RESUMO
This chapter describes an ImageJ/Fiji automated macro approach to estimate synapse densities in 2D fluorescence confocal microscopy images. The main step-by-step imaging workflow is explained, including example macro language scripts that perform all steps automatically for multiple images. Such tool provides a straightforward method for exploratory synapse screenings where hundreds to thousands of images need to be analyzed in order to render significant statistical information. The method can be adapted to any particular set of images where fixed brain slices have been immunolabeled against validated presynaptic and postsynaptic markers.
Assuntos
Encéfalo/diagnóstico por imagem , Processamento de Imagem Assistida por Computador/métodos , Animais , Encéfalo/citologia , Corantes Fluorescentes/química , Imuno-Histoquímica/métodos , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Camundongos , Microscopia Confocal/métodos , Neurônios/citologia , Software , Coloração e Rotulagem/métodos , Sinapses , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/análise , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/imunologiaRESUMO
Morphogenesis is the fundamental developmental process during which the embryo body is formed. Proper shaping of different body parts depends on cellular divisions and rearrangements in the growing embryo. Understanding three-dimensional shaping of organs is one of the basic questions in developmental biology. Here, we consider the early stages of pectoral fin development in zebrafish, which serves as a model for limb development in vertebrates, to study emerging shapes during embryogenesis. Most studies on pectoral fin are concerned with late stages of fin development when the structure is morphologically distinct. However, little is known about the early stages of pectoral fin formation because of the experimental difficulties in establishing proper imaging conditions during these stages to allow long-term live observation. In this protocol, we address the challenges of pectoral fin imaging during the early stages of zebrafish embryogenesis and provide a strategy for three-dimensional shape analysis of the fin. The procedure outlined here is aimed at studying pectoral fin during the first 24 h of its formation corresponding to the time period between 24 and 48 h of zebrafish development. The same principles could also be applied when studying three-dimensional shape establishment of other embryonic structures. We first discuss the imaging procedure and then propose strategies of extracting quantitative information regarding fin shape and dimensions.
Assuntos
Nadadeiras de Animais/diagnóstico por imagem , Embrião não Mamífero/diagnóstico por imagem , Imageamento Tridimensional/métodos , Microscopia Intravital/métodos , Imagem com Lapso de Tempo/métodos , Nadadeiras de Animais/embriologia , Animais , Animais Geneticamente Modificados , Desenvolvimento Embrionário , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Microscopia de Fluorescência/métodos , Software , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteína Vermelha FluorescenteRESUMO
This chapter provides an ImageJ/Fiji automated macro approach to remove the vitelline membrane autofluorescence in live Drosophila embryo movies acquired in a 4D (3D plus time) fashion. The procedure consists in a segmentation pipeline that can cope with different relative intensities of the vitelline membrane autofluorescence, followed by a developed algorithm that adjusts the extracted outline selection to the shape deformations that naturally occur during Drosophila embryo development. Finally, the fitted selection is used to clear the external glowing halo that, otherwise, would obscure the visualization of the internal embryo labeling upon projection or 3D rendering.
