illumination Microscopy (SPIM) yield three-dimensional (3D) data sets in need of appropriate computational methods for their analysis. The reconstruction, segmentation and registration are best approached from the 3D representation of the data set.ResultsHere we present a platform-independent framework based on Java and Java 3D for accelerated rendering of biological images. Our framework is seamlessly integrated into ImageJ, a free image processing package with a vast collection of community-developed biological image analysis tools. Our framework enriches the ImageJ software libraries with methods that greatly reduce the complexity of developing image analysis tools in an interactive 3D visualization environment. In particular, we provide high-level access to volume rendering, volume editing, surface extraction, and image annotation. The ability to rely on a library that removes the low-level details enables concentrating software development efforts on the algorithm implementation parts.ConclusionsOur framework enables biomedical image software development to be built with 3D visualization capabilities with very little effort. We offer the source code and convenient binary packages along with extensive documentation at http://3dviewer.neurofly.de.

An integrated micro- and macroarchitectural analysis of the Drosophila brain by computer-assisted serial section electron microscopy.

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.

iPSC-derived neurons from GBA1-associated Parkinson's disease patients show autophagic defects and impaired calcium homeostasis

Mutations in the acid β-glucocerebrosidase (GBA1) gene, responsible for the lysosomal storage disorder Gauchers disease (GD), are the strongest genetic risk factor for Parkinsons disease (PD) known to date. Here we generate induced pluripotent stem cells from subjects with GD and PD harbouring GBA1 mutations, and differentiate them into midbrain dopaminergic neurons followed by enrichment using fluorescence-activated cell sorting. Neurons show a reduction in glucocerebrosidase activity and protein levels, increase in glucosylceramide and α-synuclein levels as well as autophagic and lysosomal defects. Quantitative proteomic profiling reveals an increase of the neuronal calcium-binding protein 2 (NECAB2) in diseased neurons. Mutant neurons show a dysregulation of calcium homeostasis and increased vulnerability to stress responses involving elevation of cytosolic calcium. Importantly, correction of the mutations rescues such pathological phenotypes. These findings provide evidence for a link between GBA1 mutations and complex changes in the autophagic/lysosomal system and intracellular calcium homeostasis, which underlie vulnerability to neurodegeneration.

Rapid 3D light-sheet microscopy with a tunable lens

The in-vivo investigation of highly dynamic biological samples, for example the beating zebrafish heart, requires high-speed volume imaging techniques. Light-sheet microscopy is ideal for such samples as it records high-contrast images of entire planes within large samples at once. However, in order to obtain images of different planes, it has been necessary to move the sample relative to the fixed focal plane of the detection objective lens. This mechanical movement limits speed, precision and may be harmful to the sample. We have built a light-sheet microscope that uses remote focusing with an electrically tunable lens (ETL). Without moving specimen or objective we have thereby achieved flexible volume imaging at much higher speeds than previously reported. Our high-speed microscope delivers 3D snapshots of sensitive biological samples. As an example, we imaged 17 planes within a beating zebrafish heart at 510 frames per second, equivalent to 30 volume scans per second. Movements, shape changes and signals across the entire volume can be followed which has been impossible with existing reconstruction techniques.

A new ImageJ plug-in "ActogramJ" for chronobiological analyses.

While the rapid development of personal computers and high-throughput recording systems for circadian rhythms allow chronobiologists to produce huge amounts of data, the software to analyze them often lags behind. Here, we announce newly developed chronobiology software that is easy to use, compatible with many different systems, and freely available. Our system can perform the most frequently used analyses: actogram drawing, periodogram analysis, and waveform analysis. The software is distributed as a pure Java plug-in for ImageJ and so works on the 3 main operating systems: Linux, Macintosh, and Windows. We believe that this free software raises the speed of data analyses and makes studying chronobiology accessible to newcomers.

High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics

The ever-increasing speed and resolution of modern microscopes make the storage and post-processing of images challenging and prevent thorough statistical analyses in developmental biology. Here, instead of deploying massive storage and computing power, we exploit the spherical geometry of zebrafish embryos by computing a radial maximum intensity projection in real time with a 240-fold reduction in data rate. In our four-lens selective plane illumination microscope (SPIM) setup the development of multiple embryos is recorded in parallel and a map of all labelled cells is obtained for each embryo in <10 s. In these panoramic projections, cell segmentation and flow analysis reveal characteristic migration patterns and global tissue remodelling in the early endoderm. Merging data from many samples uncover stereotypic patterns that are fundamental to endoderm development in every embryo. We demonstrate that processing and compressing raw image data in real time is not only efficient but indispensable for image-based systems biology.

High-resolution reconstruction of the beating zebrafish heart

The heart′s continuous motion makes it difficult to capture high-resolution images of this organ in vivo. We developed tools based on high-speed selective plane illumination microscopy (SPIM), offering pristine views into the beating zebrafish heart. We captured three-dimensional cardiac dynamics with postacquisition synchronization of multiview movie stacks, obtained static high-resolution reconstructions by briefly stopping the heart with optogenetics and resolved nonperiodic phenomena by high-speed volume scanning with a liquid lens.

Hyperspectral light sheet microscopy

To study the development and interactions of cells and tissues, multiple fluorescent markers need to be imaged efficiently in a single living organism. Instead of acquiring individual colours sequentially with filters, we created a platform based on line-scanning light sheet microscopy to record the entire spectrum for each pixel in a three-dimensional volume. We evaluated data sets with varying spectral sampling and determined the optimal channel width to be around 5 nm. With the help of these data sets, we show that our setup outperforms filter-based approaches with regard to image quality and discrimination of fluorophores. By spectral unmixing we resolved overlapping fluorophores with up to nanometre resolution and removed autofluorescence in zebrafish and fruit fly embryos.