Wir danken Andreas Schönle, Max-Plank-Institut für biophysikalische Chemie, Deutschland und David J. Sandström, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA für die technische Beratung. Wir bedanken uns bei Frank Kötting, European Neuroscience Institute, Göttingen für den Bau der Bildgebung Kammer und die Betäubung Gerät. Diese Arbeit wurde durch Zuschüsse der Alzheimer Forschung Initiative zu TMRYZ unterstützt durch ein Stipendium des China Scholarship Council, SBH durch ein Stipendium der Graduate School for Cellular and Molecular Neuroscience, University of Tübingen unterstützt.

A) Montage der Bildgebung Kammer <ol><li> Wählen Sie eine Larve der gewählten Stufe (zB frühe 3 rd Larvenstadium verlassen eine Beobachtung im Abstand von ca. 24 Stunden bei 25 ° C bis wandernde Stufe). </li><li> Spülen Sie die Larve mit Wasser, um es von dem Kulturmedium und tupfen sie trocken zu reinigen. </li><li> Bestreichen Sie die Mitte der unteren Element der Kammer, die Region um die Larve Gesicht, mit einem dünnen Film von Halocarbonöl. </li><li> Legen Sie die Larve mit der Bauchseite mit Blick auf den Mikroskop-Objektiv in der Kammer (ermöglicht neuromuskulären Synapse (NMJ) 26 und 27 werden abgebildet) (Abbildung 1 AE). </li><li> Legen Sie die Kunststoffscheibe auf die Ölschicht mit den Luftschlitzen des Netzes nach oben zeigen (Abbildung 1 A). Bitte überprüfen Sie bei diesem Schritt: Die Höhe der Spacer ist etwa die Hälfte der Larven Durchmesser sollte die Breite des Schlitzes etwa das Doppelte des Durchmessers der Larve werden. </li><li> Legen Sie die Imaging-Kammer unter dem Mikroskop </li></ol> B) Betäubung der Larve <ol start="7"><li> Verbinden Sie die beiden Eingänge der Kammer mit einem geeigneten Gerät Betäubung / Verdampfer. Bitte überprüfen Sie up-front: Die effektive Konzentration von Desfluran in den Raum-Luft sollte nie mehr als die von Sicherheitsvorschriften angegeben! Nur ein sehr kleiner Gesamtvolumen von Desfluran ist notwendig, um Larven zu betäuben. Die Anwendung von 15% (v / v) von Desfluran 1 hat sich als guter Ausgangspunkt für die Bestimmung der idealen Konzentration, die in einem Experiment verwendet werden. Aus Sicherheitsgründen empfehlen wir, dass der Verdampfer sollte nie enthalten mehr Desfluran als für 2-4 Stunden in-vivo-Bildgebung erforderlich. </li><li> Tauchen Sie das andere Ende des Schlauches an der Kammerauslass in einem Glas Wasser, dann öffnen die Ventile steuern den Fluss des Anästhetikums in die Kammer für etwa fünf Sekunden. Bitte überprüfen Sie bei diesem Schritt: Sie Luftblasen aufsteigen in das Wasser? Wenn nicht die Kammer undicht. </li><li> Schließen Sie die Ventile für etwa drei Sekunden. </li><li> Monitor verbleibende Larven Bewegungen in das Mikroskop. Überprüfen Sie beide den Herzschlag und Muskelbewegungen. Bei Bedarf öffnen und Schließen von Ventilen, wie in Schritt 8 und 9 beschrieben, bis die Betäubung der Larve abgeschlossen ist, schließen Sie alle Ventile und starten Bildgebung. Bitte überprüfen Sie bei diesem Schritt: Residual Muskelbewegungen oder Herzschlag zeigt an, dass die Larve nicht richtig betäubt. Komplette Betäubung ist für Bilder mit hoher Auflösung. Die Larve ist in der Regel nicht länger als 15-20 Minuten narkotisiert zu einem bestimmten Zeitpunkt. </li></ol> C) Imaging <ol start="11"><li> Identifizieren Sie die richtige Position und das Image der interessierenden Struktur (Abb. 2-4). </li></ol> D) Erholung von Betäubung <ol start="12"><li> Nach der Bebilderung abgeschlossen ist lassen Sie Luft in die Kammer. Bitte überprüfen Sie bei diesem Schritt: Überprüfen Sie Herzschlag und Muskelkontraktionen der Larve durch übertragen Halogenlicht. Da Muskeln Auftraggeber Start ist es sicher, die Larve aus dem Imaging-Kammer zu entfernen. </li><li> Lösen Sie die Kammer von der Betäubung Gerät und entfernen Sie sie aus dem Mikroskop. Demontieren Sie die Kammer vorsichtig und platzieren Sie die Larve in püriert fliegen Kultivierungsmedium. </li><li> Bewahren Sie die Schüssel in einem Brutschrank bei der entsprechenden Temperatur. </li></ol> E) Zeitreihe <ol start="15"><li> Wiederholen Sie die Schritte 2-14, bis genügend Zeit Punkte erhalten worden sind. Alternative-Protokoll: Wenn die Larve ist es, mehr als einmal innerhalb von 30 Minuten-Intervall abgebildet werden es sinnvoll ist, um es in der Imaging-Kammer zu verlassen, bis der nächste Betäubung Zeitpunkt. Wiederholen Sie die Schritte 7-14, bis genügend Zeit Punkte erhalten worden sind. Bitte überprüfen Sie bei diesem Schritt: Die Larve ist nicht in der Imaging-Kammer für mehr als zwei Stunden zu einer Zeit gehalten werden, noch ist es zu betäubende länger als 15-20 Minuten zu einer Zeit. </li></ol><img src="/files/ftp_upload/2249/2249fig1.jpg" alt="Abbildung 1" /> Abbildung 1 Versammlung der Bildgebung Kammer. (A) Setzen Sie die Larve und die Kunststoff-Abstandhalter auf der Ölschicht. (B) Legen Sie eine 22 x 22 mm Deckglas auf dem Abstandshalter und setzen Sie das Plexiglas Führungsring in die Kammer, in der Nähe (C) fixieren die Lage der Larve mit dem Metallring und (D) nahe der Kammer. (E) Nun wird die Kammer bereit, auf das Mikroskop angebracht werden. <img src="/files/ftp_upload/2249/2249fig2.jpg" alt="Abbildung 2" /> Abbildung 2 Body-Wand Muskeln in Drosophila-Larven. Muskeln und NMJs wurden durch Ausdruck einer CD8-GFP-Sh Fusionsprotein 8. Die Muskeln sind dargestellt als beobachtet werden, wenn der Schwerpunkt auf der Bauchseite durch die Kutikula in die Larve. In (AC) der oberflächlichen Muskulatur, 27, wird gezeigt, in (KL) den Innenraum Muskeln, 6 und 7 dargestellt. Scale-Bar: 100 pm, ist DELTA Z zwischen den Schichten 2 um. <img src="/files/ftp_upload/2249/2249fig3.jpg" alt="Abbildung 3" /> Abbildung 3 Identität von Körper-Wand Muskeln in Drosophila-Larven. Die Identität der Muskeln in L3 Drosophila-Larven-, Segment-A3, wird angezeigt. Muskeln und NMJs wurden durch Ausdruck einer CD8-GFP-Sh Fusionsprotein 8. Die Muskeln werden angezeigt, wie beobachtet werden, wenn der Schwerpunkt auf der Bauchseite durch die Kutikula in die Larven. In AC die oberflächlichen Muskeln, 27, wird gezeigt, in KL den Innenraum Muskeln, 6 und 7 dargestellt. Maßstab: 100 um, DELTA Z zwischen den Schichten ist 2 um. <img src="/files/ftp_upload/2249/2249fig4.jpg" alt="Abbildung 4" /> Abbildung 4 Identität des neuromuskulären Synapsen von Drosophila-Larven. (AD) NMJs wurden durch die Expression eines DGluRIIA-mRFP fusion protein 1. Die NMJs gezeigt, wie beobachtet werden, wenn der Schwerpunkt auf der Bauchseite durch die Kutikula in die Larve. In (AB) die oberflächliche NMJ, 27, wird gezeigt, in CD den Innenraum NMJs, 6 und 7, gezeigt. Maßstab: 100 um, DELTA Z zwischen den Schichten ist 5 um. (E) und (F) die Identität des oberflächlichen (E) und Innenraum (F) NMJs ist nur zur Information angegeben.

<ol>
	<li>Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptor+dynamics+organizing+synapse+formation+in+vivo.">Glutamate receptor dynamics organizing synapse formation in vivo.</a> Nat Neurosci. 8, 898-905 (2005).</li><li>Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In vivo imaging of long-term changes in the Drosophila neuromuscular system [dissertation]. , (1988).</li><li>Fuger, P., Behrends, L. B., Mertel, S., Sigrist, S. J., Rasse, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+synapse+development+and+measuring+protein+dynamics+using+two-color+fluorescence+recovery+after+photo-bleaching+at+Drosophila+synapses.">Live imaging of synapse development and measuring protein dynamics using two-color fluorescence recovery after photo-bleaching at Drosophila synapses.</a> Nat Protoc. 2, 3285-3298 (2007).</li><li>Wang, X., Schwarz, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+axonal+transport+of+mitochondria.">Imaging axonal transport of mitochondria.</a> Methods in enzymology. 457, 319-333 (2009).</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=Mesoscopic+fluorescence+tomography+for+in-vivo+imaging+of+developing+Drosophila.">Mesoscopic fluorescence tomography for in-vivo imaging of developing Drosophila.</a> J Vis Exp. , (2009).</li><li>Lin, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+imaging+of+Drosophila+larva+by+multiphoton+autofluorescence+and+second+harmonic+generation+microscopy.">Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy.</a> Journal of biomedical optics. 13, 050502-050502 (2008).</li><li>Levitan, E. S., Lanni, F., Shakiryanova, D. <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+vesicle+motion+and+release+at+the+Drosophila+neuromuscular+junction.">In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction.</a> Nat Protoc. 2, 1117-1125 (2007).</li><li>Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y. &. a. m. p. ;. a. m. p., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li></ol>

Die vorgestellte Methode wurde ursprünglich entwickelt, um glutamatergen Synapsen auf den Körper Wand Muskeln Drosophila melanogaster Larven zu studieren. Die Drosophila neuromuskulären Synapse (NMJ) wird durch eine stereotype Zyto-Architektur der Muskeln und Neuronen aus und ist damit ideal für den in-vivo-Bildgebung geeignet. Allerdings ist die beschriebene Betäubung Protokoll nicht zu bildgebenden die NMJ begrenzt; die Transparenz der Drosophila-Larven erleichtert die Anpassung der beschriebenen Protokoll, um die Entwicklung von Organen Studie, die Migration von Zellen, den Transport von Fracht-und axonalen sub-zellulären Reorganisation innerhalb der Zellen.

Reagenzien: <ul><li> Desfluran (Suprane, Baxter, Unterschleißheim, Deutschland) </li><li> Halocarbonöl (zB Voltalef H10S Öl / Atofina, Puteaux, Frankreich) </li><li> Fly Kulturmedium </li></ul> Ausstattung: <ul><li> Inverted konfokalen Mikroskop </li><li> Binokulares Mikroskop (zB Stemi 2000, Carl Zeiss, Jena, Deutschland) </li><li> Kleine Pinsel als zur Art fliegt </li><li> Inkubator </li><li> Maßarbeit Bildgebung Kammer (Für mechanische Zeichnungen sehen Ref. 3) </li><li> Vaporizer / Betäubung Gerät (für Details siehe Lit.. 3) </li></ul>

in vivo imaging of intact drosophila larvae at sub-cellular resolution

Die jüngsten Verbesserungen in der optischen Bildgebung, genetisch kodierte Fluorophore und genetische Werkzeuge, die eine effiziente Einrichtung von gewünschten transgenen Tier Linien aktiviert haben biologischer Prozesse im Rahmen eines lebenden studiert werden, und in einigen Fällen sogar zu verhalten, Organismus. In diesem Protokoll werden wir beschreiben, wie intakt Drosophila-Larven, mit dem volatilen Anästhetika Desfluran, um die Entwicklung und Plastizität der synaptischen Populationen subzellulärer Auflösung 1-3 folgen zu betäuben. Während andere nützliche Methoden, um Drosophila melanogaster Larven betäuben wurden bisher 4,5,6,7,8 beschrieben, zeigt das Protokoll hier vorgestellte signifikante Verbesserungen aufgrund der folgenden kombiniert die wichtigsten Funktionen: (1) Ein sehr hohes Maß an Betäubung, auch die Herzschlag wird verhaftet Berücksichtigung lateralen Auflösung von bis zu 150 nm 1, (2) eine hohe Überlebensrate von&gt; 90% pro Zyklus Betäubung, erlaubt die Aufnahme von mehr als fünf Zeitpunkten über einen Zeitraum von Stunden bis Tagen 2 und ( 3) eine hohe Empfindlichkeit ermöglicht uns in 2 Fällen zur Untersuchung der Dynamik von Proteinen in physiologischen Konzentrationen ausgedrückt. Im Einzelnen konnten wir die postsynaptische Glutamat-Rezeptor-Untereinheit GluR-IIA über den endogenen Promotor 1 in stabilen transgenen Linien und der Exon-trap line FasII-GFP 1 ausgedrückt zu visualisieren. (4) Im Gegensatz zu anderen Methoden 4,7 können die Larven nicht nur lebendig abgebildet werden, sondern auch intakte (dh nicht seziert) ermöglicht Beobachtung über mehrere Tage 1 auftreten. Das dazugehörige Video Details der Funktion der einzelnen Teile der in-vivo-Bildgebung Kammer 2,3, die korrekte Montage der Larven, die Betäubung Verfahren, wie re-identifizieren bestimmte Positionen innerhalb einer Larve und die sichere Entfernung der Larven aus der Bildgebung Kammer.

Watch this Scientific Journal Video about In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution at JoVE.com

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Dieses Protokoll beschreibt eine zuverlässige Methode zur Betäubung und Bildgebung von intakten<em&gt; Drosophila melanogaster</em&gt; Larven. Wir nutzten die volatilen Anästhetika Desfluran, um für sich wiederholende Bildgebung bei sub-zellulärer Auflösung und Re-Identifizierung von Strukturen für bis zu ein paar Tagen ermöglichen<sup&gt; 1</sup&gt;.

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 ...

in-vivo-imaging-of-intact-drosophila-larvae-at-sub-cellular-resolution

Research

JoVE Journal

Biology

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

14.5K Aufrufe.  University of Tübingen. Dieses Protokoll beschreibt eine zuverlässige Methode zur Betäubung und Bildgebung von intakten Drosophila melanogaster Larven. Wir nutzten die volatilen Anästhetika Desfluran, um für sich wiederholende Bildgebung bei sub-zellulärer Auflösung und Re-Identifizierung von Strukturen für bis zu ein paar Tagen ermöglichen 1.

Serie: In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

Hochauflösende Messung von Odor-Driven Behavior in Drosophila-Larven

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Forschung

Biologie

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

Labeling hESCs und hMSCs mit Eisenoxid-Nanopartikel für Non-Invasive in vivo-Tracking mit MR-Bildgebung

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Hochauflösendes Video Tracking von Locomotion in Adult Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

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 ...

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 ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Imaging Zellgröße Veränderung der Lebens- Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Microgavage of Zebrafish Larvae

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results. We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to quantify transit from the intestinal lumen to extraintestinal spaces. This test can be used to verify proper execution of the microgavage procedure, and also provides a novel zebrafish assay to examine intestinal epithelial barrier integrity under different experimental conditions (e.g. genetic manipulation, drug treatment, or exposure to environmental factors). Furthermore, we show how gavage can be used to evaluate intestinal motility by gavaging fluorescent microspheres and monitoring their subsequent transit. Microgavage can be applied to deliver diverse materials such as live microorganisms, secreted microbial factors/toxins, pharmacological agents, and physiological probes. With these capabilities, the larval zebrafish microgavage method has the potential to enhance a broad range of research fields using the zebrafish model system.

We present a novel method for microgavage of larval zebrafish utilizing standard embryo microinjection and stereomicroscopy equipment. We demonstrate that microgavage is a safe and efficient technique useful for delivering controlled amounts of diverse materials specifically into the larval zebrafish intestinal lumen.

Microgavage der Zebrafischlarven

The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Live-Imaging von apoptotischen Ausverkauf während<em&gt; Drosophila</em&gt; Embryogenese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

Ex-vivo-Verfahren für High Resolution Imaging von Cilia Motilität in Nagetier Atemwegsepithelien

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Die konfokale Bildgebung von Einzel Mitochondrial Super Blinkt Intact Herz oder<em&gt; In Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...