Os autores gostariam de agradecer Alexandra Opie para assistência técnica e apoio, James Barton para fornecer vídeo narração e Ramona Flatz e Joellen Sweeney para aparecer no vídeo em anexo. Este trabalho foi financiado pela Fundação de Caridade MJ Murdock (Grant n º 2012205 para JED).

1 O sistema de microscópio estéreo Nota: Apesar de este protocolo é facilmente adaptável a praticamente qualquer sistema de microscópio estéreo acoplado a uma câmera digital com a capacidade de adquirir vídeo, são fornecidos detalhes sobre o sistema utilizado em nosso laboratório (Tabela de Materiais / Equipamentos). <ol><li> Adquirir vídeo digital utilizando um microscópio estéreo trinocular acoplado a uma câmera digital comercial. </li><li> A fim de acoplar a câmera digital comercial à porta trinocular do microscópio estéreo, remova o ½x C-mount do porto fototubo do microscópio estéreo e substituí-lo por um 1X C-mount. </li><li> Montar um acoplador câmera digital (43 mm Rosca) ao 1X C-mount. </li><li> Monte dois anéis abaixadoras, 58 mm a 48 mm, e 48 mm a 43 mm, para o acoplador câmera para colmatar a conexão da câmera digital a um acoplador kit adaptador de lente para a câmera digital. </li><li> Monte a câmera digital para o kit adaptador de lente. </li><li> Adquirir vídeo com a ampliação do microscópio e zoom óptico da câmera digital definida para uma ampliação combinada de cerca de 12X (30 quadros por segundo, 640 x 480 pixels). Nota: A ampliação do microscópio estéreo deve ser compensado de acordo com o recém-reconfigurado 1X C-mount da porta trinocular. </li></ol> 2 Imagem Drosophila terceiro instar Larvas <ol><li> Tape um marcador permanente para a placa preta palco de um microscópio estéreo acoplado a uma câmera digital, de modo que o lado da tampa marcador ocupa cerca de ⅓ a ¼ do campo de visão vertical observado no monitor LCD da câmera. Use tops de marcadores como o palco para realizar imagem larval porque eles vêm em uma variedade de cores que pode ser usado para código de cor e diferenciar os genótipos de larvas sendo fotografada. </li><li> Delimitar o campo de visão observada no monitor LCD da câmara digital na superfície do topo do marcador com uma ponta finamarcador. </li><li> Selecione uma terceira larva instar a imagem. Os critérios para a selecção de larvas de terceiro instar é o comprimento do corpo, a emergência da fonte de alimentação durante a fase larval do ciclo de vida, a presença dos anteriores e posteriores espiráculos, e a estrutura dos ganchos inferiores do aparelho de boca 11. Garantir a larva está limpo, lavando-o bem em água. </li><li> Iluminar o palco marcador permanente topo a partir de cima com a luz a partir de um sistema de iluminação de fibra óptica. Ajuste o ângulo da luz incidente para proporcionar a iluminação ideal. </li><li> Concentre o microscópio sobre a borda de topo marcador permanente. Comece a aquisição de vídeo digital. </li><li> Coloque a larva do lado da tampa de marcador de aproximadamente 75 ° a partir do eixo vertical, fora do campo de visão, sendo o anterior da larva virado para o campo de visão (figura 1). Observação: Colocação da larva no lado da tampa de marcador permite que a câmara da ficha movimento the larva de uma perspectiva lateral. Ela ajuda a manter a larva úmido com água, para que não caia do lado da tampa do marcador. Deve ser tomado cuidado, porém, para não usar muita água, como quantidades excessivas vai aderir à larva como rasteja em todo o campo. </li><li> Gentilmente cutucar a larva com um pequeno pincel para forçá-lo a engatinhar em todo o campo de visão. Seja paciente como as larvas raramente cooperam e muitas vezes tem que ser devolvido ao ponto de partida muitas vezes antes que rastejar em frente do campo. </li><li> Grave aproximadamente 10-15 min de vídeo ininterrupto e cultura digital e remover todas as filmagens desnecessária pós-aquisição com software de edição de vídeo digital. </li></ol> 3 Imagens Adulto Drosophila <ol><li> Coloque um único adulto Drosophila num descartável 1,5 ml tina espectroscópica de poliestireno. Nota: CO 2 para anestesia de adulto Drosophila imediatamente antes de uma behavprotocolo de análise ioral pode comprometer os resultados 12. É recomendado que adultos Drosophila dispor de um prazo de 24 horas para se recuperar de CO 2 para anestesia antes de executar em um teste comportamental 13. </li><li> Ligue o final da cuvete com uma pequena bola de algodão. Garantir a bola de algodão é embalado apertado o suficiente para ocupar o espaço grande boné e limita a voar para o compartimento volume reduzido da tina. </li><li> Coloque a tina na placa de fase branca de um microscópio estéreo e alinhar adequadamente o compartimento de volume reduzido da tina com o campo de visão observada no monitor LCD da câmara digital. </li><li> Iluminar o cadinho a partir de cima com a luz a partir de um sistema de iluminação de fibra óptica. Ajuste o ângulo da luz incidente para proporcionar a iluminação ideal. </li><li> Concentre-se o microscópio e começar a adquirir vídeo digital. </li><li> Grave aproximadamente 30-45 min de vídeo ininterrupto e cultura digital e remover todos os desnecessárioscenas pós-aquisição com software de edição de vídeo digital. </li></ol>

<ol>
	<li>Zhang, B., Freeman, M. R., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila neurobiology: a laboratory manual. , (2010).</li><li>Frank, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+for+studying+synaptic+development,+function,+and+plasticity+using+Drosophila+as+a+model+system.">New approaches for studying synaptic development, function, and plasticity using Drosophila as a model system.</a> J Neurosci. 33, 17560-17568 (2013).</li><li>Mudher, A., Newman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila : a toolbox for the study of neurodegenerative disease. , (2008).</li><li>Bilen, J., Bonini, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+human+neurodegenerative+disease.">Drosophila as a model for human neurodegenerative disease.</a> Annu Rev Genet. 39, 153-171 (2005).</li><li>Jakubowski, B. R., Longoria, R. A., Shubeita, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+throughput+and+sensitive+method+correlates+neuronal+disorder+genotypes+to+Drosophila+larvae+crawling+phenotypes.">A high throughput and sensitive method correlates neuronal disorder genotypes to Drosophila larvae crawling phenotypes.</a> Fly (Austin). 6, 303-308 (2012).</li><li>Caldwell, J. C., Miller, M. M., Wing, S., Soll, D. R., Eberl, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+larval+locomotion+in+Drosophila+chordotonal+organ+mutants).">Dynamic analysis of larval locomotion in Drosophila chordotonal organ mutants).</a> Proc Natl Acad Sci U S A. 100, 16053-16058 (2003).</li><li>Jahn, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+early+locomotor+abnormalities+in+a+Drosophila+model+of+Alzheimer's+disease.">Detection of early locomotor abnormalities in a Drosophila model of Alzheimer's disease.</a> J Neurosci Methods. 197, 186-189 (2011).</li><li>Donelson, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+positional+tracking+for+long-term+analysis+of+Drosophila+sleep+and+locomotion+using+the+&amp;#34;tracker&amp;#34;+program.">High-resolution positional tracking for long-term analysis of Drosophila sleep and locomotion using the &amp;#34;tracker&amp;#34; program.</a> PLoS ONE. 7, e37250 (2012).</li><li>Slawson, J. B., Kim, E. Z., Griffith, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+video+tracking+of+locomotion+in+adult+Drosophila+melanogaster.">High-resolution video tracking of locomotion in adult Drosophila melanogaster.</a> J Vis Exp. (24), (2009).</li><li>Colomb, J., Reiter, L., Blaszkiewicz, J., Wessnitzer, J., Brembs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+tracking+and+analysis+of+adult+Drosophila+locomotion+in+Buridan's+paradigm+with+and+without+visual+targets.">Open source tracking and analysis of adult Drosophila locomotion in Buridan's paradigm with and without visual targets.</a> PLoS ONE. 7, e42247 (2012).</li><li>Demerec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Drosophila. , (1965).</li><li>Barron, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaesthetising+Drosophila+for+behavioural+studies.">Anaesthetising Drosophila for behavioural studies.</a> J Insect Physiol. 46, 439-442 (2000).</li><li>Greenspan, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fly pushing : the theory and practice of Drosophila genetics.. , (2004).</li><li>Duncan, J. E., Lytle, N. K., Zuniga, A., Goldstein, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Microtubule+Regulatory+Protein+Stathmin+Is+Required+to+Maintain+the+Integrity+of+Axonal+Microtubules+in+Drosophila.">The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila.</a> 8, e683244 (2013).</li><li>Belmont, L. D., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+protein+that+interacts+with+tubulin+dimers+and+increases+the+catastrophe+rate+of+microtubules.">Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules.</a> Cell. 84, 623-631 (1996).</li><li>Cassimeris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oncoprotein+18/stathmin+family+of+microtubule+destabilizers.">The oncoprotein 18/stathmin family of microtubule destabilizers.</a> Curr Opin Cell Biol. 14, 18-24 (2002).</li></ol>

Os autores declaram que não existem interesses conflitantes.

Força Drosophila melanogaster &#39;s como um sistema modelo para estudar a função do sistema nervoso deriva em grande parte da convergência das poderosas ferramentas genéticas disponíveis e da ampla gama de ensaios comportamentais robustos desenvolvidos. Aqui apresentamos uma técnica de microscopia simples e amplamente acessível para a aquisição de vídeo digital de alta qualidade de Drosophila adulto e fenótipos locomotiva larvais de uma perspectiva lateral. Temos utilizado com sucesso esta...

A fruta comum mosca Drosophila melanogaster é um poderoso sistema de modelo experimental para o estudo da função do sistema nervoso 1-3. Conservação evolutiva da estrutura e função do sistema nervoso, com os seres humanos, assim como a facilidade da manipulação genética e uma vasta variedade de ferramentas genéticas torna o organismo Drosophila estreia para modelar doenças neurodegenerativas humano 4. As mutações genéticas que causam disfunção do sistema nervoso, muitas vezes resultam em larvas mutante viável e adulto de Drosophila com locomoção diminuída. Fenótipos observados no sistema nervoso mutantes defeituosos incluem taxa de locomoção, coordenação aberrante, e os movimentos espásticas em adultos, assim como os défices de contracção peristáltica da musculatura da parede do corpo, e paralisia parcial de larvas reduzida. Estes fenótipos foram explorados no desenvolvimento de telas genéticas de alto rendimento e ensaios de locomoção das larvas 5 mutante, 6 e adulto de Drosophila 7-10 destina-se a quantificar a disfunção locomoção e a identificação de genes necessários para a função do sistema nervoso. Embora essas abordagens são extremamente úteis para quantificar comportamentos locomotiva larvas e adultos, eles não conseguem transmitir informações qualitativas sobre cada comportamento aberrante específico. Por exemplo, enquanto as larvas de terceiro estádio mutante pode apresentar parâmetros de locomoção alterados em um ensaio comportamental, pode não ser claro se este é o resultado de alterações nas contrações peristálticas rítmicas durante o ciclo de rastreamento, em geral falta de coordenação ou paralisia parcial do corpo posterior musculatura da parede. Aqui nós descrevemos uma técnica de microscopia simples e amplamente acessível para a aquisição de vídeo digital de alta qualidade de Drosophila adulto e fenótipos locomotiva larvais de uma perspectiva lateral. O vídeo digital adquirido de uma perspectiva lateral, permite a observação direta e análise de distinções sutis em locomotive comportamentos de uma orientação mais informativa visão lateral.

<table><tbody><tr><td>Trinocular Stereozoom Microscope</td><td>Olympus Corporation</td><td>SZ6145TR</td><td>&amp;frac12; C-mount was removed and replaced with 1X C-mount</td></tr><tr><td>1X C-mount</td><td>Leeds Precision Instruments</td><td>LSZ-1XCMT2</td><td></td></tr><tr><td>Digital Camera Coupler (43 mm thread)</td><td>Qioptiq Imaging Solutions</td><td>25-70-10-02</td><td></td></tr><tr><td>58 mm to 48 mm Step Down Ring</td><td>B&amp;amp;H Video</td><td>GBSDR5848</td><td></td></tr><tr><td>48 mm to 43 mm Step Down Ring</td><td>B&amp;amp;H Video</td><td>GBSDR4843</td><td></td></tr><tr><td>Lensmate Adapter Kit for Canon G10</td><td>LensMateOnline.com</td><td></td><td></td></tr><tr><td>Canon PowerShot G10 Digital Camera</td><td>Canon U.S.A., Inc.</td><td></td><td></td></tr><tr><td>1.5 ml Spectroscopic Polysterene Cuvette</td><td>Denville Scientific</td><td>U8650-4</td><td></td></tr></tbody></table>

acquisition of high-quality digital video of drosophila larval and adult behaviors from a lateral perspective

Drosophila melanogaster é um poderoso sistema de modelo experimental para o estudo da função do sistema nervoso. As mutações genéticas que causam a disfunção do sistema nervoso muitas vezes produzem larvas viáveis ​​e adultos que têm locomoção fenótipos defeituosos que são difíceis de descrever adequadamente com texto ou representar completamente com uma imagem fotográfica única. Atuais modos de publicação científica, no entanto, apoiar a apresentação de mídia de vídeo digital como material suplementar para acompanhar um manuscrito. Aqui nós descrevemos uma técnica de microscopia simples e amplamente acessível para a aquisição de vídeo digital de alta qualidade de ambos os fenótipos de Drosophila larvas e adultos a partir de uma perspectiva lateral. Vídeo de locomoção das larvas e adultos a partir de uma visão lateral é vantajosa porque permite a observação e análise das distinções sutis e variações nos comportamentos aberrantes de locomotivas. Temos utilizado com sucesso a técnica de visualizar e quantificar aberrant rastejando comportamentos em larvas de terceiro estádio, além de fenótipos e comportamentos mutantes adultos incluindo o grooming.

Nós utilizamos com sucesso desta técnica para adquirir e quantificar o fenótipo comportamental larval associada com a perda de função do gene stathmin (Figura 2) 14. O gene codifica uma proteína stathmin regulamentar microtúbulos que particiona dímeros de tubulina de piscinas de tubulina solúvel, e liga os microtúbulos e promove a sua desmontagem 15,16. Stathmin função é necessária para manter a integridade dos microtúbulos nos axónios dos nervos p...

Watch this Scientific Journal Video about Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective at JoVE.com

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Aqui nós descrevemos uma técnica de microscopia simples e amplamente acessível para a aquisição de vídeo digital de alta qualidade de Drosophila adulto e fenótipos mutantes larvais de uma perspectiva lateral.

Aquisição de Vídeo de Alta Qualidade Digital de<em&gt; Drosophila</em&gt; Larvais e adultas Comportamentos de uma perspectiva lateral

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

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Research

JoVE Journal

Neuroscience

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

8.7K visualizações.  Willamette University.  Aqui nós descrevemos uma técnica de microscopia simples e amplamente acessível para a aquisição de vídeo digital de alta qualidade de Drosophila adulto e fenótipos mutantes larvais de uma perspectiva lateral. 

Vídeo: Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Vivo-imaging da<em&gt; Drosophila</em&gt; Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Biologia do Desenvolvimento

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

De alta resolução de vídeo de Rastreamento Locomotion em Adult Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Biologia

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but effective method to observe and document the behavior or morphology of flies. Flies were placed in a translucent observation chamber c.a. &#216;15 x 5mm (no food inside) or &#216;15 x 12 mm (with an 8 mm-high piece of food inside). After covering with an ultraviolet (UV)/clear filter with high light transmittance, the chamber was placed under a 5-50x zoom stereo microscope, and mini light-emitting diode (LED) video lights were placed on both sides of the microscope to illuminate the chamber to obtain uniform, soft, bright, and nearly shadow-free light. Then, a compact digital camera with 3-5x optical zoom, which can record 1080 P high-definition or higher resolution video (at a frame rate of &#8805;30 fps), was connected to the eyepiece of microscope through a bracket, and photographs or videos were taken through the eyepiece. By adjusting the zoom knob of the zoom stereo microscope, it was very easy to track the flies and take panoramic or detailed close-up images as needed, while the camera recorded everything under the microscope. Because the flies can stay at any position in the chamber, they can be observed and recorded from all directions. The photographs or videos taken are of good image quality. This method can be used both for scientific research and teaching.

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

This protocol describes how to make a simple adult Drosophila behavior observation chamber, and how to take high-definition photographs/videos of the morphology or behavior of different types of fruit flies in the observation chamber through relatively simple and affordable methods.

Um sistema de imagem de alta definição ajustável para estudos comportamentais de adultos drosophila

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but ...

Comportamento

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Imagem e Análise da Dinâmica de Orientação e Crescimento tecidual na Epitélia Drosophila em Desenvolvimento durante os estágios pupal

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Uma técnica simples para avaliar a atividade locomotora em Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Na imagem in vivo de larvas Drosophila Intact no Sub-celular Resolução

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

<meta charset="utf8"></head><body>Imagem ao vivo estendida da morfogênese da perna de pupa de Drosophila sem pupúrio

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which fate-determined cells form three-dimensional organismal shapes remains unclear. Recent advances in confocal microscopy have facilitated efforts to observe cell dynamics during development through live imaging. The Drosophila melanogaster pupa is ideal for live imaging due to its immobility, transparent pupal cuticle, and the availability of fluorescent reporter lines. A primary challenge for imaging is the puparium, the cuticle surrounding the pupa, which obstructs optical imaging. While previous methods involved either partial or complete removal of the puparium, maintaining pupal viability for extended periods after this procedure has remained challenging. Here, a simple method is presented for days-long live imaging of the Drosophila leg during the pupal stage, involving complete puparium removal. The method includes removing the puparium from a pupa adhered to double-sided tape, followed by assembling a small chamber on a glass-bottom dish to enclose the pupa and a drop of water. This setup is straightforward, reliable, and supports extended pupal survival by preventing desiccation. Long-term live imaging of the Drosophila pupa has significantly contributed to capturing how the adult leg undergoes dramatic three-dimensional structural changes over 2-3 days. These changes include the transient formation of an intriguing structure (the Parthenon-like structure) by epithelial cells, rapid tissue narrowing, joint formation, and bristle elongation. This method is applicable to the observation of various tissues and can potentially be combined with other techniques, such as optical gene induction, to advance the understanding of cell dynamics during the final shape formation of tissues in the pupal stage.

<meta charset="utf8"></head><body>Autor em destaque: Descobrindo estruturas epiteliais dinâmicas durante o desenvolvimento de Drosophila

Here, a protocol is presented for long-term live imaging of the Drosophila leg during the pupal stage, involving complete removal of the puparium. This protocol can also be applied to other tissues.

Imagem ao vivo de longo prazo do desenvolvimento da perna de pupa de Drosophila após a remoção do pupário

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which ...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

Tempo-lapso de imagem ao vivo e quantificação da dinâmica da filial dendrítica rápida no desenvolvimento de neurônios de Drosophila

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

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