C. Vinegoni reconoce el apoyo de los Institutos Nacionales de Salud (NIH) de subvención 1-SR1-EB006432.

Las poblaciones de Drosophila melanogaster utilizados en este experimento son los siguientes: <ul style="list-style-type:none;"><li> elav-Gal4 (FBti0002575) </li><li> AP-Gal4md544 (FBal0051787) </li><li> UAS-srcEGFP (FBti0013990) </li><li> w1118 (FBal0018186) </li></ul> Para todos los experimentos, el sistema UAS-Gal4 se utilizó para sobreexpresar las buenas prácticas agrarias en el tejido de interés (es decir, las glándulas salivares o los discos del ala). Más específicamente, las moscas hembras transgénicas con la adecuada Gal4 fueron cruzadas a UAS-EGFP compañeros de transgénicos. Las cruces fueron criados a 25 ° C en un incubador humidificado. Cuando las pupas de comenzar a llenar los viales (aproximadamente 5-6 días después del inicio de la cruz), que fueron seleccionados para la fluorescencia de GFP en la etapa de blanco prepupal (0-1 horas después de la formación pupa) y recogidos por tomografía. <ol><li> Establecer cruzamientos apropiados y posterior a 25 ° C </li><li> 5-6 días después del inicio de la cruz, seleccione la progenie fluorescentes para imágenes. Utilice una brocha húmeda para la eliminación suave de la prepupas blancos desde los laterales del vial. </li><li> Ponga prepupas en una gota de agua o PBS coloca en una placa de Petri para eliminar las partículas de alimentos y el pegamento que se refiere al caso de pupa y hace que la muestra autofluorescentes. Limpie suavemente con un pincel. Si el envejecimiento de las pupas es necesario, lugar elegido prepupas blanco en una placa de Petri con una toalla de papel húmeda (una cámara húmeda) o en los lados de una cubeta limpia con la comida volar. Esto asegurará que no hay suficiente humedad, para permitir el desarrollo adecuado de los animales. </li></ol> Drosophila montaje <ol><li> Pegue la parte inferior de la caja de pupa en un tubo capilar de vidrio (800 micras de diámetro) de tal manera que la mosca anterior / posterior del eje se orienta paralelo al tubo. </li><li> Fijar el tubo capilar verticalmente en un escenario de rotación. </li><li> Ajuste el eje de inclinación de la fase de rotación para que el eje de rotación de modo que sea paralelo a las columnas de píxeles de la imagen CCD. </li></ol> Determinación adelante Modelo <ol><li> Fijar una pupa en un vaso capilar y montaje como se describió anteriormente </li><li> Llenar un tubo capilar de sílice (100 micrones, OD) con un tinte fluorescente (Cy5.5) </li><li> Inserte el tubo capilar dentro de la pupa a 45 grados con respecto al eje anterior / posterior </li><li> Iluminar la pupa con un haz de láser de excitación con una lente de apertura numérica baja. Recoge imágenes de fluorescencia transiluminación más de 360 ​​grados de rotación de la pupa a lo largo de su eje vertical. </li><li> Ajustar los datos experimentales con un modelo apropiado hacia adelante </li></ol> Imágenes de las glándulas salivales <ol><li> Montar una pupa expresar GFP en las glándulas salivales como se describió anteriormente </li><li> Iluminar con un haz de excitación y se acumulan en la transiluminación la señal de fluorescencia de GFP en 360 grados de rotación de la pupa a lo largo de su eje vertical. </li><li> Reconstruir los datos obtenidos utilizando el modelo de avance </li></ol> Time-lapse de imágenes <ol><li> Montar un blanco GFP prepupa expresar en el disco imaginal de ala </li><li> Mantener el ambiente húmedo y de imagen a temperatura ambiente para evitar la deshidratación de la pupa. </li><li> Coloque una velocidad de obturación para evitar la iluminación continua </li><li> Iluminar con un haz de excitación y se acumulan en la transiluminación la señal de fluorescencia de GFP en 360 grados de rotación de la pupa a lo largo de su eje vertical. </li><li> Reconstruir los datos obtenidos utilizando el modelo a seguir. </li></ol> Histología <ol><li> Diseccionar las glándulas salivales del SNC / discos ojo en 1x PBS. </li><li> Para reparar tejidos en formol al 4%, 1x PEM (tuberías de 100 mm, 1 mM EGTA, 1 mM MgCl 2) de 20 minutos a temperatura ambiente. </li><li> Quitar la fijación y montaje en Vectashield con DAPI (Vector) </li><li> Imagen con un microscopio de fluorescencia. </li></ol> Time-Lapse Histología <ol><li> Recoger prepupas blanca y la que en una cámara húmeda. </li><li> Recoger las pupas en las distintas fases </li><li> Pinchazo de la envoltura pupal dos veces (0 horas después de la formación pupario) con una aguja fina (insectos pin 000, FST) antes de la fijación. </li><li> Fix pupas en el 8% de formaldehído, 1x PEM por 6-8 horas a temperatura ambiente con agitación suave. </li><li> Después de la eliminación de la fijación y enjuague con PBS 1x, cortar la pupa fija con una cuchilla de dos piezas perpendiculares al eje A / P; </li><li> Sumergir las dos piezas en Vectashield con DAPI (Vector) y montaje en portaobjetos cubreobjetos (Lab-Tek) para imágenes. </li><li> Adquirir imágenes con un microscopio confocal de fluorescencia. </li></ol><img src="/files/ftp_upload/1510/1510fig1thumb.jpg" alt="la figura 1" border="0" /> Figura 1. Por favor, <a href="https://www.jove.com/files/ftp_upload/1510/1510fig1.jpg">haga clic aquí</a> para ver alArger versión de la figura 1.

<ol>
	<li>Ntziachristos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+and+listening+to+light:+the+evolution+of+whole-body+photonic+imaging.">Looking and listening to light: the evolution of whole-body photonic imaging.</a> Nat. Biotechnol. 23, 313-320 (2005).</li><li>J, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography+as+a+tool+for+3D+microscopy+and+gene+expression+studies.">Optical projection tomography as a tool for 3D microscopy and gene expression studies.</a> Science. 296, 541-545 (2002).</li><li>Dodt, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultramicroscopy:+three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nat. Methods. 4, 331-336 (2007).</li><li>Vinegoni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+Drosophila+melanogaster+pupae+with+mesoscopic+fluorescence+tomography.">In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography.</a> Nat. Methods. 5, 45-47 (2008).</li></ol>

The authors have nothing to disclose.

En vivo-reconstrucciones de la envoltura pupal y las glándulas salivales que expresan GFP-de una D. melanogaster prepupa (Barra de escala, de 500 micras) con la histología correspondiente (azul, DAPI, de color verde, la fluorescencia de GFP) se muestra en la Fig. 1 (a). Time-lapse serie de imágenes de D. melanogaster ala discos imaginales se muestra en la Fig. 1 (b). Las imágenes se adquieren a partir de un espécimen vivo sola, en cuatro diferentes puntos de tiempo (0,3.0, 3.5 y 6.5 horas). En la ...

mesoscopic fluorescence tomography for in-vivo imaging of developing drosophila

Visualizar el desarrollo de la formación de órganos, así como progresión de la enfermedad y el tratamiento a menudo depende en gran medida la capacidad de interrogar ópticamente cambios moleculares y funcionales en los organismos vivos intactos. La mayoría de los métodos ópticos actuales imágenes son inadecuadas para las imágenes en las dimensiones que se encuentran entre los límites de la penetración de la microscopía óptica moderna (0,5-1mm) y los límites impuestos por difusión de macroscopía óptica (&gt; 1 cm) [1]. Por lo tanto, muchos organismos modelo importantes, por ejemplo, los insectos, embriones de animales o en las extremidades de animales pequeños, siguen siendo inaccesibles para los in-vivo de imágenes ópticas. Aunque hay un creciente interés hacia el desarrollo de métodos de resolución de nanómetros de imagen óptica, no ha habido muchos esfuerzos exitosos en el mejoramiento de la profundidad de penetración de imágenes. La capacidad de realizar en vivo de imágenes de microscopía allá de los límites es, de hecho, se reunió con las dificultades asociadas con la dispersión de fotones presentes en los tejidos. Los recientes esfuerzos para embriones toda la imagen, por ejemplo, [2,3] requieren un tratamiento químico especial de la muestra, para borrar de la dispersión, un procedimiento que hace que sean adecuadas sólo para la post-mortem de imágenes. Estos métodos sin embargo la evidencia la necesidad de que las muestras de imágenes más grandes que los que habitualmente se permite el microscopio de dos fotones o confocal, sobre todo en la biología del desarrollo y de descubrimiento de fármacos. Hemos desarrollado una nueva técnica de imagen óptica llamada tomografía de fluorescencia mesoscópicas [4], que apropiado para no invasiva en vivo de imágenes en dimensiones de 1 mm a 5 mm. La resolución del método de intercambios para la profundidad de la penetración, pero ofrece un rendimiento de imagen sin precedentes, tomografía y se ha desarrollado para añadir el tiempo como una nueva dimensión en las observaciones de la biología del desarrollo (y posiblemente otras áreas de la investigación biológica), impartiendo la capacidad de la imagen de la evolución de la fluorescencia etiquetados respuestas a través del tiempo. Como tal, puede acelerar los estudios de las dependencias morfológicos o funcionales de las mutaciones del gen o los estímulos externos, y lo más importante puede capturar la imagen completa del desarrollo o la función del tejido, permitiendo longitudinal lapso de tiempo de visualización del mismo organismo, en desarrollo. La técnica utiliza un microscopio de laboratorio modificados y multi-proyección de la iluminación para recoger datos en las proyecciones de 360 ​​grados. Se aplica la simplificación de Fermi a la solución de Fokker-Planck de la ecuación de transporte de fotones, en combinación con los principios de la óptica geométrica con el fin de construir un plan de inversión realista adecuada para el rango mesoscópico. Esto permite a los in-vivo de todo el cuerpo de visualización de la falta de transparencia de estructuras tridimensionales en las muestras de hasta varios milímetros de tamaño. Hemos demostrado la actuación en vivo de la técnica por imágenes en tres dimensiones de estructuras de desarrollo de los tejidos de Drosophila en vivo y después de la morfogénesis de las alas en la Drosophila pupas opaco en tiempo real de más de seis horas consecutivas.

Watch this Scientific Journal Video about Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila at JoVE.com

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscópica fluorescencia tomografía opera más allá de los límites de penetración de los tejidos de seccionamiento microscopía de fluorescencia. La técnica se basa en la iluminación multi-proyección y una descripción del transporte de fotones. Se demuestra en vivo de todo el cuerpo visualización en 3D de la morfogénesis de las buenas prácticas agrarias-que expresa ala discos imaginales en<em&gt; Drosophila melanogaster.</em

Mesoscópica fluorescencia Tomografía por En vivo Imágenes de desarrollo Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

mesoscopic-fluorescence-tomography-for-vivo-imaging-developing

Research

JoVE Journal

Biology

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

10.6K vistas.  Massachusetts General Hospital. Mesoscópica fluorescencia tomografía opera más allá de los límites de penetración de los tejidos de seccionamiento microscopía de fluorescencia. La técnica se basa en la iluminación multi-proyección y una descripción del transporte de fotones. Se demuestra en vivo de todo el cuerpo visualización en 3D de la morfogénesis de las buenas prácticas agrarias-que expresa ala discos imaginales en Drosophila melanogaster.

Video: Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

La electroporación in vivo sola célula en el cerebro intacto desarrollo

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Investigación

Biología

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

La normalización nació para tomografía de fluorescencia de proyección óptica de imágenes de todo corazón

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse de imágenes de mitosis después de la transfección de siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Preparación de desarrollo y de adultos Drosophila Los cerebros y retinas de imágenes en vivo

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

En vivo-La centrifugación de embriones de Drosophila

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

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 ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Análisis rápido y exploración de microscopía de fluorescencia de Imágenes

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Drosophila Preparación e Imagen longitudinal de la función cardiaca in vivo utilizando microscopía de coherencia óptica (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /(T1)Magnetic Resonance Imaging

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible synthetic procedures. In this case, starting from FeCl3 and citrate trisodium salt, iron oxide nanoparticles coated with citric acid are obtained in 10 min in the microwave. These nanoparticles present a small core size of 4.2 &#177; 1.1 nm and a hydrodynamic size of 7.5 &#177; 2.1 nm. Moreover, they have a high longitudinal relaxivity (r1) value of 11.9 mM-1&#183;s-1 and a modest transversal relaxivity value (r2) of 22.9 mM-1&#183;s-1, which results in a low r2/r1 ratio of 1.9. These values enable positive contrast generation in magnetic resonance imaging (MRI) instead of negative contrast, commonly used with iron oxide nanoparticles. In addition, if a 68GaCl3 elution from a 68Ge/68Ga generator is added to the starting materials, a nano-radiotracer doped with 68Ga is obtained. The product is obtained with a high radiolabeling yield (&#62; 90%), regardless of the initial activity used. Furthermore, a single purification step renders the nano-radiomaterial ready to be used in vivo.

Synthesis of 68Ga Core-doped Iron Oxide Nanoparticles for Dual Positron Emission Tomography /T1Magnetic Resonance Imaging

Here, we present a protocol to obtain 68Ga core-doped iron oxide nanoparticles via fast microwave-driven synthesis. The methodology renders PET/(T1)MRI nanoparticles with radiolabeling efficiencies higher than 90% and radiochemical purity of 99% in a 20-min synthesis.

Síntesis de 68Ga dopado a base de óxido de hierro nanopartículas de doble emisión de positrones/proyección de imagen de resonancia magnética (T1)

Here, we describe a microwave synthesis to obtain iron oxide nanoparticles core-doped with 68Ga. Microwave technology enables fast and reproducible ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.