Los autores desean agradecer Alexandra Opie de asistencia y apoyo técnico, James Barton para proporcionar narración de vídeo, y Ramona Flatz y Joellen Sweeney por aparecer en el video adjunto. Este trabajo fue apoyado por el MJ Murdock Charitable Trust (Grant No. 2012205 a JED).

1. El Sistema de Microscopio estéreo Nota: Aunque este protocolo es fácilmente adaptable a prácticamente cualquier sistema de microscopio estéreo acoplado a una cámara digital con la capacidad de adquisición de vídeo, los detalles se proporcionan en el sistema utilizado en nuestro laboratorio (Tabla de Materiales / Equipos). <ol><li> Adquirir vídeo digital usando un microscopio estéreo trinocular acoplado a una cámara digital comercial. </li><li> Para acoplar la cámara digital comercial al puerto trinocular del microscopio estereoscópico, retire el ½ x C-monte del puerto phototube del microscopio estereoscópico y reemplazarlo con un C-mount 1X. </li><li> Montar un acoplador de la cámara digital (43 mm de rosca) a la C-mount 1X. </li><li> Monte dos anillos reductores, 58 mm a 48 mm, y 48 mm a 43 mm, hasta el acoplador de la cámara para cerrar la conexión del acoplador de la cámara digital a un kit adaptador de lente para la cámara digital. </li><li> Monte la cámara digital al kit adaptador de lente. </li><li> Adquirir video con el microscopio óptico y el zoom óptico de la serie cámara digital para un aumento combinado de aproximadamente 12 aumentos (30 fotogramas por segundo, 640 x 480 píxeles). Nota: El aumento del microscopio estéreo debe ser compensado de acuerdo con la recién reconfigurada 1X C-monte del puerto trinocular. </li></ol> 2. Imaging Drosophila larvas de tercer estadio <ol><li> Cinta un marcador permanente a la placa de la platina negro de un microscopio estereoscópico acoplado a una cámara digital, de manera que el lado de la tapa marcador ocupa aproximadamente ⅓ a ¼ del campo de visión vertical observada en el monitor LCD de la cámara. Utilice tapas de marcadores como la etapa para realizar las imágenes de las larvas porque vienen en una variedad de colores que se pueden utilizar para el código de color y diferenciar los genotipos de las larvas a ser visualizada. </li><li> Delimita el campo de visión observada en el monitor LCD de la cámara digital en la superficie de la parte superior con un marcador de punta finamarcador. </li><li> Seleccionar un tercer instar de la larva de la imagen. Los criterios para seleccionar las larvas de tercer instar era la longitud del cuerpo, la salida de la fuente de alimento durante la fase larvaria del ciclo de vida, la presencia de espiráculos anterior y posterior, y la estructura de los ganchos inferiores del aparato de la boca 11. Asegúrese de que la larva es limpio lavándolo bien con agua. </li><li> Ilumine la etapa superior marcador permanente desde arriba con la luz de un sistema de iluminación por fibra óptica. Ajuste el ángulo de la luz incidente para proporcionar la iluminación óptima. </li><li> Enfoque del microscopio en el borde de la parte superior marcador permanente. Comience la adquisición de vídeo digital. </li><li> Coloque la larva en el lado de la tapa de marcador de aproximadamente 75 ° fuera del eje vertical, justo fuera del campo de visión, con la parte anterior de la larva que mira hacia el campo de visión (Figura 1). Nota: La colocación de la larva en el lado de la tapa de marcador permite a la cámara para registrar el movimiento de le larva desde una perspectiva lateral. Ayuda a mantener la larva húmedo con agua para que no se caen del lado de la tapa del marcador. Se debe tener cuidado, sin embargo, no usar demasiada agua como cantidades excesivas se adherirán a la larva que se arrastra a través del campo. </li><li> Clave suavemente y empujar a la larva con un pincel pequeño para coaccionar a arrastrarse por el campo de visión. Sea paciente como las larvas raramente cooperar ya menudo tienen que ser devueltos al punto de partida muchas veces antes de que se arrastran directamente a través del campo. </li><li> Record aproximadamente 10-15 minutos de ininterrumpida video digital y de cultivos y retirar todo el material innecesario después de la adquisición con el software de edición de vídeo digital. </li></ol> 3. Imaging Drosophila Adulto <ol><li> Coloque un solo adulto de Drosophila en una desechable de 1,5 ml cubeta de poliestireno espectroscópico. Nota: CO 2 para la anestesia de adultos Drosophila inmediatamente antes de un comporprotocolo de análisis conductuales puede comprometer los resultados 12. Se recomienda que los adultos Drosophila disponer de un plazo de 24 horas para recuperarse de CO 2 para la anestesia antes de realizar una prueba de comportamiento 13. </li><li> Conecte el extremo de la cubeta con una pequeña bola de algodón. Asegúrese de que la bola de algodón está lleno suficientemente apretado para ocupar el espacio de gran capitalización y limita la marcha para el compartimiento de reducción del volumen de la cubeta. </li><li> Colocar la cubeta en la placa blanca etapa de un microscopio estéreo y alinear correctamente el compartimiento de volumen reducido de la cubeta con el campo de visión observada en el monitor LCD de la cámara digital. </li><li> Iluminar la cubeta desde arriba con la luz de un sistema de iluminación de fibra óptica. Ajuste el ángulo de la luz incidente para proporcionar la iluminación óptima. </li><li> Enfoque el microscopio y comenzar a adquirir de vídeo digital. </li><li> Record aproximadamente 30-45 min de ininterrumpida imágenes de vídeo digital y de cultivos y eliminar todos los innecesariosimágenes post-adquisición con el software de edición de vídeo digital. </li></ol>

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	<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. 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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>

Los autores han declarado que no existen conflictos de intereses.

Fuerza Drosophila melanogaster &#39;s como un sistema modelo para el estudio de la función del sistema nervioso proviene en gran medida de la convergencia de las poderosas herramientas genéticas disponibles y la amplia gama de ensayos de comportamiento robusto desarrollado. Aquí presentamos una técnica sencilla y ampliamente accesible de microscopía para la adquisición de vídeo digital de alta calidad de Drosophila adulta y fenotipos de la locomotora de larvas desde una perspectiva lateral. Hemo...

La fruta común mosca Drosophila melanogaster es un potente sistema de modelo experimental para el estudio de la función del sistema nervioso 1-3. Conservación evolutiva de la estructura y función del sistema nervioso con los seres humanos, así como la facilidad de la manipulación genética y una amplia gama de herramientas genéticas hace Drosophila el organismo estreno para modelar enfermedades neurodegenerativas humano 4. Las mutaciones genéticas que causan la disfunción del sistema nervioso a menudo resultan en larvas mutante viable y adultos de Drosophila con una alteración de la locomoción. Fenotipos observados en el sistema nervioso mutantes defectuosos incluyen la frecuencia de la locomoción, la coordinación aberrante, y los movimientos espásticos en adultos, así como déficits en la contracción peristáltica musculatura de la pared corporal, y parálisis parcial de las larvas reducen. Estos fenotipos se han explotado en el desarrollo de pantallas genéticos de alto rendimiento y ensayos de locomoción de larvas mutante 5, 6 y adultos 7-10 Drosophila dirigido a cuantificar el deterioro locomoción y la identificación de genes necesarios para la función del sistema nervioso. Si bien estos enfoques son extremadamente útiles para la cuantificación de los comportamientos locomotores larvas y adultos, fracasan para transmitir información cualitativa acerca de cada comportamiento aberrante específico. Por ejemplo, mientras que las larvas de tercer estadio mutante puede exhibir parámetros de locomoción alterados en un ensayo de comportamiento, puede ser poco claro si este es el resultado de alteraciones en las contracciones peristálticas rítmicas durante el ciclo de rastreo, la falta general de coordinación, o parálisis parcial del cuerpo posterior musculatura de la pared. Aquí se describe una técnica simple y ampliamente accesible de microscopía para la adquisición de vídeo digital de alta calidad de Drosophila adulta y fenotipos de la locomotora de larvas desde una perspectiva lateral. El vídeo digital adquirido desde una perspectiva lateral permite la observación directa y el análisis de las diferencias sutiles en locomotive comportamientos desde una orientación de vista lateral más informativo.

<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 es un potente sistema de modelo experimental para el estudio de la función del sistema nervioso. Las mutaciones genéticas que causan la disfunción del sistema nervioso a menudo producen larvas viables y adultos que tienen fenotipos de locomoción defectuosos que son difíciles de describir adecuadamente con el texto o completamente representar con una sola imagen fotográfica. Modos actuales de la publicación científica, sin embargo, apoyan la presentación de los medios de comunicación de vídeo digital como material complementario para acompañar a un manuscrito. Aquí se describe una técnica simple y ampliamente accesible de microscopía para la adquisición de vídeo digital de alta calidad tanto de Drosophila fenotipos larvas y adultos desde una perspectiva lateral. Vídeo de la locomoción de larvas y adultos desde una vista lateral es ventajoso porque permite la observación y el análisis de las distinciones sutiles y variaciones en los comportamientos aberrantes de locomotoras. Hemos utilizado con éxito la técnica para visualizar y cuantificar aberrant arrastrándose comportamientos en larvas de tercer estadio, además de fenotipos mutantes adultas y comportamientos que incluyen la preparación.

Hemos utilizado con éxito esta técnica para adquirir y cuantificar el comportamiento de las larvas fenotipo asociado con la pérdida de la función del gen estatmina (Figura 2) 14. El gen codifica una proteína estatmina reguladora de microtúbulos que divide los dímeros de tubulina de piscinas de tubulina soluble, y se une a los microtúbulos y promueve su desmontaje 15,16. Se requiere la función Estatmina para mantener la integridad de los microtúbulos en l...

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

Aquí se describe una técnica de microscopía simple y ampliamente accesibles para la adquisición de vídeo digital de alta calidad de Drosophila adulta y fenotipos mutantes larvas desde una perspectiva lateral.

Adquisición de vídeo de alta calidad digital de<em&gt; Drosophila</em&gt; Comportamientos larvas y adultos desde una perspectiva lateral

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

acquisition-high-quality-digital-video-drosophila-larval-adult

Research

JoVE Journal

Neuroscience

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

8.7K vistas.  Willamette University.  Aquí se describe una técnica de microscopía simple y ampliamente accesibles para la adquisición de vídeo digital de alta calidad de Drosophila adulta y fenotipos mutantes larvas desde una perspectiva lateral. 

Video: 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.

Live-proyección de imagen de la<em&gt; Drosophila</em&gt; Pupal Ojo

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

Investigación

Biología del Desarrollo

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.

Vídeo de alta resolución de Seguimiento de la locomoción en adultos de 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 ...

Biología

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.

Un sistema de imágenes ajustable de alta definición para estudios conductuales de adultos con 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 ...

Comportamiento

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.

Imágenes y análisis de la orientación del tejido y la dinámica del crecimiento en el desarrollo de la Drosophila Epithelia durante las etapas pupales

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.

Una técnica simple para evaluar la actividad locomotora en 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.

Imágenes in vivo de larvas de Drosophila intactos a nivel sub-celular Resolución

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

<meta charset="utf8"></head><body>Imágenes en vivo extendidas de la morfogénesis de las patas de la pupa de Drosophila sin pupario

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 destacado: Descubriendo estructuras epiteliales dinámicas durante el desarrollo 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.

Imágenes en vivo a largo plazo del desarrollo de la pata de la pupa de Drosophila después de la extracción del pupario

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.

Imágenes en vivo de lapso de tiempo y cuantificación de la dinámica de ramas dendríticas rápidas en el desarrollo de neuronas 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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Imágenes en vivo de lapso de tiempo y cuantificación de la dinámica de ramas dendríticas rápidas en el desarrollo de neuronas drosophila