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PURPOSE: The presence of metal implants can produce artifacts and distort Hounsfield units (HU) in patient computed tomography (CT) images. The purpose of this work was to characterize a novel metal artifact reduction (MAR) algorithm for reconstruction of CBCT images obtained by the HyperSight imaging system. METHODS: Three tissue-equivalent phantoms were fitted with materials commonly used in medical applications. The first consisted of a variety of metal samples centered within a solid water block, the second was an Advanced Electron Density phantom with metal rods, and the third consisted of hip prostheses positioned within a water tank. CBCT images of all phantoms were acquired and reconstructed using the MAR and iCBCT Acuros algorithms on the HyperSight system. The signal-to-noise ratio (SNR), artifact index (AI), structural similarity index measure (SSIM), peak signal-to-noise ratio (PSNR), and mean-square error (MSE) were computed to assess the image quality in comparison to artifact-free reference images. The mean HU at various VOI positions around the cavity was calculated to evaluate the artifact dependence on distance and angle from the center of the cavity. The artifact volume of the phantom (excluding the cavity) was estimated by summing the volume of all voxels with HU values outside the 5th and 95th percentiles of the phantom CBCT with no artifact. RESULTS: The SNR, AI, SSIM, PSNR, and MSE metrics demonstrated significantly higher similarity to baseline when using MAR compared to iCBCT Acuros for all high-density materials, except for aluminum. Mean HU returned to expected solid water background at a shorter distance from metal sample in the MAR images, and the standard deviation remained lower for the MAR images at all distances from the insert. The artifact volume decreased using the novel MAR algorithm for all metal samples excluding aluminum (p < 0.001) and all hip prostheses (p < 0.05). CONCLUSION: Varian's HyperSight MAR reconstruction algorithm shows a reduction in metal artifact metrics, motivating the use of MAR reconstruction for patients with metal implants.

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The aim of the present study was to develop and validate a quantitative image analysis (IA) algorithm to aid pathologists in assessing bright-field HER2 in situ hybridization (ISH) tests in solid cancers. A cohort of 80 sequential cases (40 HER2-negative and 40 HER2-positive) were evaluated for HER2 gene amplification with bright-field ISH. We developed an IA algorithm using the ISH Module from HALO software to automatically quantify HER2 and CEP17 copy numbers per cell as well as the HER2/CEP17 ratio. We observed a high correlation of HER2/CEP17 ratio, an average of HER2 and CEP17 copy number per cell between visual and IA quantification (Pearson's correlation coefficient of 0.842, 0.916, and 0.765, respectively). IA was able to count from 124 cells to 47,044 cells (median of 5565 cells). The margin of error for the visual quantification of the HER2/CEP17 ratio and of the average of HER2 copy number per cell decreased from a median of 0.23 to 0.02 and from a median of 0.49 to 0.04, respectively, in IA. Curve estimation regression models showed that a minimum of 469 or 953 invasive cancer cells per case is needed to reach an average margin of error below 0.1 for the HER2/CEP17 ratio or for the average of HER2 copy number per cell, respectively. Lastly, on average, a case took 212.1 s to execute the IA, which means that it evaluates about 130 cells/s and requires 6.7 s/mm2. The concordance of the IA software with the visual scoring was 95%, with a sensitivity of 90% and a specificity of 100%. All four discordant cases were able to achieve concordant results after the region of interest adjustment. In conclusion, this validation study underscores the usefulness of IA in HER2 ISH testing, displaying excellent concordance with visual scoring and significantly reducing margins of error.

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PURPOSE: To investigate the correlation between morphological lesions and functional indicators in eyes with neovascular age-related macular degeneration (nAMD). METHODS: This was a prospective observational study of treatment-naïve nAMD eyes. Various morphological lesions and impaired retinal structures were manually measured at baseline and month-3 in three-dimensional optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) images, including the volumes (mm3) of macular neovascularization (MNV), avascular subretinal hyperreflective material (avascular SHRM), subretinal fluid (SRF), intraretinal fluid (IRF), serous pigment epithelial detachment (sPED) and the impaired area (mm2) of ellipsoid zone (EZ), external limiting membrane (ELM) and outer nuclear layer (ONL). RESULTS: Sixty-three eyes were included. The volume of avascular SHRM showed persistent positive associations with the area of EZ damage, both at baseline, month-3, and change values (all P < 0.001). Poor BCVA (month-3) was associated with larger volumes of baseline IRF (ß = 0.377, P < 0.001), avascular SHRM (ß = 0.306, P = 0.032), and ELM impairment area (ß = 0.301, P = 0.036) in multivariate model. EZ and ELM impairment were primarily associated with baseline avascular SHRM (ß = 0.374, p = 0.003; ß = 0.388, P < 0.001, respectively), while ONL impairment primarily associated with MNV (ß = 0.475, P < 0.001). CONCLUSION: The utilization of three-dimensional measurements elucidates the intrinsic connections among various lesions and functional outcomes. In particular, avascular SHRM plays an important role in prognosis of nAMD.

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Correlative light and electron microscopy (CLEM) methods are powerful methods that combine molecular organization (from light microscopy) with ultrastructure (from electron microscopy). However, CLEM methods pose high cost/difficulty barriers to entry and have very low experimental throughput. Therefore, we have developed an indirect correlative light and electron microscopy (iCLEM) pipeline to sidestep the rate-limiting steps of CLEM (i.e., preparing and imaging the same samples on multiple microscopes) and correlate multiscale structural data gleaned from separate samples imaged using different modalities by exploiting biological structures identifiable by both light and electron microscopy as intrinsic fiducials. We demonstrate here an application of iCLEM, where we utilized gap junctions and mechanical junctions between muscle cells in the heart as intrinsic fiducials to correlate ultrastructural measurements from transmission electron microscopy (TEM), and focused ion beam scanning electron microscopy (FIB-SEM) with molecular organization from confocal microscopy and single molecule localization microscopy (SMLM). We further demonstrate how iCLEM can be integrated with computational modeling to discover structure-function relationships. Thus, we present iCLEM as a novel approach that complements existing CLEM methods and provides a generalizable framework that can be applied to any set of imaging modalities, provided suitable intrinsic fiducials can be identified.

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Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP-RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

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We previously constructed TheCellVision.org, a central repository for visualizing and mining data from yeast high-content imaging projects. At its inception, TheCellVision.org housed two high-content screening (HCS) projects providing genome-scale protein abundance and localization information for the budding yeast Saccharomyces cerevisiae, as well as a comprehensive analysis of the morphology of its endocytic compartments upon systematic genetic perturbation of each yeast gene. Here, we report on the expansion of TheCellVision.org by the addition of two new HCS projects and the incorporation of new global functionalities. Specifically, TheCellVision.org now hosts images from the Cell Cycle Omics project, which describes genome-scale cell cycle-resolved dynamics in protein localization, protein concentration, gene expression, and translational efficiency in budding yeast. Moreover, it hosts PIFiA, a computational tool for image-based predictions of protein functional annotations. Across all its projects, TheCellVision.org now houses >800,000 microscopy images along with computational tools for exploring both the images and their associated datasets. Together with the newly added global functionalities, which include the ability to query genes in any of the hosted projects using either yeast or human gene names, TheCellVision.org provides an expanding resource for single-cell eukaryotic biology.

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Quantitative characterisation of morphology and shape parameters of pozzolanic materials, as a fundamental problem of characterisation of pozzolanic materials, has received significant consideration in literature. Thus far, previous research works have not paid much attention to the circularity, roundness and solidity of pozzolanic materials including waste brick powder (WBP). This research makes a significant contribution on identification of circularity, roundness and solidity of WBP particles under milling conditions using quantitative image analysis. In particular, the goal was to interrogate the ball milling treatment variables for generating WBP using scanning electron microscopy (SEM) and image analysis. Under the milling conditions of changing sample masses introduced in ball mill, the average circularity values for the specimens were approximately 0.6 whilst the average solidity values for the specimens were approximately 0.71. Moreover, the average roundness values for the specimens were nearly 0.51. It was shown that the trends of shape parameters of WBP under changing fineness levels were not significant. The values of circularity, solidity and roundness in this study therefore collaborate to support the discoveries of hidden shape characteristics of WBP specimens and can tackle the overall behaviour of cement-based composites containing WBP. Quantitative image analysis was therefore observed to be capable of inheriting detailed information from SEM micrographs and remains one of the most outstanding approaches of generating shape parameters.

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Caenorhabditis elegans (C. elegans) is an excellent model system to study neurodegenerative diseases, such as Parkinson's disease, as it enables analysis of both neuron morphology and function in live animals. Multiple structural changes in neurons, such as cephalic dendrite morphological abnormalities, have been considered hallmarks of neurodegeneration in this model, but their relevance to changes in neuron function are not entirely clear. We sought to test whether hallmark morphological changes associated with chemically induced dopaminergic neuron degeneration, such as dendrite blebbing, breakage, and loss, are indicative of neuronal malfunction and result in changes in behavior. We adapted an established dopaminergic neuronal function assay by measuring paralysis in the presence of exogenous dopamine, which revealed clear differences between cat-2 dopamine deficient mutants, wildtype worms, and dat-1 dopamine abundant mutants. Next, we integrated an automated image processing algorithm and a microfluidic device to segregate worm populations by their cephalic dendrite morphologies. We show that nematodes with dopaminergic dendrite degeneration markers, such as blebbing or breakage, paralyze at higher rates in a dopamine solution, providing evidence that dopaminergic neurodegeneration morphologies are correlated with functional neuronal outputs.

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Folic acid (FA)-induced acute kidney injury (FA-AKI) is an increasingly prevalent rodent disease model involving the injection of a high dose of FA that culminates in renal FA crystal deposition and injury. However, the literature characterizing the FA-AKI model is sparse and dated in part due to the absence of a well-described methodology for the visualization and quantification of renal FA crystals. Using widely available materials and tools, we developed a straightforward and crystal-preserving histological protocol that can be coupled with automated imaging for renal FA crystal visualization and generated an automated macro for downstream crystal content quantification. The applicability of the method was demonstrated by characterizing the model in male and female C57BL6/JRj mice after 3 and 30 h of FA treatment. Kidneys from both sexes and timepoints showed a bimodal distribution of FA crystal deposition in the cortical and medullary regions while, compared with males, females exhibited higher renal FA crystal content at the 30-h timepoint accompanied by greater kidney weight and higher plasma urea. Despite comparable plasma phosphate concentrations, FA-AKI resulted in a substantially more elevated plasma intact fibroblast growth factor 23 (FGF23) in females, reflected by a similar pattern in osseous Fgf23 mRNA expression. Therefore, the presented method constitutes a valuable tool for the quantification of renal FA crystals, which can aid the mechanistic characterization of the FA-AKI model and serves as a means to control for confounding changes in FA crystallization when using the model for investigating early and prophylactic AKI therapeutic interventions.NEW & NOTEWORTHY Here, we describe a novel method for the visualization and quantification of renal folic acid (FA) crystals in the rodent FA-induced acute kidney injury (FA-AKI) model. The protocol involves a straightforward histological approach followed by fully automated imaging and quantification steps. Applicability was confirmed by showing that the FA-AKI model is sex-dependent. The method can serve as a tool to aid in characterizing FA-AKI and to control for studies investigating prophylactic therapeutic avenues using FA-AKI.

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In-situ liquid cell transmission electron microscopy (LCTEM) with electrical biasing capabilities has emerged as an invaluable tool for directly imaging electrode processes with high temporal and spatial resolution. However, accurately quantifying structural changes that occur on the electrode and subsequently correlating them to the applied stimulus remains challenging. Here, we present structural dissimilarity (DSSIM) analysis as segmentation-free video processing algorithm for locally detecting and quantifying structural change occurring in LCTEM videos. In this study, DSSIM analysis is applied to two in-situ LCTEM videos to demonstrate how to implement this algorithm and interpret the results. We show DSSIM analysis can be used as a visualization tool for qualitative data analysis by highlighting structural changes which are easily missed when viewing the raw data. Furthermore, we demonstrate how DSSIM analysis can serve as a quantitative metric and efficiently convert 3-dimensional microscopy videos to 1-dimenional plots which makes it easy to interpret and compare events occurring at different timepoints in a video. In the analyses presented here, DSSIM is used to directly correlate the magnitude and temporal scale of structural change to the features of the applied electrical bias. ImageJ, Python, and MATLAB programs, including a user-friendly interface and accompanying documentation, are published alongside this manuscript to make DSSIM analysis easily accessible to the scientific community.

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Ventral actin stress fibers (SFs) are a subset of actin SFs that begin and terminate at focal adhesion (FA) complexes. Ventral SFs can transmit forces from and to the extracellular matrix and serve as a prominent mechanosensing and mechanotransduction machinery for cells. Therefore, quantitative analysis of ventral SFs can lead to deeper understanding of the dynamic mechanical interplay between cells and their extracellular matrix (mechanoreciprocity). However, the dynamic nature and organization of ventral SFs challenge their quantification, and current quantification tools mainly focus on all SFs present in cells and cannot discriminate between subsets. Here we present an image analysis-based computational toolbox, called SFAlab, to quantify the number of ventral SFs and the number of ventral SFs per FA, and provide spatial information about the locations of the identified ventral SFs. SFAlab is built as an all-in-one toolbox that besides analyzing ventral SFs also enables the identification and quantification of (the shape descriptors of) nuclei, cells, and FAs. We validated SFAlab for the quantification of ventral SFs in human fetal cardiac fibroblasts and demonstrated that SFAlab analysis i) yields accurate ventral SF detection in the presence of image imperfections often found in typical fluorescence microscopy images, and ii) is robust against user subjectivity and potential experimental artifacts. To demonstrate the usefulness of SFAlab in mechanobiology research, we modulated actin polymerization and showed that inhibition of Rho kinase led to a significant decrease in ventral SF formation and the number of ventral SFs per FA, shedding light on the importance of the RhoA pathway specifically in ventral SF formation. We present SFAlab as a powerful open source, easy to use image-based analytical tool to increase our understanding of mechanoreciprocity in adherent cells.

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Introduction: Computer-based texture analysis provides objective data that can be extracted from medical images, including ultrasound images. One popular methodology involves the generation of a gray-level co-occurrence matrix (GLCM) from the image, and from that matrix, texture fractures can be extracted. Methods: We performed texture analysis on 280 ultrasound testicular images obtained from 70 dogs and explored the resulting texture data, by means of principal component analysis (PCA). Results: Various abnormal lesions were identified subjectively in 35 of the 280 cropped images. In 16 images, pinpoint-to-small, well-defined, hyperechoic foci were identified without acoustic shadowing. These latter images were classified as having "microliths." The remaining 19 images with other lesions and areas of non-homogeneous testicular parenchyma were classified as "other." In the PCA scores plot, most of the images with lesions were clustered. These clustered images represented by those scores had higher values for the texture features entropy, dissimilarity, and contrast, and lower values for the angular second moment and energy in the first principal component. Other data relating to the dogs, including age and history of treatment for prostatomegaly or chemical castration, did not show clustering on the PCA. Discussion: This study illustrates that objective texture analysis in testicular ultrasound correlates to some of the visual features used in subjective interpretation and provides quantitative data for parameters that are highly subjective by human observer analysis. The study demonstrated a potential for texture analysis in prediction models in dogs with testicular abnormalities.

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The shoot apical meristem is the plant tissue that produces the plant aerial organs such as flowers and leaves. To better understand how the shoot apical meristem develops and adapts to the environment, imaging developing shoot meristems expressing fluorescence reporters through laser confocal microscopy is becoming increasingly important. Yet, there are not many computational pipelines enabling a systematic and high-throughput characterization of the produced microscopy images. This chapter provides a simple method to analyze 3D images obtained through laser scanning microscopy and quantitatively characterize radially or axially symmetric 3D fluorescence domains expressed in a tissue or organ by a reporter. Then, it presents different computational pipelines aiming at performing high-throughput quantitative image analysis of gene expression in plant inflorescence and floral meristems. This methodology has notably enabled the quantitative characterization of how stem cells respond to environmental perturbations in the Arabidopsis thaliana inflorescence meristem and will open new avenues in the use of quantitative analysis of gene expression in shoot apical meristems. Overall, the presented methodology provides a simple framework to analyze quantitatively gene expression domains from 3D confocal images at the tissue and organ level, which can be applied to shoot meristems and other organs and tissues.

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Introduction: Imaging of human clinical formalin-fixed paraffin-embedded (FFPE) tissue sections provides insights into healthy and diseased states and therefore represents a valuable resource for basic research, as well as for diagnostic and clinical purposes. However, conventional light microscopy does not allow to observe the molecular details of tissue and cell architecture due to the diffraction limit of light. Super-resolution microscopy overcomes this limitation and provides access to the nanoscale details of tissue and cell organization. Methods: Here, we used quantitative multicolor stimulated emission depletion (STED) nanoscopy to study the nanoscale distribution of the nuclear phosphatidylinositol 4,5-bisphosphate (nPI(4,5)P2) with respect to the nuclear speckles (NS) marker SON. Results: Increased nPI(4,5)P2 signals were previously linked to human papillomavirus (HPV)-mediated carcinogenesis, while NS-associated PI(4,5)P2 represents the largest pool of nPI(4,5)P2 visualized by staining and microscopy. The implementation of multicolor STED nanoscopy in human clinical FFPE skin and wart sections allowed us to provide here the quantitative evidence for higher levels of NS-associated PI(4,5)P2 in HPV-induced warts compared to control skin. Discussion: These data expand the previous reports of HPV-induced increase of nPI(4,5)P2 levels and reveal for the first time the functional, tissue-specific localization of nPI(4,5)P2 within NS in clinically relevant samples. Moreover, our approach is widely applicable to other human clinical FFPE tissues as an informative addition to the classical histochemistry.

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Obesity and type 2 diabetes are major health burdens for which no effective therapy is available today. One treatment strategy could be to balance the metabolic functions of adipose tissue by regulating gene expressions using miRNAs. Here, we have loaded two anti-adipogenic miRNAs (miR26a and miR27a) into a pegylated lipid nanoparticle (PEG-LNP) formulation by a single-step microfluidic-assisted synthesis step. For the miRNA-loaded LNPs, the following system properties were determined: particle size, zeta potential, miRNA complexation efficiency, and cytotoxicity. We have used a human preadipocyte cell line to address the transfection efficiency and biological effects of the miRNA candidates at the gene and protein level. Our findings revealed that the upregulation of miR27a in preadipocytes inhibits adipogenesis by the downregulation of PPARγ and the reduction of lipid droplet formation. In contrast, miR26a transfection in adipocytes induced white adipocyte browning detected as the upregulation of uncoupling protein 1 (UCP1) as a marker of non-shivering thermogenesis. We conclude that the selective delivery of miRNAs by PEG-LNPs to adipocytes could offer new perspectives for the treatment of obesity and related metabolic diseases.

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Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based on fluorescence microscopy with conventional resolution, however, suggest only modest compaction differences (∼2-10×) between the active nuclear compartment (ANC) and inactive nuclear compartment (INC). Here, we present maps of nuclear landscapes with true-to-scale DNA densities, ranging from <5 to >300 Mbp/µm3. Maps are generated from individual human and mouse cell nuclei with single-molecule localization microscopy at ∼20 nm lateral and ∼100 nm axial optical resolution and are supplemented by electron spectroscopic imaging. Microinjection of fluorescent nanobeads with sizes corresponding to macromolecular assemblies for transcription into nuclei of living cells demonstrates their localization and movements within the ANC and exclusion from the INC.

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Collective cell movements contribute to tissue development and repair and spread metastatic disease. In epithelia, cohesive cell movements require reorganization of adherens junctions and the actomyosin cytoskeleton. However, the mechanisms that coordinate cell-cell adhesion and cytoskeletal remodeling during collective cell migration in vivo are unclear. We investigated the mechanisms of collective cell migration during epidermal wound healing in Drosophila embryos. Upon wounding, the cells adjacent to the wound internalize cell-cell adhesion molecules and polarize actin and the motor protein non-muscle myosin II to form a supracellular cable around the wound that coordinates cell movements. The cable anchors at former tricellular junctions (TCJs) along the wound edge, and TCJs are reinforced during wound closure. We found that the small GTPase Rap1 was necessary and sufficient for rapid wound repair. Rap1 promoted myosin polarization to the wound edge and E-cadherin accumulation at TCJs. Using embryos expressing a mutant form of the Rap1 effector Canoe/Afadin that cannot bind Rap1, we found that Rap1 signals through Canoe for adherens junction remodeling, but not for actomyosin cable assembly. Instead, Rap1 was necessary and sufficient for RhoA/Rho1 activation at the wound edge. The RhoGEF Ephexin localized to the wound edge in a Rap1-dependent manner, and Ephexin was necessary for myosin polarization and rapid wound repair, but not for E-cadherin redistribution. Together, our data show that Rap1 coordinates the molecular rearrangements that drive embryonic wound healing, promoting actomyosin cable assembly through Ephexin-Rho1, and E-cadherin redistribution through Canoe, thus enabling rapid collective cell migration in vivo.

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Neuroblastoma is the most common extracranial, malignant, solid tumor found in children. In more than one-third of cases, the tumor is in an advanced stage, with limited resectability. The treatment options include resection, with or without (neo-/) adjuvant therapy, and conservative therapy, the latter even with curative intent. Contrast-enhanced MRI is used for staging and therapy monitoring. Diffusion-weighted imaging (DWI) is often included. DWI allows for a calculation of the apparent diffusion coefficient (ADC) for quantitative assessment. Histological tumor characteristics can be derived from ADC maps. Monitoring the response to treatment is possible using ADC maps, with an increase in ADC values in cases of a response to therapy. Changes in the ADC value precede volume reduction. The usual criteria for determining the response to therapy can therefore be supplemented by ADC values. While these changes have been observed in neuroblastoma, early changes in the ADC value in response to therapy are less well described. In this study, we evaluated whether there is an early change in the ADC values in neuroblastoma under therapy; if this change depends on the form of therapy; and whether this change may serve as a prognostic marker. We retrospectively evaluated neuroblastoma cases treated in our institution between June 2007 and August 2014. The examinations were grouped as 'prestaging'; 'intermediate staging'; 'final staging'; and 'follow-up'. A classification of "progress", "stable disease", or "regress" was made. For the determination of ADC values, regions of interest were drawn along the borders of all tumor manifestations. To calculate ADC changes (∆ADC), the respective MRI of the prestaging was used as a reference point or, in the case of therapies that took place directly after previous therapies, the associated previous staging. In the follow-up examinations, the previous examination was used as a reference point. The ∆ADC were grouped into ∆ADCregress for regressive disease, ∆ADCstable for stable disease, and ∆ADC for progressive disease. In addition, examinations at 60 to 120 days from the baseline were grouped as er∆ADCregress, er∆ADCstable, and er∆ADCprogress. Any differences were tested for significance using the Mann-Whitney test (level of significance: p < 0.05). In total, 34 patients with 40 evaluable tumor manifestations and 121 diffusion-weighted MRI examinations were finally included. Twenty-seven patients had INSS stage IV neuroblastoma, and seven had INSS stage III neuroblastoma. A positive N-Myc expression was found in 11 tumor diseases, and 17 patients tested negative for N-Myc (with six cases having no information). 26 patients were assigned to the high-risk group according to INRG and eight patients to the intermediate-risk group. There was a significant difference in mean ADC values from the high-risk group compared to those from the intermediate-risk group, according to INRG. The differences between the mean ∆ADC values (absolute and percentage) according to the course of the disease were significant: between ∆ADCregress and ∆ADCstable, between ∆ADCprogress and ∆ADCstable, as well as between ∆ADCregress and ∆ADCprogress. The differences between the mean er∆ADC values (absolute and percentage) according to the course of the disease were significant: between er∆ADCregress and er∆ADCstable, as well as between er∆ADCregress and er∆ADCprogress. Forms of therapy, N-Myc status, and risk groups showed no further significant differences in mean ADC values and ∆ADC/er∆ADC. A clear connection between the ADC changes and the response to therapy could be demonstrated. This held true even within the first 120 days after the start of therapy: an increase in the ADC value corresponds to a probable response to therapy, while a decrease predicts progression. Minimal or no changes were seen in cases of stable disease.

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WNT/CTNNB1 signaling plays a critical role in the development of all multicellular animals. Here, we include both the embryonic stages, during which tissue morphogenesis takes place, and the postnatal stages of development, during which tissue homeostasis occurs. Thus, embryonic development concerns lineage development and cell fate specification, while postnatal development involves tissue maintenance and regeneration. Multiple tools are available to researchers who want to investigate, and ideally visualize, the dynamic and pleiotropic involvement of WNT/CTNNB1 signaling in these processes. Here, we discuss and evaluate the decisions that researchers need to make in identifying the experimental system and appropriate tools for the specific question they want to address, covering different types of WNT/CTNNB1 reporters in cells and mice. At a molecular level, advanced quantitative imaging techniques can provide spatio-temporal information that cannot be provided by traditional biochemical assays. We therefore also highlight some recent studies to show their potential in deciphering the complex and dynamic mechanisms that drive WNT/CTNNB1 signaling.

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