scholarly journals SynapseJ: an automated, synapse identification macro for ImageJ

2021 ◽  
Author(s):  
Juan Felipe Moreno Manrique ◽  
Parker R. Voit ◽  
Kathryn E. Windsor ◽  
Aamuktha R. Karla ◽  
Sierra R. Rodriguez ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Open Source ◽  
Fluorescent Protein ◽  
Imaging Techniques ◽  
Yellow Fluorescent Protein ◽  
Synaptic Proteins ◽  
Scaffolding Protein ◽  
Substantia Nigra Pars Compacta ◽  
Distinct Advantage ◽  
Specific Cell

While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. These methods are applied to analysis of a presynaptic marker, bassoon, with two different postsynaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Using gephyrin as an inhibitory, postsynaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental neuron (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation. 

scholarly journals SynapseJ: An Automated, Synapse Identification Macro for ImageJ

2021 ◽  
Vol 15 ◽  
Author(s):  
Juan Felipe Moreno Manrique ◽  
Parker R. Voit ◽  
Kathryn E. Windsor ◽  
Aamuktha R. Karla ◽  
Sierra R. Rodriguez ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Open Source ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Synaptic Proteins ◽  
Pedunculopontine Tegmental Nucleus ◽  
Scaffolding Protein ◽  
Substantia Nigra Pars Compacta ◽  
Distinct Advantage ◽  
Specific Cell

While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. These methods are applied to analysis of a pre-synaptic marker, bassoon, with two different post-synaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Using gephyrin as an inhibitory, post-synaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental nucleus (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation.

Distribution dynamics and functional importance of NHERF1 in regulation of Mrp-2 trafficking in hepatocytes

2014 ◽  
Vol 307 (8) ◽  
pp. C727-C737 ◽  
Author(s):  
Serhan Karvar ◽  
Jo Suda ◽  
Lixin Zhu ◽  
Don C. Rockey
Keyword(s):  
Binding Site ◽  
Deletion Mutant ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Secretory Response ◽  
Scaffolding Protein ◽  
Pdz Domains ◽  
Canalicular Membrane ◽  
Infected Cells ◽  
And Function

Na+/H+ exchanger regulatory factor 1 (NHERF1) is a multifunctional scaffolding protein that interacts with receptors and ion transporters in its PDZ domains and with the ezrin-radixin-moesin (ERM) family of proteins in its COOH terminus. The role of NHERF1 in hepatocyte function remains largely unknown. We examine the distribution and physiological significance of NHERF1 and multidrug resistance-associated protein 2 (Mrp-2) in hepatocytes. A WT radixin binding site mutant (F355R) and NHERF1 PDZ1 and PDZ2 domain adenoviral mutant constructs were tagged with yellow fluorescent protein and expressed in polarized hepatocytes to study localization and function of NHERF1. Cellular distribution of NHERF1 and radixin was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate (CMFDA) assay was used to characterize Mrp-2 function. Similar to Mrp-2, WT NHERF1 and the NHERF1 PDZ2 deletion mutant were localized to the canalicular membrane. In contrast, the radixin binding site mutant (F355R) and the NHERF1 PDZ1 deletion mutant, which interacts poorly with Mrp-2, were rarely associated with the canalicular membrane. Knockdown of NHERF1 led to dramatically impaired CMFDA secretory response. Use of CMFDA showed that the NHERF1 PDZ1 and F355R mutants were devoid of a secretory response, while WT NHERF1-infected cells exhibited increased secretion of glutathione-methylfluorescein. The data indicate that NHERF1 interacts with Mrp-2 via the PDZ1 domain of NHERF1 and, furthermore, that NHERF1 is essential for maintaining the localization and function of Mrp-2.

scholarly journals Structural Abnormalities in Neurons Are Sufficient To Explain the Clinical Disease and Fatal Outcome of Experimental Rabies in Yellow Fluorescent Protein-Expressing Transgenic Mice

2007 ◽  
Vol 82 (1) ◽  
pp. 513-521 ◽  
Author(s):  
Courtney A. Scott ◽  
John P. Rossiter ◽  
R. David Andrew ◽  
Alan C. Jackson
Keyword(s):  
Electron Microscopy ◽  
Cerebral Cortex ◽  
Virus Infection ◽  
Hind Limb ◽  
Pyramidal Neurons ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Fatal Outcome ◽  
Histopathological Changes ◽  
Rabies Virus Infection

ABSTRACT Under natural conditions and in some experimental models, rabies virus infection of the central nervous system causes relatively mild histopathological changes, without prominent evidence of neuronal death despite its lethality. In this study, the effects of rabies virus infection on the structure of neurons were investigated with experimentally infected transgenic mice expressing yellow fluorescent protein (YFP) in neuronal subpopulations. Six-week-old mice were inoculated in the hind-limb footpad with the CVS strain of fixed virus or were mock infected with vehicle (phosphate-buffered saline). Brain regions were subsequently examined by light, epifluorescent, and electron microscopy. In moribund CVS-infected mice, histopathological changes were minimal in paraffin-embedded tissue sections, although mild inflammatory changes were present. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase-3 immunostaining showed only a few apoptotic cells in the cerebral cortex and hippocampus. Silver staining demonstrated the preservation of cytoskeletal integrity in the cerebral cortex. However, fluorescence microscopy revealed marked beading and fragmentation of the dendrites and axons of layer V pyramidal neurons in the cerebral cortex, cerebellar mossy fibers, and axons in brainstem tracts. At an earlier time point, when mice displayed hind-limb paralysis, beading was observed in a few axons in the cerebellar commissure. Toluidine blue-stained resin-embedded sections from moribund YFP-expressing animals revealed vacuoles within the perikarya and proximal dendrites of pyramidal neurons in the cerebral cortex and hippocampus. These vacuoles corresponded with swollen mitochondria under electron microscopy. Vacuolation was also observed ultrastructurally in axons and in presynaptic nerve endings. We conclude that the observed structural changes are sufficient to explain the severe clinical disease with a fatal outcome in this experimental model of rabies.

scholarly journals In vivo visualization of uterine mast cells by two-photon microscopy

Reproduction ◽  
2014 ◽  
Vol 147 (6) ◽  
pp. 781-788 ◽  
Author(s):  
Franziska Schmerse ◽  
Katja Woidacki ◽  
Monika Riek-Burchardt ◽  
Peter Reichardt ◽  
Axel Roers ◽  
...  
Keyword(s):  
Mast Cells ◽  
Intravital Microscopy ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Specific Cell ◽  
Enhanced Yellow Fluorescent Protein ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Custom Made

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for the study ofin vivobehavior of these cells. We have recently reported that uterine mast cells (uMCs) are important for implantation and placentation. However, theirin vivolocalization in uterus before and during pregnancy is unknown. Herein, we report the direct observation of uMCsin vivousing double-transgenic C57BL/6JMcpt5-Cre ROSA26-EYFPmice with high expression of enhanced yellow fluorescent protein in MC protease 5 (Cma1(Mcpt5))-expressing cells by intravital two-photon microscopy. We were able to monitor MCs livein uteroduring the murine estrous cycle and at different days of pregnancy. We demonstrated that uMCs accumulated during the receptive phase of the female (estrus) and persisted in large numbers at early pregnancy stages and around mid-gestation and declined in number in non-pregnant animals at diestrus. This intravital microscopy technique, including a custom-made microscope stage and the adaption of the surgical procedure, allowed the access of the uterus and implantations for imaging. The introduced application of intravital microscopy to C57BL/6J-Mcpt5-Cre ROSA26-EYFPmice offers a novel and powerfulin vivoapproach to further address the evident relevance of uMCs to reproductive processes with obvious clinical implications.

scholarly journals Both daughter cells traffic and exocytose membrane at the cleavage furrow during mammalian cytokinesis

2008 ◽  
Vol 181 (7) ◽  
pp. 1047-1054 ◽  
Author(s):  
John W. Goss ◽  
Derek K. Toomre
Keyword(s):  
Membrane Trafficking ◽  
Fluorescent Protein ◽  
Imaging Techniques ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Virus Protein ◽  
Temperature Sensitive ◽  
Cleavage Furrow ◽  
Microscopy Imaging ◽  
Daughter Cells

Membrane trafficking during cytokinesis is not well understood. We used advanced live cell imaging techniques to track exocytosis of single vesicles to determine whether constitutively exocytosed membrane is focally delivered to the cleavage furrow. Ultrasensitive three-dimensional confocal time-lapse imaging of the temperature-sensitive membrane cargo protein vesicular stomatitis virus protein–yellow fluorescent protein revealed that vesicles from both daughter cells traffic out of the Golgi and into the furrow, following curvilinear paths. Immunolocalization and photobleaching experiments indicate that individual vesicles accumulate at the midbody and generate a reserve vesicle pool that is distinct from endosomal and lysosomal compartments. Total internal reflection fluorescence microscopy imaging provided direct evidence that Golgi-derived vesicles from both daughter cells not only traffic to the furrow region but dock and fuse there, supporting a symmetrically polarized exocytic delivery model. In contrast, quantitative analysis of midbody abscission showed inheritance of the midbody remnant by one daughter cell, indicating that cytokinesis is composed of both symmetrical and asymmetrical stages.

scholarly journals Toward A Reproducible, Scalable Framework for Processing Large Neuroimaging Datasets

2019 ◽  
Author(s):  
Erik C. Johnson ◽  
Miller Wilt ◽  
Luis M. Rodriguez ◽  
Raphael Norman-Tenazas ◽  
Corban Rivera ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Open Source ◽  
Data Storage ◽  
Large Scale ◽  
Imaging Techniques ◽  
Scientific Discovery ◽  
Imaging Data ◽  
Considerable Difficulty ◽  
X Ray ◽  
The Brain

ABSTRACTEmerging neuroimaging datasets (collected through modalities such as Electron Microscopy, Calcium Imaging, or X-ray Microtomography) describe the location and properties of neurons and their connections at unprecedented scale, promising new ways of understanding the brain. These modern imaging techniques used to interrogate the brain can quickly accumulate gigabytes to petabytes of structural brain imaging data. Unfortunately, many neuroscience laboratories lack the computational expertise or resources to work with datasets of this size: computer vision tools are often not portable or scalable, and there is considerable difficulty in reproducing results or extending methods. We developed an ecosystem of neuroimaging data analysis pipelines that utilize open source algorithms to create standardized modules and end-to-end optimized approaches. As exemplars we apply our tools to estimate synapse-level connectomes from electron microscopy data and cell distributions from X-ray microtomography data. To facilitate scientific discovery, we propose a generalized processing framework, that connects and extends existing open-source projects to provide large-scale data storage, reproducible algorithms, and workflow execution engines. Our accessible methods and pipelines demonstrate that approaches across multiple neuroimaging experiments can be standardized and applied to diverse datasets. The techniques developed are demonstrated on neuroimaging datasets, but may be applied to similar problems in other domains.

scholarly journals BigDataProcessor2: A free and open-source Fiji plugin for inspection and processing of TB sized image data

2020 ◽  
Author(s):  
Christian Tischer ◽  
Ashis Ravindran ◽  
Sabine Reither ◽  
Rainer Pepperkok ◽  
Nils Norlin
Keyword(s):  
Electron Microscopy ◽  
Open Source ◽  
Imaging Techniques ◽  
Image Data ◽  
Light Sheet ◽  
Sensor Technology ◽  
Time Points ◽  
Light Sheet Microscopy ◽  
Link Type ◽  
Image Datasets

SummaryModern bioimaging and related areas such as sensor technology has seen tremendous development the last years allowing several contemporary imaging techniques, electron microscopy (EM) and light sheet microscopy in particular, to generate datasets frequently reaching the size of several terabytes (TB). As a consequence, even seemingly simple data operations such as cropping, chromatic- and drift-corrections and even visualisation, poses challenges when applied to thousands of time points or tiles. To address this we developed BigDataProcessor2 – a Fiji plugin facilitating processing workflows for TB sized image datasets.Availability and implementationBigDataProcessor2 is available as a Fiji plugin via the BigDataProcessor update site. The application is implemented in Java and the code is publicly available on GitHub (https://github.com/bigdataprocessor/bigdataprocessor2)[email protected], [email protected]

scholarly journals Toward a scalable framework for reproducible processing of volumetric, nanoscale neuroimaging datasets

GigaScience ◽  
2020 ◽  
Vol 9 (12) ◽  
Author(s):  
Erik C Johnson ◽  
Miller Wilt ◽  
Luis M Rodriguez ◽  
Raphael Norman-Tenazas ◽  
Corban Rivera ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Open Source ◽  
Data Storage ◽  
Large Scale ◽  
Imaging Techniques ◽  
Scientific Discovery ◽  
Imaging Data ◽  
Considerable Difficulty ◽  
X Ray ◽  
The Brain

Abstract Background Emerging neuroimaging datasets (collected with imaging techniques such as electron microscopy, optical microscopy, or X-ray microtomography) describe the location and properties of neurons and their connections at unprecedented scale, promising new ways of understanding the brain. These modern imaging techniques used to interrogate the brain can quickly accumulate gigabytes to petabytes of structural brain imaging data. Unfortunately, many neuroscience laboratories lack the computational resources to work with datasets of this size: computer vision tools are often not portable or scalable, and there is considerable difficulty in reproducing results or extending methods. Results We developed an ecosystem of neuroimaging data analysis pipelines that use open-source algorithms to create standardized modules and end-to-end optimized approaches. As exemplars we apply our tools to estimate synapse-level connectomes from electron microscopy data and cell distributions from X-ray microtomography data. To facilitate scientific discovery, we propose a generalized processing framework, which connects and extends existing open-source projects to provide large-scale data storage, reproducible algorithms, and workflow execution engines. Conclusions Our accessible methods and pipelines demonstrate that approaches across multiple neuroimaging experiments can be standardized and applied to diverse datasets. The techniques developed are demonstrated on neuroimaging datasets but may be applied to similar problems in other domains.

scholarly journals BigDataProcessor2: a free and open-source Fiji plugin for inspection and processing of TB sized image data

2021 ◽  
Author(s):  
Christian Tischer ◽  
Ashis Ravindran ◽  
Sabine Reither ◽  
Nicolas Chiaruttini ◽  
Rainer Pepperkok ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Open Source ◽  
Imaging Techniques ◽  
Image Data ◽  
Light Sheet ◽  
Supplementary Information ◽  
Sensor Technology ◽  
Supplementary Data ◽  
Time Points ◽  
Light Sheet Microscopy

Abstract Summary Modern bioimaging and related areas such as sensor technology have undergone tremendous development over the last few years. As a result, contemporary imaging techniques, particularly electron microscopy (EM) and light sheet microscopy, can frequently generate datasets attaining sizes of several terabytes (TB). As a consequence, even seemingly simple data operations such as cropping, chromatic- and drift-corrections and even visualisation, poses challenges when applied to thousands of time points or tiles. To address this we developed BigDataProcessor2—a Fiji plugin facilitating processing workflows for TB sized image datasets. Availability and implementation BigDataProcessor2 is available as a Fiji plugin via the BigDataProcessor update site. The application is implemented in Java and the code is publicly available on GitHub (https://github.com/bigdataprocessor/bigdataprocessor2). Supplementary information Supplementary data are available at Bioinformatics online.

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