C. Vinegoni dankt für die Unterstützung von National Institutes of Health (NIH) gewähren 1-RO1-EB006432.

Die Drosophila melanogaster Aktien in diesem Experiment verwendet, sind folgende: <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> Für alle Versuche wurde die UAS-Gal4-System zu überexprimieren GFP in das Gewebe von Interesse (dh der Speicheldrüsen oder der Flügel-Discs) verwendet. Genauer gesagt wurden weibliche Fliegen transgenen mit der entsprechenden Gal4 zu UAS-EGFP transgenen Kameraden gekreuzt. Die Kreuze wurden bei 25 ° C in einem befeuchteten Inkubator aufgezogen. Wenn Puppen bevölkern die Fläschchen (ca. 5-6 Tage nach Beginn des Kreuzes) zu starten, wurden sie für GFP-Fluoreszenz auf die weiße prepupal Bühne (0-1 Stunden nach Puparium Bildung) ausgewählt und gesammelt für Tomographie. <ol><li> Richten Sie geeignete Kreuzungen und hinten bei 25 ° C </li><li> 5-6 Tage nach der Einweihung des Kreuzes, wählen fluoreszierenden Nachkommen für die Bildgebung. Verwenden Sie einen feuchten Pinsel für die schonende Entfernung der weißen prepupae von den Seiten des Fläschchens. </li><li> Put prepupae in einem Tropfen Wasser oder PBS in eine Petrischale Speisereste und der Leim, der das Puppenstadium Cover und macht die Probe autofluoreszierenden entfernen. Reinigen Sie mit einem Pinsel. Wenn die Alterung der Puppen ist notwendig, statt weiß prepupae in einer Petrischale mit einem feuchten Papiertuch (einer feuchten Kammer) oder an den Seiten eine saubere Flasche mit fliegen Lebensmittel ausgewählt. Dadurch wird sichergestellt, dass genügend Feuchtigkeit, um die ordnungsgemäße Entwicklung des Tieres zu ermöglichen. </li></ol> Drosophila Montage <ol><li> Kleben Sie den unteren Teil der Puppenhülle in eine Glaskapillare (800 Mikrometer Durchmesser), so dass die Fliege anterior / posterior Achse parallel zur Röhre. </li><li> Fix die Kapillare vertikal auf einem Rotations-Bühne. </li><li> Passen Sie die Kippachse des Rotations-Bühne, um für die Drehachse parallel zu der Pixel-Spalten der bildgebenden CCD. </li></ol> Bestimmung Vorwärts-Modell <ol><li> Fix eine Puppe in eine Kapillare aus Glas und Montage wie zuvor beschrieben </li><li> Füllen Sie eine Silica-Kapillare (100 Mikron, OD) mit einem fluoreszierenden Farbstoff (Cy5.5) </li><li> Legen Sie die Kapillare im Inneren der Puppe bei 45 Grad in Bezug auf den vorderen / hinteren Achse </li><li> Illuminate die Puppe mit einer Anregung Laserstrahl mit geringer numerischer Apertur Linse. Sammeln Fluoreszenz Transilluminationsbilder über 360 Grad drehbare die Puppe um seine Hochachse. </li><li> Fit der experimentellen Daten mit einem geeigneten Vorwärtsmodell </li></ol> Imaging Speicheldrüsen <ol><li> Montieren Sie eine Puppe, die GFP in den Speicheldrüsen, wie zuvor beschrieben </li><li> Illuminate mit einem Anregungsstrahl und sammeln sich in Durchleuchtung der GFP-Fluoreszenz-Signal über 360 Grad drehbare die Puppe um seine Hochachse. </li><li> Rekonstruieren Sie die erfassten Daten unter Verwendung der Vorwärtsmodell </li></ol> Time-Lapse Imaging <ol><li> Montieren Sie einen weißen Vorpuppe, die GFP in den Flügel Imaginalscheiben </li><li> Halten Sie die Imaging-Umgebung feucht und bei Raumtemperatur, um eine Dehydratation der Puppe zu vermeiden. </li><li> Legen Sie einen Auslöser, um kontinuierliche Beleuchtung zu vermeiden </li><li> Illuminate mit einem Anregungsstrahl und sammeln sich in Durchleuchtung der GFP-Fluoreszenz-Signal über 360 Grad drehbare die Puppe um seine Hochachse. </li><li> Rekonstruieren Sie die erfassten Daten unter Verwendung der nach vorne Modell. </li></ol> Histologie <ol><li> Dissect Speicheldrüsen CNS / Auge-Discs in 1x PBS. </li><li> Fix Gewebe in 4% Formaldehyd, 1x PEM (100mm Rohre, 1 mM EGTA, 1 mM MgCl 2) für 20min bei Raumtemperatur. </li><li> Entfernen Befestigung und montieren in Vectashield mit DAPI (Vector) </li><li> Bild mit einem Fluoreszenzmikroskop. </li></ol> Time-Lapse Histologie <ol><li> Sammeln Sie weiß prepupae und Bühne sie in einer feuchten Kammer. </li><li> Sammeln Puppen in verschiedenen Entwicklungsstadien </li><li> Prick das Puppenstadium Fall zweimal (0 Std. Nach Puparium Formation) mit einer feinen Nadel (Insekten Stift 000, FST) vor der Fixierung. </li><li> Fix Puppen in 8% Formaldehyd, 1x PEM für 6-8 Stunden bei Raumtemperatur unter leichtem Schütteln. </li><li> Nach dem Entfernen der Fixierung und Spülen mit 1x PBS, schneiden Sie die feste Puppe mit einer scharfen Klinge in zwei Teile senkrecht zur A / P-Achse; </li><li> Tauchen Sie die beiden Stücke in Vectashield mit DAPI (Vector) und mounten auf Deckglas Objektträger (Lab-Tek) für die Bildgebung. </li><li> Erwerben Sie Bilder mit einem Fluoreszenz-konfokalen Mikroskop. </li></ol><img src="/files/ftp_upload/1510/1510fig1thumb.jpg" alt="Abbildung 1" border="0" /> Abbildung 1. Bitte <a href="https://www.jove.com/files/ftp_upload/1510/1510fig1.jpg">klicken Sie hier</a> , um al sehenarger Version von Abbildung 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-Rekonstruktionen der Puppenhülle und die GFP-exprimierenden Speicheldrüsen der D. melanogaster Vorpuppe (Scale bar, 500 Mikron) mit den entsprechenden Histologie (blau, DAPI-Färbung, grün, GFP-Fluoreszenz) ist in Abb. 1 (a) gezeigt. Zeitraffer-Serie Abbildung von D. melanogaster Flügel Imaginalscheiben ist in Abb. 1 (b) gezeigt. Die Bilder sind aus einem einzigen lebenden Exemplars erworben, an vier verschiedenen Zeitpunkten (0,3.0, 3,5 und 6,5 Stunden). In der ersten Spalte einer Projekti...

mesoscopic fluorescence tomography for in-vivo imaging of developing drosophila

Visualisierung der Entwicklung Organbildung sowie Progression und Behandlung von Krankheiten oft in hohem Maße von der Fähigkeit, optisch verhören molekulare und funktionelle Veränderungen in intakten lebenden Organismen. Die meisten existierenden Verfahren der optischen Bildgebung sind unzureichend für die Bildgebung bei Dimensionen, die zwischen dem Eindringen Grenzen der modernen Lichtmikroskopie (0,5-1mm) und die Verbreitung auferlegten Grenzen der optischen Makroskopie liegen (&gt; 1cm) [1]. So viele wichtige Modellorganismen, zB Insekten-, Tier-Embryonen oder Kleintier Extremitäten, bleiben unzugänglich für in-vivo optische Bildgebung. Zwar gibt es zunehmendes Interesse an der Entwicklung von Nanometer-Auflösung optische bildgebende Verfahren gibt es nicht viele erfolgreiche Anstrengungen bei der Verbesserung der bildgebenden Eindringtiefe worden. Die Fähigkeit zur in-vivo-Bildgebung über Mikroskopie Grenzen durchzuführen, ist in der Tat mit den Schwierigkeiten mit Photonenstreuung im Gewebe verbunden erfüllt. Die jüngsten Bemühungen zur Bild gesamten Embryos zum Beispiel [2,3] erfordern spezielle chemische Behandlung der Probe, um sie von Streuung klar, ein Verfahren, das sie sich nur für Post-mortem-Bildgebung ermöglicht. Diese Methoden jedoch Beweise, dass für die Bildgebung größere Exemplare als die, die in der Regel durch Zwei-Photonen-oder konfokalen Mikroskopie, vor allem in der Entwicklungsbiologie und in der Wirkstoffforschung erlaubt. Wir haben einen neuen optischen Bildgebungsverfahren namens Mesoscopic Fluoreszenz-Tomographie entwickelt [4], die geeignet für non-invasive In-vivo-Bildgebung bei Abmessungen von 1mm-5mm. Die Methode Austausch Auflösung für Eindringtiefe, sondern bietet eine beispiellose tomographischen Imaging-Leistung und es wurde entwickelt, um die Zeit als neue Dimension in der Entwicklungsbiologie Beobachtungen (und möglicherweise auch in anderen Bereichen der biologischen Forschung) hinzufügen, indem Sie die Vermittlung der Fähigkeit zur Abbildung der Entwicklung der Fluoreszenz- getaggt Reaktionen im Laufe der Zeit. Als solche kann sie beschleunigen Studien der morphologischen oder funktionellen Abhängigkeiten von Gen-Mutationen oder äußere Reize und können wichtiger ist, fangen die vollständigen Überblick über die Entwicklung oder die Gewebe-Funktion, indem sie Längs-Zeitraffer-Visualisierung der gleichen, sich entwickelnden Organismus. Die Technik nutzt eine modifizierte Labormikroskop und Multi-Projektion Beleuchtung, Daten mit 360-Grad-Projektionen zu sammeln. Es gilt das Fermi Vereinfachung Fokker-Plank-Lösung des Photons Transport Gleichung, mit der geometrischen Optik Prinzipien kombiniert, um eine realistische Inversionsschema für mesoskopischen Bereich zu bauen. Dies ermöglicht in-vivo Ganzkörper-Visualisierung von nicht-transparenten dreidimensionale Strukturen in Proben bis zu einigen Millimetern Größe. Wir haben die in-vivo-Leistung der Technik durch bildgebende dreidimensionale Strukturen gezeigt, die Entwicklung Drosophila Gewebe in-vivo und nach der Morphogenese der Flügel in der undurchsichtigen Drosophila Puppen in Echtzeit über sechs Stunden hintereinander.

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

Mesoscopic Fluoreszenz-Tomographie arbeitet darüber hinaus das Eindringen Grenzen der Gewebe-Schnitte Fluoreszenzmikroskopie. Die Technik basiert auf Multi-Projektion Beleuchtung und ein Photon Transport Beschreibung basiert. Wir zeigen in-vivo Ganzkörper-3D-Visualisierung der Morphogenese von GFP-exprimierenden Flügel Imaginalscheiben in<em&gt; Drosophila melanogaster.</em

Mesoscopic Fluoreszenz-Tomographie für In-vivo Imaging der Entwicklungsländer 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 Aufrufe.  Massachusetts General Hospital. Mesoscopic Fluoreszenz-Tomographie arbeitet darüber hinaus das Eindringen Grenzen der Gewebe-Schnitte Fluoreszenzmikroskopie. Die Technik basiert auf Multi-Projektion Beleuchtung und ein Photon Transport Beschreibung basiert. Wir zeigen in-vivo Ganzkörper-3D-Visualisierung der Morphogenese von GFP-exprimierenden Flügel Imaginalscheiben in Drosophila melanogaster.

Serie: 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.

Single Cell Elektroporation in vivo innerhalb der intakten sich entwickelnde Gehirn

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

Forschung

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.

Geboren Normalisierung für Fluoreszenz-Optical Projection Tomographie für ganzem Herzen Imaging

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.

Zeitraffer-Imaging der Mitose Nach siRNA-Transfektion

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.

Erstellung von Entwicklungs-und Adult Drosophila Brains und retinae für Live Imaging

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-Zentrifugation von Drosophila Embryonen

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.

In-vivo-Imaging von Intact Drosophila-Larven auf subzellulärer Auflösung

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.

Schnelle Analyse und Exploration von Fluoreszenzmikroskopie 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 Vorbereitung und Longitudinal Imaging der Herzfunktion in vivo unter Verwendung optischer Kohärenzmikroskopie (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.

Synthese von 68-Ga-Kern-dotierte Eisenoxid-Nanopartikeln für Dual Positronen-Emissions-Tomographie / (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 ...

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.