Technologies bringing young Zebrafish from a niche field to the limelight

Fundamental life science and pharmaceutical research are continually striving to provide physiologically relevant context for their biological studies. Zebrafish present an opportunity for high-content screening (HCS) to bring a true in vivo model system to screening studies. Zebrafish embryos and young larvae are an economical, human-relevant model organism that are amenable to both genetic engineering and modification, and direct inspection via microscopy. The use of these organisms entails unique challenges that new technologies are overcoming, including artificial intelligence (AI). In this perspective article, we describe the state-of-the-art in terms of automated sample handling, imaging, and data analysis with zebrafish during early developmental stages. We highlight advances in orienting the embryos, including the use of robots, microfluidics, and creative multi-well plate solutions. Analyzing the micrographs in a fast, reliable fashion that maintains the anatomical context of the fluorescently labeled cells is a crucial step. Existing software solutions range from AI-driven commercial solutions to bespoke analysis algorithms. Deep learning appears to be a critical tool that researchers are only beginning to apply, but already facilitates many automated steps in the experimental workflow. Currently, such work has permitted the cellular quantification of multiple cell types in vivo, including stem cell responses to stress and drugs, neuronal myelination and macrophage behavior during inflammation and infection. We evaluate pro and cons of proprietary versus open-source methodologies for combining technologies into fully automated workflows of zebrafish studies. Zebrafish are poised to charge into HCS with ever-greater presence, bringing a new level of physiological context

Fundamental noise dynamics in cascaded-order Brillouin lasers

The dynamics of cascaded-order Brillouin lasers make them ideal for applications such as rotation sensing, highly coherent optical communications, and low-noise microwave signal synthesis. Remark- ably, when implemented at the chip-scale, recent experimental studies have revealed that Brillouin lasers can operate in the fundamental linewidth regime where optomechanical and quantum noise sources dominate. To explore new opportunities for enhanced performance, we formulate a simple model to describe the physics of cascaded Brillouin lasers based on the coupled mode dynamics governed by electrostriction and the fluctuation-dissipation theorem. From this model, we obtain analytical formulas describing the steady state power evolution and accompanying noise properties, including expressions for phase noise, relative intensity noise and power spectra for beat notes of cascaded laser orders. Our analysis reveals that cascading modifies the dynamics of intermediate laser orders, yielding noise properties that differ from single-mode Brillouin lasers. These modifications lead to a Stokes order linewidth dependency on the coupled order dynamics and a broader linewidth than that predicted with previous single order theories. We also derive a simple analytical expression for the higher order beat notes that enables calculation of the Stokes linewidth based on only the relative measured powers between orders instead of absolute parameters, yielding a method to measure cascaded order linewidth as well as a prediction for sub-Hz operation. We validate our results using stochastic numerical simulations of the cascaded laser dynamics.Comment: 18 pages, 9 figure

A versatile, automated and high-throughput drug screening platform for zebrafish embryos

Zebrafish provide a unique opportunity for drug screening in living animals, with the fast developing, transparent embryos allowing for relatively high-throughput, microscopy-based screens. However, the limited availability of rapid, flexible imaging and analysis platforms has limited the use of zebrafish in drug screens. We have developed an easy-to-use, customisable automated screening procedure suitable for high-throughput phenotype-based screens of live zebrafish. We utilised the WiScan® Hermes High Content Imaging System to rapidly acquire brightfield and fluorescent images of embryos, and the WiSoft® Athena Zebrafish Application for analysis, which harnesses an Artificial Intelligence-driven algorithm to automatically detect fish in brightfield images, identify anatomical structures, partition the animal into regions, and exclusively select the desired side-oriented fish. Our initial validation combined structural analysis with fluorescence images to enumerate GFP-tagged haematopoietic stem and progenitor cells in the tails of embryos, which correlated with manual counts. We further validated this system to assess the effects of genetic mutations and x-ray irradiation in high content using a wide range of assays. Further, we performed simultaneous analysis of multiple cell types using dual fluorophores in high throughput. In summary, we demonstrate a broadly applicable and rapidly customisable platform for high-content screening in zebrafish

Eph-ephrin signaling modulated by polymerization and condensation of receptors

Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization–condensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour

Income Migration and the Spatial Redistribution of Poverty and Income in the Mountain West Region during the 1990s

The purpose of this research is to understand how rapid population growth in the Mountain West region in the U.S. during the 1990s has affected the spatial distribution of income and poverty. Population growth in the Mountain West during the 1990s was principally through the mechanism of internal and international migration. In previous research we found that much of this growth, and the income growth that went along with it, was highly concentrated in only a few, high-amenity counties. In this paper we use IRS/Census Bureau data on income migration to identify migration of different income groups and how their migration during the 1990s has affected spatial patterns of income and poverty. Counties with the lowest levels of poverty had the largest increase in poverty during the decade. However, somewhat paradoxically, these counties also experienced the largest increase in income due to outmigration of lower income people. In general, migration patterns between 1995 and 2000 within and from beyond the Mountain West region tended to reinforce existing spatial patterns of poverty with higher income people moving to lower poverty counties and lower income people moving to higher poverty counties.Migrations et redistribution spatiale des revenus et de la pauvreté dans l'Ouest américain durant les années 1990. L'objectif de cette recherche est de comprendre comment la rapide croissance de population de l'Ouest intérieur des États-Unis durant les années 1990 a affecté la distribution spatiale des revenus et de la pauvreté. La croissance de population dans l'Ouest montagneux est due principalement aux mouvements migratoires interrégionaux et internationaux. Dans une recherche précédente, nous avions montré qu'une bonne partie de cette croissance, et la croissance des revenus qui l'accompagnait, était fortement concentrée dans quelques comtés bien dotés en aménités. Dans cet article, nous utilisons des données du recensement pour identifier la migration de divers groupes selon leur niveau de revenus, et comment leur migration dans les années 1990 a pu affecter les schémas spatiaux de pauvreté et de revenus. Les comtés aux taux de pauvreté les plus faibles ont connu la plus forte croissance des niveaux de pauvreté, bien qu'ayant aussi enregistré les plus forts accroissements des revenus du fait de départs de catégories pauvres. De manière générale, les schémas migratoires de la période 1995-2000 dans l'Ouest montagneux ont eu tendance à renforcer les schémas préexistants : les migrants plus aisés s'installent dans les comtés à faible pauvreté et les migrants les moins fortunés dans des comtés à plus fort taux de pauvreté.Shumway J. Matthew, Otterstrom Samuel M. Income Migration and the Spatial Redistribution of Poverty and Income in the Mountain West Region during the 1990s . In: Espace, populations, sociétés, 2003-1. Diversité des populations d'Amérique du Nord. pp. 15-28

Environmental Hazards as Disamenities: Selective Migration and Income Change in the United States from 2000-2010

An emerging area of migration research is the complex relationship between migration and environmental hazards, broadly defined. Environmental hazards are best viewed through a vulnerability lens, which has two components. The first is exposure - the frequency and duration of the hazardous events-and the second is adaptive capacity - the ability of communities to mitigate, deflect, or absorb the effects of exposure. Because migration is selective of individuals and places, it changes both the population's size and composition, thus affecting its exposure and adaptive capacity. In this article we examine how migration varies among sets of counties that experience significantly different exposures to all environmental hazards in the United States. We create an environmental hazards impact index in an attempt to measure the impacts of environmental hazards at the county level over a period of years. We found that counties that experience the greatest impacts from environmental hazards are losing income as a result of the migration. In counties with the highest impacts, income is lost through both net outmigration as well as income loss through out-migrants having higher incomes than in-migrants