C. Vinegoni riconosce il sostegno di National Institutes of Health (NIH) concedere 1-RO1-EB006432.

Le scorte di Drosophila melanogaster utilizzati in questo esperimento sono i seguenti: <ul style="list-style-type:none;"><li> ELAV-GAL4 (FBti0002575) </li><li> ap-Gal4md544 (FBal0051787) </li><li> SUP-srcEGFP (FBti0013990) </li><li> w1118 (FBal0018186) </li></ul> Per tutti gli esperimenti, le scuole universitarie professionali-GAL4 sistema è stato utilizzato per iperespressione di GFP nel tessuto di interesse (ad esempio le ghiandole salivari o dischi di ala). Più in particolare, le mosche femmina transgenici con l&#39;apposito GAL4 furono incrociati a UAS-EGFP compagni transgenici. Le croci sono stati allevati a 25 ° C in un incubatore umidificato. Quando pupe iniziare popolare le fiale (circa 5-6 giorni dopo l&#39;inizio della croce), sono stati selezionati per la fluorescenza GFP in fase di bianco prepupal (0-1 ore dopo la formazione puparium) e raccolti per la tomografia di imaging. <ol><li> Impostare attraversa appropriati e posteriore a 25 ° C </li><li> 5-6 giorni dopo l&#39;inizio della croce, selezionare progenie fluorescenti per l&#39;imaging. Utilizzare un pennello umido per la rimozione delicata della prepupae bianco dai lati della fiala. </li><li> Mettere prepupae in una goccia d&#39;acqua o PBS posto in una capsula di Petri per rimuovere le particelle di cibo e la colla che copre il caso pupa e rende il campione autofluorescenti. Pulire delicatamente con un pennello. Se l&#39;invecchiamento delle pupe è necessario, luogo scelto prepupae bianco in una capsula di Petri con un tovagliolo di carta umido (camera umida) o ai lati di una cuvetta pulita con il cibo volare. Questo farà sì che vi sia umidità sufficiente, per consentire un corretto sviluppo dell&#39;animale. </li></ol> Drosophila montaggio <ol><li> Colla nella parte inferiore del case pupa in un tubo capillare di vetro (800 micron di diametro) in modo che la mosca anteriore / posteriore asse è orientato parallelamente al tubo. </li><li> Fissare il tubo capillare in verticale su un palcoscenico di rotazione. </li><li> Regolare l&#39;inclinazione dell&#39;asse della fase di rotazione in modo che l&#39;asse di rotazione deve essere parallela alle colonne pixel del CCD. </li></ol> Modello di determinazione in avanti <ol><li> Fissare una pupa in un capillare di vetro e montare come descritto in precedenza </li><li> Riempire un tubo capillare di silice (100 micron, OD) con un colorante fluorescente (Cy5.5) </li><li> Inserire il tubo capillare all&#39;interno della pupa a 45 gradi rispetto alla anteriore / posteriore asse </li><li> Illuminare la pupa con un fascio laser di eccitazione con una lente a bassa apertura numerica. Raccogliere immagini transilluminazione fluorescenza a 360 gradi di rotazione la pupa lungo il suo asse verticale. </li><li> Montare i dati sperimentali con un modello appropriato in avanti </li></ol> Le ghiandole salivari Imaging <ol><li> Montare un GFP pupa che esprime nelle ghiandole salivari, come descritto prima </li><li> Illuminare con un fascio di eccitazione e raccogliere in transilluminazione il segnale di fluorescenza GFP su 360 gradi di rotazione la pupa lungo il suo asse verticale. </li><li> Ricostruire i dati acquisiti utilizzando il modello in avanti </li></ol> Time-Lapse Imaging <ol><li> Montare un GFP bianco prepupa esprimendo nei dischi ala immaginale </li><li> Mantenere l&#39;ambiente umido e di imaging a temperatura ambiente per evitare la disidratazione della pupa. </li><li> Posizionare un otturatore per evitare l&#39;illuminazione continua </li><li> Illuminare con un fascio di eccitazione e raccogliere in transilluminazione il segnale di fluorescenza GFP su 360 gradi di rotazione la pupa lungo il suo asse verticale. </li><li> Ricostruire i dati acquisiti utilizzando il modello in avanti. </li></ol> Istologia <ol><li> Sezionare SNC ghiandole salivari / dischi occhio in 1x PBS. </li><li> Fissare i tessuti nel 4% di formaldeide, 1x PEM (Tubi 100mM, 1MM EGTA, 1mm MgCl 2) per 20 minuti a temperatura ambiente. </li><li> Rimuovere fissaggio e montaggio in Vectashield con DAPI (Vector) </li><li> Immagine con un microscopio a fluorescenza. </li></ol> Time-Lapse Istologia <ol><li> Raccogliere prepupae bianco e lo stadio in una camera umida. </li><li> Raccogliere pupe a diversi stadi di sviluppo </li><li> Cazzo il caso pupa due volte (0 ore dopo la formazione Puparium) con un ago sottile (insetti pin 000, FST) prima di fissazione. </li><li> Fissare pupe in 8% di formaldeide, 1x PEM per 6-8 ore a temperatura ambiente e scuotendo con cautela. </li><li> Dopo la rimozione di fissazione e risciacquo con PBS 1x, tagliare il fisso pupa con una lama affilata in due pezzi perpendicolari all&#39;asse A / P; </li><li> Immergere i due pezzi in Vectashield con DAPI (Vector) e montare su vetrini coprioggetti (Lab-Tek) per l&#39;imaging. </li><li> Acquisire le immagini con un microscopio a fluorescenza confocale. </li></ol><img src="/files/ftp_upload/1510/1510fig1thumb.jpg" alt="figura 1" border="0" /> Figura 1. Si prega di <a href="https://www.jove.com/files/ftp_upload/1510/1510fig1.jpg">cliccare qui</a> per vedere alarger versione della 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.

In vivo ricostruzioni del caso pupa e la GFP che esprimono ghiandole salivari di una D. melanogaster prepupa (Scala bar, 500 micron) con l&#39;istologia corrispondente (blu, colorazione DAPI, verde, GFP fluorescenza) è mostrato in Fig. 1 (a). Time-lapse imaging di serie D. melanogaster ala dischi immaginale è mostrata in Fig.1 (b). Le immagini vengono acquisite da un singolo esemplare dal vivo, in quattro momenti diversi (0,3.0, 3,5 e 6,5 ore). Nella prima colonna una proiezione a 0 gradi rispetto al...

mesoscopic fluorescence tomography for in-vivo imaging of developing drosophila

Visualizzazione formazione degli organi di sviluppo così come progession e trattamento della malattia spesso si basa pesantemente sulla capacità di interrogare otticamente cambiamenti molecolari e funzionali negli organismi viventi intatti. La maggior parte delle attuali metodi di imaging ottico sono inadeguati per l&#39;imaging a dimensioni che si trovano tra i limiti di penetrazione della moderna microscopia ottica (0,5-1mm) e la diffusione imposto limiti di macroscopia ottica (&gt; 1 cm) [1]. Così, molti organismi modello importante, ad esempio insetti, embrioni animali o delle estremità piccolo animale, restano inaccessibili per in vivo imaging ottico. Anche se vi è un crescente interesse verso lo sviluppo di metodi di risoluzione nanometrica imaging ottico, non ci sono stati molti gli sforzi di successo per migliorare la profondità di penetrazione di imaging. La capacità di eseguire l&#39;imaging in vivo oltre i limiti microscopia è infatti incontrato con le difficoltà associate con dispersione di fotoni presenti nei tessuti. Recenti tentativi di embrioni intera immagine per esempio [2,3] richiedono un trattamento speciale chimica del campione, per eliminarli dallo scattering, una procedura che li rende adatti solo per la post-mortem di imaging. Questi metodi tuttavia evidenza la necessità di esemplari immagini più grandi di quelle normalmente consentite dalla microscopia a due fotoni o confocale, in particolare in biologia dello sviluppo e nella scoperta della droga. Abbiamo sviluppato una nuova tecnica di imaging ottico chiamato mesoscopici fluorescenza Tomografia [4], che appropriato per non invasiva in vivo immagini a dimensioni di 1mm-5mm. La risoluzione metodo scambi per profondità di penetrazione, ma offre prestazioni di imaging tomografico senza precedenti ed è stato sviluppato per aggiungere il tempo come una nuova dimensione nelle osservazioni biologia dello sviluppo (e forse altre aree di ricerca biologica), donando la possibilità di immagine l&#39;evoluzione della fluorescenza tagged risposte nel tempo. Come tale, può accelerare gli studi delle dipendenze morfologiche o funzionali a mutazioni genetiche o stimoli esterni, e può, soprattutto, acquisire il quadro completo dello sviluppo o della funzione dei tessuti, permettendo longitudinale time-lapse visualizzazione degli stessi, organismo di sviluppo. La tecnica utilizza un microscopio da laboratorio modificati e multi-proiezione di illuminazione per raccogliere i dati a 360 gradi proiezioni. Si applica la semplificazione Fermi di Fokker-Plank soluzione dell&#39;equazione di trasporto di fotoni, in combinazione con i principi dell&#39;ottica geometrica al fine di costruire un piano realistico inversione adatti per la gamma mesoscopici. Ciò consente di in-vivo di tutto il corpo visualizzazione di non trasparente strutture tridimensionali in campioni fino a diversi millimetri di dimensione. Abbiamo dimostrato in vivo le prestazioni della tecnica di imaging di strutture tridimensionali di sviluppare tessuti Drosophila in vivo e seguendo la morfogenesi delle ali nel opaco Drosophila pupe in tempo reale oltre sei ore consecutive.

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

Mesoscopici fluorescenza tomografia opera oltre i limiti di penetrazione del tessuto-sezionamento microscopia a fluorescenza. La tecnica è basata su multi-proiezione di illuminazione e una descrizione di trasporto fotone. Abbiamo dimostrato in vivo di tutto il corpo visualizzazione 3D della morfogenesi della GFP che esprimono ala dischi immaginale in<em&gt; Drosophila melanogaster.</em

Mesoscopici fluorescenza per Tomografia In-vivo Imaging di sviluppo 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 Views.  Massachusetts General Hospital. Mesoscopici fluorescenza tomografia opera oltre i limiti di penetrazione del tessuto-sezionamento microscopia a fluorescenza. La tecnica è basata su multi-proiezione di illuminazione e una descrizione di trasporto fotone. Abbiamo dimostrato in vivo di tutto il corpo visualizzazione 3D della morfogenesi della GFP che esprimono ala dischi immaginale in 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.

Cella singola elettroporazione in vivo all&#39;interno del cervello intatto in via di sviluppo

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

Ricerca

Biologia

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 normalizzazione nata per fluorescenza Tomografia Ottica proiezione per l&#39;imaging tutto il cuore

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 imaging di mitosi dopo la trasfezione di 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.

Preparazione di Sviluppo e degli adulti Drosophila Brains e retina per l&#39;imaging dal 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.

In vivo centrifugazione di embrioni di 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.

Imaging in vivo di intatto Larve di Drosophila a livello sub-cellulare Risoluzione

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.

Analisi rapida ed esplorazione di microscopia a fluorescenza Immagini

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 preparazione e imaging longitudinale della funzione cardiaca in vivo utilizzando Optical Coherence Microscopy (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.

Sintesi di nanoparticelle Core-drogati di ossido di ferro di 68Ga per Dual tomografia a emissione di positroni/Imaging di risonanza magnetica (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.