C. Vinegoni reconnaît le soutien de National Institutes of Health (NIH) accorder une-SR1-EB006432.

Les stocks de Drosophila melanogaster utilisés dans cette expérience sont les suivantes: <ul style="list-style-type:none;"><li> elav-Gal4 (FBti0002575) </li><li> AP-Gal4md544 (FBal0051787) </li><li> SAMU-srcEGFP (FBti0013990) </li><li> w1118 (FBal0018186) </li></ul> Pour toutes les expériences, le système UAS-Gal4 a été utilisé pour surexprimer la GFP dans les tissus d&#39;intérêt (c&#39;est à dire des glandes salivaires ou les disques d&#39;aile). Plus précisément, les mouches femelles transgéniques avec les Gal4 appropriés ont été croisés pour SAMU-EGFP compagnons transgéniques. Les croisements ont été élevés à 25 ° C dans un incubateur humidifié. Lorsque les nymphes commencent peupler les flacons (environ 5-6 jours après le début de la croix), ils ont été sélectionnés pour fluorescence de la GFP au stade blanche prépupal (0-1 h après la formation puparium) et collectées pour la tomographie par imagerie. <ol><li> Mettre en place des croisements appropriés et à l&#39;arrière à 25 ° C </li><li> 5-6 jours après l&#39;initiation de la croix, sélectionnez la descendance fluorescentes pour l&#39;imagerie. Utilisez un pinceau humide pour enlever en douceur de l&#39;prénymphes blanches sur les côtés de la fiole. </li><li> Mettez prénymphes dans une goutte d&#39;eau ou du PBS placé dans une boîte de Pétri pour enlever les particules de nourriture et de la colle qui couvre le cas de nymphe et rend le spécimen autofluorescente. Nettoyez délicatement avec un pinceau. Si le vieillissement de la pupe est nécessaire, endroit choisi prénymphes blanc dans une boîte de Pétri avec une serviette en papier humide (une chambre humide) ou sur les côtés d&#39;une cuvette propre avec de la nourriture volée. Cela permettra d&#39;assurer qu&#39;il ya suffisamment d&#39;humidité, afin de permettre le bon développement de l&#39;animal. </li></ol> Drosophile montage <ol><li> Collez la partie inférieure de la coque de nymphose dans un tube capillaire en verre (800 microns de diamètre) de telle sorte que antérieure / postérieure de la mouche axe est orienté parallèlement au tube. </li><li> Fixer le tube capillaire à la verticale sur une étape de rotation. </li><li> Ajuster l&#39;axe de basculement de la phase de rotation pour que l&#39;axe de rotation parallèle à des colonnes de pixels de l&#39;imagerie CCD. </li></ol> Détermination Forward modèle <ol><li> Fixer une chrysalide dans un capillaire en verre et montez comme décrit précédemment </li><li> Remplir un tube capillaire de silice (100 microns, DO) avec un colorant fluorescent (Cy5.5) </li><li> Insérez le tube capillaire à l&#39;intérieur de la pupe à 45 degrés par rapport à l&#39;axe antérieur / postérieur </li><li> Illuminer la nymphe avec un faisceau laser d&#39;excitation avec un objectif d&#39;ouverture numérique faible. Collecter des images de fluorescence transillumination plus de 360 ​​degrés de rotation de la pupe long de son axe vertical. </li><li> Ajuster les données expérimentales avec un modèle approprié avant </li></ol> Glandes salivaires Imaging <ol><li> Monter une nymphe exprimer la GFP dans les glandes salivaires, comme décrit précédemment </li><li> Éclairer avec un faisceau d&#39;excitation et de recueillir en transillumination du signal de fluorescence GFP plus de 360 ​​degrés de rotation de la pupe long de son axe vertical. </li><li> Reconstruire les données acquises en utilisant le modèle de l&#39;avant </li></ol> Imagerie time-lapse <ol><li> Monter une GFP blanche prénymphe exprimer dans les disques imaginaux de l&#39;aile </li><li> Gardez à l&#39;environnement d&#39;imagerie et humide à température ambiante pour éviter la déshydratation de la nymphe. </li><li> Placer un obturateur pour éviter un éclairage continu </li><li> Éclairer avec un faisceau d&#39;excitation et de recueillir en transillumination du signal de fluorescence GFP plus de 360 ​​degrés de rotation de la pupe long de son axe vertical. </li><li> Reconstruire les données acquises en utilisant le modèle de l&#39;avant. </li></ol> Histologie <ol><li> Disséquer les glandes salivaires du SNC / disques oeil dans du PBS 1X. </li><li> Fixer les tissus dans le formol à 4%, 1x PEM (Pipes 100 mM, 1 mM EGTA, 1 mM MgCl 2) pendant 20min à température ambiante. </li><li> Retirer de fixation et de montage dans Vectashield au DAPI (Vector) </li><li> Image avec un microscope à fluorescence. </li></ol> Time-Lapse Histologie <ol><li> Recueillir prénymphes blancs et les mettre en scène dans une chambre humide. </li><li> Recueillir les pupes à différents stades de développement </li><li> Piquer la coque de nymphose deux fois (0 h après la formation puparium) avec une aiguille fine (insecte broches 000, FST) avant la fixation. </li><li> Fix pupes dans formaldéhyde à 8%, 1x PEM pour 6-8 heures à température ambiante avec agitation douce. </li><li> Après le retrait de fixation et de rinçage avec du PBS 1x, couper la nymphe fixes avec une lame tranchante en deux morceaux perpendiculairement à l&#39;axe A / P; </li><li> Immerger les deux pièces en Vectashield au DAPI (Vector) et monter sur des lames couvre-objet (Lab-Tek) pour l&#39;imagerie. </li><li> Acquérir les images avec un microscope à fluorescence confocale. </li></ol><img src="/files/ftp_upload/1510/1510fig1thumb.jpg" alt="Figure 1" border="0" /> Figure 1. S&#39;il vous plaît <a href="https://www.jove.com/files/ftp_upload/1510/1510fig1.jpg">cliquez ici</a> pour voir alVersion Arger de la figure 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.

In vivo reconstructions de l&#39;affaire de nymphe et les glandes salivaires exprimant la GFP d&#39;un D. melanogaster prénymphe (barre d&#39;échelle, 500 microns) avec l&#39;histologie correspondante (en bleu, la coloration DAPI; vert, fluorescence de la GFP) est montré dans la figure 1 (a). Time-lapse série d&#39;imagerie de D. melanogaster aile disques imaginaux est indiqué dans la Fig.1 (b). Les images sont acquises à partir d&#39;un seul spécimen vivant, à quatre points de temps différent...

mesoscopic fluorescence tomography for in-vivo imaging of developing drosophila

Visualiser la formation des organes en développement ainsi que progession et le traitement de la maladie repose souvent lourdement sur la capacité à interroger optiquement les changements moléculaires et fonctionnelles dans les organismes vivants intacts. La plupart des méthodes existantes d&#39;imagerie optique sont insuffisants pour l&#39;imagerie à des dimensions qui se trouvent entre les limites de pénétration de la microscopie optique moderne (0,5-1mm) et les limites imposées par diffusion de la macroscopie optique (&gt; 1 cm) [1]. Ainsi, de nombreux organismes modèles importants, par exemple insectes, des embryons animaux ou des extrémités du petit animal, restent inaccessibles pour l&#39;imagerie in vivo optique. Bien qu&#39;il y ait un intérêt croissant envers le développement de nanomètre résolution méthodes d&#39;imagerie optique, il n&#39;ya pas eu beaucoup d&#39;efforts réussi à améliorer la profondeur de pénétration d&#39;imagerie. La capacité d&#39;effectuer l&#39;imagerie in vivo au delà des limites de microscopie est en fait rencontré des difficultés liées à la diffusion de photons présents dans les tissus. Les efforts récents pour embryons image entière par exemple [2,3] nécessitent un traitement chimique spécial de l&#39;échantillon, pour les faire disparaître de la diffusion, une procédure qui permet de les utiliser uniquement pour l&#39;imagerie post-mortem. Ces méthodes cependant des preuves de la nécessité pour les spécimens d&#39;imagerie plus grandes que celles habituellement autorisés par microscopie à deux photons ou confocale, en particulier dans la biologie du développement et de découverte de médicaments. Nous avons développé une nouvelle technique d&#39;imagerie optique de fluorescence nommée mésoscopique tomographie [4], qui convient pour les non-invasives d&#39;imagerie in vivo à des dimensions de 1mm à 5mm. La méthode de résolution des échanges pour la profondeur de pénétration, mais offre sans précédent de l&#39;imagerie tomographique performance et il a été développé pour ajouter du temps comme une dimension nouvelle dans les observations de biologie du développement (et éventuellement d&#39;autres domaines de la recherche biologique) en donnant la possibilité à l&#39;image de l&#39;évolution de la fluorescence taggés réponses au fil du temps. Comme telle, elle peut accélérer les études des dépendances morphologiques ou fonctionnelles sur les mutations des gènes ou des stimuli extérieurs, et peut surtout, capter l&#39;image complète du développement ou de la fonction des tissus en permettant longitudinale time-lapse de visualisation de la même, l&#39;organisme en développement. La technique utilise un microscope de laboratoire modifié et multi-projection d&#39;illumination pour recueillir des données à 360 degrés des projections. Elle s&#39;applique à la simplification de Fermi à la solution de Fokker-Plank de l&#39;équation de transport des photons, combinée avec les principes de l&#39;optique géométrique afin de construire un système d&#39;inversion réalistes adapté à une large échelle mésoscopique. Cela permet in vivo du corps entier de visualisation de la non-transparence des structures tridimensionnelles dans des échantillons de plusieurs millimètres dans la taille. Nous avons démontré la performance in vivo de la technique par imagerie en trois dimensions des structures de développement de tissus in vivo chez la drosophile et en suivant la morphogenèse des ailes dans l&#39;opaque pupes de drosophile en temps réel de plus de six heures consécutives.

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

Mésoscopique de fluorescence de tomographie opère au-delà des limites de la pénétration des tissus sectionnement microscopie à fluorescence. La technique est basée sur la multi-projection de l&#39;illumination et une description de transport des photons. Nous démontrons in vivo du corps entier de visualisation 3D de la morphogenèse des ailes exprimant la GFP dans les disques imaginaux<em&gt; Drosophila melanogaster.</em

Mésoscopique fluorescence tomographie pour In vivo L&#39;imagerie du développement Drosophile</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 vues.  Massachusetts General Hospital. Mésoscopique de fluorescence de tomographie opère au-delà des limites de la pénétration des tissus sectionnement microscopie à fluorescence. La technique est basée sur la multi-projection de l'illumination et une description de transport des photons. Nous démontrons in vivo du corps entier de visualisation 3D de la morphogenèse des ailes exprimant la GFP dans les disques imaginaux Drosophila melanogaster.

Vidéo: 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.

L&#39;électroporation cellule unique in vivo dans le cerveau intact développement

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

Recherche

Biologie

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.

Normalisation Né pour tomographie par fluorescence de projection optique pour l&#39;imagerie cardiaque entier

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.

Imagerie time-lapse de la mitose après transfection 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.

Préparation de développement et de l&#39;adulte Drosophile Cerveaux et la rétine d&#39;imagerie en temps réel

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.

In vivo d&#39;embryons de drosophile Centrifugation

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.

L&#39;imagerie in vivo chez la drosophile larves Intact au sub-cellulaire Résolution

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.

Analyse rapide et exploration de microscopie par fluorescence Images

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 Préparation et imagerie longitudinale de la fonction cardiaque in vivo en utilisant cohérence optique Microscopie (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.

Synthèse de 68Ga dopée à base d’oxyde de fer nanoparticules pour tomographie par émission de double/imagerie de résonance magnétique (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.