46 research outputs found
Microtome-integrated microscope system for high sensitivity tracking of in-resin fluorescence in blocks and ultrathin sections for correlative microscopy
Many areas of biological research demand the combined use of different imaging modalities to cover a wide range of magnifications and measurements or to place fluorescent patterns into an ultrastructural context. A technically difficult problem is the efficient specimen transfer between different imaging modalities without losing the coordinates of the regions-of-interest (ROI). Here, we report a new and highly sensitive integrated system that combines a custom designed microscope with an ultramicrotome for in-resin-fluorescence detection in blocks, ribbons and sections on EM-grids. Although operating with long-distance lenses, this system achieves a very high light sensitivity. Our instrumental set-up and operating workflow are designed to investigate rare events in large tissue volumes. Applications range from studies of individual immune, stem and cancer cells to the investigation of non-uniform subcellular processes. As a use case, we present the ultrastructure of a single membrane repair patch on a muscle fiber in intact muscle in a whole animal context
Roles of retinoic acid receptors and of Hox genes in the patterning of the teeth and of the jaw skeleton.
Retinoic acid receptors and transcriptional factors encoded by Hox genes play key roles in vertebrate development and belong to an integrated functional network. To investigate the actual functions of these molecules during ontogenesis and in particular in the patterning of the cranial neural crest cells giving rise to the teeth and to the jaw bones, we have generated null mutant mice lacking functional retinoic acid receptors or Hox genes by gene targeting in embryonic stem cells.journal articleresearch support, non-u.s. gov'treview1995 Febimporte
Fast fluorescence microscopy for imaging the dynamics of embryonic development
Live imaging has gained a pivotal role in developmental biology since it increasingly allows real-time observation of cell behavior in intact organisms. Microscopes that can capture the dynamics of ever-faster biological events, fluorescent markers optimal for in vivo imaging, and, finally, adapted reconstruction and analysis programs to complete data flow all contribute to this success. Focusing on temporal resolution, we discuss how fast imaging can be achieved with minimal prejudice to spatial resolution, photon count, or to reliably and automatically analyze images. In particular, we show how integrated approaches to imaging that combine bright fluorescent probes, fast microscopes, and custom post-processing techniques can address the kinetics of biological systems at multiple scales. Finally, we discuss remaining challenges and opportunities for further advances in this field
Low Complexity Regularization of Linear Inverse Problems
Inverse problems and regularization theory is a central theme in contemporary
signal processing, where the goal is to reconstruct an unknown signal from
partial indirect, and possibly noisy, measurements of it. A now standard method
for recovering the unknown signal is to solve a convex optimization problem
that enforces some prior knowledge about its structure. This has proved
efficient in many problems routinely encountered in imaging sciences,
statistics and machine learning. This chapter delivers a review of recent
advances in the field where the regularization prior promotes solutions
conforming to some notion of simplicity/low-complexity. These priors encompass
as popular examples sparsity and group sparsity (to capture the compressibility
of natural signals and images), total variation and analysis sparsity (to
promote piecewise regularity), and low-rank (as natural extension of sparsity
to matrix-valued data). Our aim is to provide a unified treatment of all these
regularizations under a single umbrella, namely the theory of partial
smoothness. This framework is very general and accommodates all low-complexity
regularizers just mentioned, as well as many others. Partial smoothness turns
out to be the canonical way to encode low-dimensional models that can be linear
spaces or more general smooth manifolds. This review is intended to serve as a
one stop shop toward the understanding of the theoretical properties of the
so-regularized solutions. It covers a large spectrum including: (i) recovery
guarantees and stability to noise, both in terms of -stability and
model (manifold) identification; (ii) sensitivity analysis to perturbations of
the parameters involved (in particular the observations), with applications to
unbiased risk estimation ; (iii) convergence properties of the forward-backward
proximal splitting scheme, that is particularly well suited to solve the
corresponding large-scale regularized optimization problem
Brain Struct Funct
Opioid receptors are G protein-coupled receptors (GPCRs) that modulate brain function at all levels of neural integration, including autonomic, sensory, emotional and cognitive processing. Mu (MOR) and delta (DOR) opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular levels remains unsolved. To challenge the hypothesis of MOR/DOR heteromerization in the brain, we generated redMOR/greenDOR double knock-in mice and report dual receptor mapping throughout the nervous system. Data are organized as an interactive database offering an opioid receptor atlas with concomitant MOR/DOR visualization at subcellular resolution, accessible online. We also provide co-immunoprecipitation-based evidence for receptor heteromerization in these mice. In the forebrain, MOR and DOR are mainly detected in separate neurons, suggesting system-level interactions in high-order processing. In contrast, neuronal co-localization is detected in subcortical networks essential for survival involved in eating and sexual behaviors or perception and response to aversive stimuli. In addition, potential MOR/DOR intracellular interactions within the nociceptive pathway offer novel therapeutic perspectives
Automatic Tumor-Stroma Separation in Fluorescence TMAs Enables the Quantitative High-Throughput Analysis of Multiple Cancer Biomarkers
The upcoming quantification and automation in biomarker based histological tumor evaluation will require computational methods capable of automatically identifying tumor areas and differentiating them from the stroma. As no single generally applicable tumor biomarker is available, pathology routinely uses morphological criteria as a spatial reference system. We here present and evaluate a method capable of performing the classification in immunofluorescence histological slides solely using a DAPI background stain. Due to the restriction to a single color channel this is inherently challenging. We formed cell graphs based on the topological distribution of the tissue cell nuclei and extracted the corresponding graph features. By using topological, morphological and intensity based features we could systematically quantify and compare the discrimination capability individual features contribute to the overall algorithm. We here show that when classifying fluorescence tissue slides in the DAPI channel, morphological and intensity based features clearly outpace topological ones which have been used exclusively in related previous approaches. We assembled the 15 best features to train a support vector machine based on Keratin stained tumor areas. On a test set of TMAs with 210 cores of triple negative breast cancers our classifier was able to distinguish between tumor and stroma tissue with a total overall accuracy of 88%. Our method yields first results on the discrimination capability of features groups which is essential for an automated tumor diagnostics. Also, it provides an objective spatial reference system for the multiplex analysis of biomarkers in fluorescence immunohistochemistry
Unlike for Human Monocytes after LPS Activation, Release of TNF-α by THP-1 Cells Is Produced by a TACE Catalytically Different from Constitutive TACE
Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine today identified as a key mediator of several chronic inflammatory diseases. TNF-α, initially synthesized as a membrane-anchored precursor (pro-TNF-α), is processed by proteolytic cleavage to generate the secreted mature form. TNF-α converting enzyme (TACE) is currently the first and single protease described as responsible for the inducible release of soluble TNF-α.Here, we demonstrated the presence on THP-1 cells as on human monocytes of a constitutive proteolytical activity able to cleave pro-TNF-α. Revelation of the cell surface TACE protein expression confirmed that the observed catalytic activity is due to TACE. However, further studies using effective and innovative TNF-α inhibitors, as well as a highly selective TACE inhibitor, support the presence of a catalytically different sheddase activity on LPS activated THP-1 cells. It appears that this catalytically different TACE protease activity might have a significant contribution to TNF-α release in LPS activated THP-1 cells, by contrast to human monocytes where the TACE activity remains catalytically unchanged even after LPS activation.On the surface of LPS activated THP-1 cells we identified a releasing TNF-α activity, catalytically different from the sheddase activity observed on human monocytes from healthy donors. This catalytically-modified TACE activity is different from the constitutive shedding activity and appears only upon stimulation by LPS
Gain of affinity point mutation in the serotonin receptor gene 5-HT2Dro accelerates germband extension movements during Drosophila gastrulation.
International audienceSerotonin (5-HT) not only works as a neurotransmitter in the nervous system, but also as a morphogenetic factor during early embryogenesis. In Drosophila, a previous report showed that embryos that lack the 5-HT(2Dro) receptor locus, display abnormal gastrulation movements. In this work, we screened for point mutations in the 5-HT(2Dro) receptor gene. We identified one point mutation that generates a gain of serotonin affinity for the receptor and affects germband extension: 5-HT(2Dro) (C1644). Embryos homozygous for this point mutation display a fourfold increase in the maximal speed of ectodermal cell movements during the rapid phase of germband extension. Homozygous 5-HT(2Dro) (C1644) embryos present a cuticular phenotype, including a total lack of denticle belt. Identification of this gain of function mutation shows the participation of serotonin in the regulation of the cell speed movements during the germband extension and suggests a role of serotonin in the regulation of cuticular formation during early embryogenesis