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1.  Software Framework for Controlling Unsupervised Scientific Instruments 
PLoS ONE  2016;11(8):e0161671.
Science outreach and communication are gaining more and more importance for conveying the meaning of today’s research to the general public. Public exhibitions of scientific instruments can provide hands-on experience with technical advances and their applications in the life sciences. The software of such devices, however, is oftentimes not appropriate for this purpose. In this study, we describe a software framework and the necessary computer configuration that is well suited for exposing a complex self-built and software-controlled instrument such as a microscope to laymen under limited supervision, e.g. in museums or schools. We identify several aspects that must be met by such software, and we describe a design that can simultaneously be used to control either (i) a fully functional instrument in a robust and fail-safe manner, (ii) an instrument that has low-cost or only partially working hardware attached for illustration purposes or (iii) a completely virtual instrument without hardware attached. We describe how to assess the educational success of such a device, how to monitor its operation and how to facilitate its maintenance. The introduced concepts are illustrated using our software to control eduSPIM, a fluorescent light sheet microscope that we are currently exhibiting in a technical museum.
doi:10.1371/journal.pone.0161671
PMCID: PMC5003368  PMID: 27570966
2.  eduSPIM: Light Sheet Microscopy in the Museum 
PLoS ONE  2016;11(8):e0161402.
Light Sheet Microscopy in the Museum
Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology.
Design Principles of an Educational Light Sheet Microscope
To maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously.
The eduSPIM Design Is Tailored Easily to Fit Numerous Applications
The universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided.
doi:10.1371/journal.pone.0161402
PMCID: PMC4999180  PMID: 27560188
3.  Hyperspectral light sheet microscopy 
Nature Communications  2015;6:7990.
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.
Multicolour information is required to study the complex interplay of biological tissues. Here, Jahr et al. acquire spectral information at high resolution for each pixel in a hyperspectral light sheet microscope, while maintaining its perpendicular illumination and low phototoxicity.
doi:10.1038/ncomms8990
PMCID: PMC4569691  PMID: 26329685
4.  Real-time multi-view deconvolution 
Bioinformatics  2015;31(20):3398-3400.
Summary: In light-sheet microscopy, overall image content and resolution are improved by acquiring and fusing multiple views of the sample from different directions. State-of-the-art multi-view (MV) deconvolution simultaneously fuses and deconvolves the images in 3D, but processing takes a multiple of the acquisition time and constitutes the bottleneck in the imaging pipeline. Here, we show that MV deconvolution in 3D can finally be achieved in real-time by processing cross-sectional planes individually on the massively parallel architecture of a graphics processing unit (GPU). Our approximation is valid in the typical case where the rotation axis lies in the imaging plane.
Availability and implementation: Source code and binaries are available on github (https://github.com/bene51/), native code under the repository ‘gpu_deconvolution’, Java wrappers implementing Fiji plugins under ‘SPIM_Reconstruction_Cuda’.
Contact: bschmid@mpi-cbg.de or huisken@mpi-cbg.de
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btv387
PMCID: PMC4595906  PMID: 26112291
5.  Optical tomography complements light sheet microscopy for in toto imaging of zebrafish development 
Development (Cambridge, England)  2015;142(5):1016-1020.
Fluorescently labeled structures can be spectrally isolated and imaged at high resolution in living embryos by light sheet microscopy. Multimodal imaging techniques are now needed to put these distinct structures back into the context of the surrounding tissue. We found that the bright-field contrast of unstained specimens in a selective plane illumination microscopy (SPIM) setup can be exploited for in vivo tomographic reconstructions of the three-dimensional anatomy of zebrafish, without causing phototoxicity. We report multimodal imaging of entire zebrafish embryos over several hours of development, as well as segmentation, tracking and automatic registration of individual organs.
Summary: Bright-field imaging of unstained specimens during selective plane illumination microscopy can provide in vivo tomographic reconstruction of zebrafish anatomy.
doi:10.1242/dev.116970
PMCID: PMC4352980  PMID: 25655702
SPIM; Fluorescence; Light sheet microscopy; Optical tomography; Time-lapse imaging; Zebrafish
6.  Fiji - an Open Source platform for biological image analysis 
Nature methods  2012;9(7):10.1038/nmeth.2019.
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 novel 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.
doi:10.1038/nmeth.2019
PMCID: PMC3855844  PMID: 22743772
8.  Derivation and Expansion Using Only Small Molecules of Human Neural Progenitors for Neurodegenerative Disease Modeling 
PLoS ONE  2013;8(3):e59252.
Phenotypic drug discovery requires billions of cells for high-throughput screening (HTS) campaigns. Because up to several million different small molecules will be tested in a single HTS campaign, even small variability within the cell populations for screening could easily invalidate an entire campaign. Neurodegenerative assays are particularly challenging because neurons are post-mitotic and cannot be expanded for implementation in HTS. Therefore, HTS for neuroprotective compounds requires a cell type that is robustly expandable and able to differentiate into all of the neuronal subtypes involved in disease pathogenesis. Here, we report the derivation and propagation using only small molecules of human neural progenitor cells (small molecule neural precursor cells; smNPCs). smNPCs are robust, exhibit immortal expansion, and do not require cumbersome manual culture and selection steps. We demonstrate that smNPCs have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons (MNs) and midbrain dopaminergic neurons (mDANs) as well as neural crest lineages, including peripheral neurons and mesenchymal cells. These properties are so far only matched by pluripotent stem cells. Finally, to demonstrate the usefulness of smNPCs we show that mDANs differentiated from smNPCs with LRRK2 G2019S are more susceptible to apoptosis in the presence of oxidative stress compared to wild-type. Therefore, smNPCs are a powerful biological tool with properties that are optimal for large-scale disease modeling, phenotypic screening, and studies of early human development.
doi:10.1371/journal.pone.0059252
PMCID: PMC3606479  PMID: 23533608
9.  Serum and Cerebrospinal Fluid Levels of Transthyretin in Lewy Body Disorders with and without Dementia 
PLoS ONE  2012;7(10):e48042.
Parkinson’s disease (PD) without (non-demented, PDND) and with dementia (PDD), and dementia with Lewy bodies (DLB) are subsumed under the umbrella term Lewy body disorders (LBD). The main component of the underlying pathologic substrate, i.e. Lewy bodies and Lewy neurites, is misfolded alpha-synuclein (Asyn), and - in particular in demented LBD patients - co-occurring misfolded amyloid-beta (Abeta). Lowered blood and cerebrospinal fluid (CSF) levels of transthyretin (TTR) - a clearance protein mainly produced in the liver and, autonomously, in the choroid plexus - are associated with Abeta accumulation in Alzheimer’s disease. In addition, a recent study suggests that TTR is involved in Asyn clearance. We measured TTR protein levels in serum and cerebrospinal fluid of 131 LBD patients (77 PDND, 26 PDD, and 28 DLB) and 72 controls, and compared TTR levels with demographic and clinical data as well as neurodegenerative markers in the CSF. Five single nucleotide polymorphisms of the TTR gene which are considered to influence the ability of the protein to carry its ligands were also analyzed. CSF TTR levels were significantly higher in LBD patients compared to controls. Post-hoc analysis demonstrated that this effect was driven by PDND patients. In addition, CSF TTR levels correlated negatively with CSF Abeta1–42, total tau and phospho-tau levels. Serum TTR levels did not significantly differ among the studied groups. There were no relevant associations between TTR levels and genetic, demographic and clinical data, respectively. These results suggest an involvement of the clearance protein TTR in LBD pathophysiology, and should motivate to elucidate TTR-related mechanisms in LBD in more detail.
doi:10.1371/journal.pone.0048042
PMCID: PMC3485000  PMID: 23133543
10.  BoneJ: free and extensible bone image analysis in ImageJ 
Bone  2010;47(6):1076-1079.
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.
doi:10.1016/j.bone.2010.08.023
PMCID: PMC3193171  PMID: 20817052
bone; tomography; image; open source; software; morphometry
11.  An Integrated Micro- and Macroarchitectural Analysis of the Drosophila Brain by Computer-Assisted Serial Section Electron Microscopy 
PLoS Biology  2010;8(10):e1000502.
A new software package allows for dense electron microscopy reconstructions of neuronal networks in the fruit fly brain, and reveals specific differences in microcircuits between insects and vertebrates.
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.
Author Summary
Brains contain a vast number of connections between neurons, termed synapses. The precise patterns of these synaptic contacts form the structural underpinning of electrical microcircuits responsible for animal behavior. Due to their small size, synaptic contacts can be conclusively shown using only high-resolution electron microscopy (EM). Therefore, complete series of ultrathin sections are required to reconstruct neuronal microcircuitry. The acquisition and analysis of EM sections (with 15,000 sections per millimeter of tissue) is practical only by computer-assisted means. In this article, we demonstrate the utility of the software package TrakEM2 to model interconnections of nerve fibers from consecutive EM sections and to efficiently reconstruct the neural networks encountered in different parts of a small brain, the early larval brain of the fruit fly Drosophila melanogaster. Neuronal networks are composed of patterns of axons and dendrites (neuronal extensions that transmit and receive signals, respectively), and using TrakEM2, we describe the most common motifs they form. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect brains.
doi:10.1371/journal.pbio.1000502
PMCID: PMC2950124  PMID: 20957184
12.  A high-level 3D visualization API for Java and ImageJ 
BMC Bioinformatics  2010;11:274.
Background
Current 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.
Results
Here 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.
Conclusions
Our 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.
doi:10.1186/1471-2105-11-274
PMCID: PMC2896381  PMID: 20492697
13.  High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics 
Nature Communications  2013;4:2207.
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.
Systematic large-scale analysis of embryonic development requires the processing of large amounts of microscopy data. Here Schmid et al. solve this problem by developing a high-speed imaging system that projects zebrafish embryos onto a ‘world map’ in real time, revealing characteristic migration patterns in the early endoderm.
doi:10.1038/ncomms3207
PMCID: PMC3731668  PMID: 23884240

Results 1-13 (13)