Real-time monitoring of an endogenous Fgf8a gradient attests to its role as a morphogen during zebrafish gastrulation.

Morphogen gradients impart positional information to cells in a homogenous tissue field. Fgf8a, a highly conserved growth factor, has been proposed to act as a morphogen during zebrafish gastrulation. However, technical limitations have so far prevented direct visualization of the endogenous Fgf8a gradient and confirmation of its morphogenic activity. Here, we monitor Fgf8a propagation in the developing neural plate using a CRISPR/Cas9-mediated EGFP knock-in at the endogenous fgf8a locus. By combining sensitive imaging with single-molecule fluorescence correlation spectroscopy, we demonstrate that Fgf8a, which is produced at the embryonic margin, propagates by diffusion through the extracellular space and forms a graded distribution towards the animal pole. Overlaying the Fgf8a gradient curve with expression profiles of its downstream targets determines the precise input-output relationship of Fgf8a-mediated patterning. Manipulation of the extracellular Fgf8a levels alters the signaling outcome, thus establishing Fgf8a as a bona fide morphogen during zebrafish gastrulation. Furthermore, by hindering Fgf8a diffusion, we demonstrate that extracellular diffusion of the protein from the source is crucial for it to achieve its morphogenic potential.

QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy.

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.

Crocetin Prevents RPE Cells from Oxidative Stress through Protection of Cellular Metabolic Function and Activation of ERK1/2.

Age-related macular degeneration (AMD) is a leading cause for visual impairment in aging populations with limited established therapeutic interventions available. Oxidative stress plays an essential role in the pathogenesis of AMD, damaging the retinal pigment epithelium (RPE), which is essential for the function and maintenance of the light-sensing photoreceptors. This study aimed to evaluate the effects of crocetin, one of the main components of Saffron, on an in vitro RPE model of tert-butyl hydroperoxide (TBHP) induced oxidative stress using ARPE19 cells. The effects of crocetin were assessed using lactate de-hydrogenase (LDH) and ATP assays, as well as immunocytochemistry for cell morphology, junctional integrity, and nuclear morphology. The mechanism of crocetin action was determined via assessment of energy production pathways, including mitochondrial respiration and glycolysis in real-time as well as investigation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and distribution. Our results show that crocetin pre-treatment protects ARPE19 cells from TBHP-induced LDH release, intracellular ATP depletion, nuclear condensation, and disturbance of junctional integrity and cytoskeleton. The protective effect of crocetin is mediated via the preservation of energy production pathways and activation of ERK1/2 in the first minutes of TBHP exposure to potentiate survival pathways. The combined data suggest that a natural antioxidant, such as crocetin, represents a promising candidate to prevent oxidative stress in RPE cells and might halt or delay disease progression in AMD.

Correction: Karimi, P., et al. Crocetin Prevents RPE Cells from Oxidative Stress through Protection of Cellular Metabolic Function and Activation of ERK1/2. Int. J. Mol. Sci. 2020, 21, 2949.

The authors wish to make the following correction to Figure 6B of this article [...].

Corrigendum: Bessel Beam Illumination Reduces Random and Systematic Errors in Quantitative Functional Studies Using Light-Sheet Microscopy.

[This corrects the article DOI: 10.3389/fncel.2018.00315.].

Bessel Beam Illumination Reduces Random and Systematic Errors in Quantitative Functional Studies Using Light-Sheet Microscopy.

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a method of choice to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20-fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams.

IR spectroscopic methods for the investigation of the CO release from CORMs.

Carbon monoxide (CO) is a toxic gas for mammals, and despite this fact, it is naturally produced in these organisms and has been proven to be beneficial in medical treatments, too. Therefore, CO-releasing molecules (CORMs) are intensively developed to administer and dose CO for physiological applications. Nearly all of these compounds are metal carbonyl complexes, which have been synthesized and investigated. However, for most of these CORMs, the exact reaction mechanisms of CO release is not completely elucidated, although it is of utmost importance. The widely used myoglobin assay for testing the CO release has several disadvantages, and therefore, different methods have to be applied to characterize CORMs. In this work, different setups of IR absorption spectroscopy are used to analyze and quantify the CO release during the decay of various CORMs: IR spectroscopy of the gas phase is applied to follow the CO liberation, and attenuated total reflection (ATR) IR spectroscopy is used to record the decay of the metal carbonyl. IR spectroscopy supported by DFT calculations yields valuable insights in the CO release reaction mechanism. The focus is set on two different CORMs: CORM-2 (Ru2(CO)(6)Cl(4)) and on the photoactive CORM-S1 (photoCORM [Fe(CO)2(SCH2CH2NH2)2]). Our results indicate that the CO liberation from CORM-2 strongly depends on sodium dithionite, which is required for the commonly applied myoglobin assay and that CORM-S1 loses all its bound CO molecules upon irradiation with blue light.