Name: in vivo imaging of intact drosophila larvae at sub-cellular resolution
Uploaded: 2010-09-10T17:00:00
Duration: 17 min 51 s
Description: Watch this Scientific Journal Video about In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution at JoVE.com

We thank Andreas Sch&ouml;nle, Max-Plank-Institute for Biophysical Chemistry, Germany and David J. Sandstrom, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA for technical advice. We thank Frank K&ouml;tting, European Neuroscience Institute, G&ouml;ttingen for constructing the imaging chamber and the anesthetization device. This work was supported by grants from the Alzheimer Forschung Initiative to T.M.R. Y.Z. was supported by a fellowship of the China Scholarship Council, S.B.H. by a fellowship of the Graduate School for Cellular and Molecular Neuroscience, University of T&uuml;bingen.

A) Assembly of the imaging chamber
<ol>
	<li>Select a larva of chosen stage (e.g. early 3rd instar larvae leaving an observation interval of about 24 hours at 25&deg;C until wandering stage).</li>
	<li>Rinse the larva with water to clean it of the culture medium and dab it dry.</li>
	<li>Coat the center of the bottom element of the chamber, the region to face the larva, with a thin film of halocarbon oil.</li>
	<li>Put the larva with the ventral side facing the microscope objective in the chamber (allows neuromuscular junction (NMJ) 26 and 27 to be imaged) (Figure 1 A-E).</li>
	<li>Place the plastic spacer onto the oil layer, with the air slots of the grid face upwards (Figure 1 A).
Please verify at this step:
The height of the spacer is roughly half the larval diameter, the width of the slit should be about twice the diameter of the larva.</li>
	<li>Place the imaging chamber on the microscope</li>
</ol>
B) Anesthetization of the larva
<ol start="7">
	<li>Connect the two inlets of the chamber with an appropriate anesthetization device/vaporizer. Please verify up-front: The effective concentration of desflurane in the room-air should never exceed that specified by safety regulations! Only a very small total volume of desflurane is needed to anesthetize larvae. The application of 15% (v/v) of desflurane1 has proven to be a good starting point for determining the ideal concentration to be used in an experiment. For safety reasons we suggest that the vaporizer should never contain more desflurane than needed for 2-4 hours of in vivo imaging.</li>
	<li>Immerse the other end of the tube attached to the chamber outlet in a glass of water then open the valves controlling the flow of the anesthetic into the chamber for about five seconds. Please verify at this step: Do air bubbles ascend in the water? If not the chamber is leaky.</li>
	<li>Close the valves for about three seconds.</li>
	<li>Monitor residual larval movements in the microscope. Check both the heartbeat and muscle movements. If necessary open and close valves as described in step 8 and 9 until anesthetization of the larva is complete, then close all valves and start imaging. Please verify at this step: Residual muscle movement or heartbeat indicates that the larva is not properly anesthetized. Complete anesthetization is vital for high resolution images. The larva should normally not be anesthetized for longer than 15-20 minutes at a given time.</li>
</ol>
C) Imaging
<ol start="11">
	<li>Identify the correct position and image the structure of interest (Figure 2-4).</li>
</ol>
D) Recovery from anesthetization
<ol start="12">
	<li>After imaging is completed let air into the chamber. Please verify at this step: Check heartbeat and muscle contractions of the larva by transmitted halogen light. As muscles start contracting it is safe to remove the larva from the imaging chamber.</li>
	<li>Detach the chamber from the anesthetization device and remove it from the microscope. Disassemble the chamber carefully and place the larva in mashed fly cultivation medium.</li>
	<li>Store the dish in an incubator at the appropriate temperature.</li>
</ol>
E) Time series
<ol start="15">
	<li>Repeat the steps 2-14 until sufficient time points have been obtained. Alternative protocol: If the larva is to be imaged more than once within a 30 minute interval it might be practical to leave it in the imaging chamber until the next anesthetization time point. Repeat the steps 7-14 until sufficient time points have been obtained. Please verify at this step: The larva is not to be kept in the imaging chamber for more than two hours at a time, nor is it to be anesthetized for longer than 15-20 minutes at a time.</li>
</ol>

<img src="/files/ftp_upload/2249/2249fig1.jpg" alt="Figure 1" /> Figure 1 Assembly of the imaging chamber. (A) Place the larva and the plastic spacer onto the oil layer. (B) Place a 22 x 22 mm cover slip on the spacer and insert the plexiglas guide ring into the chamber, next (C) fix the position of the larva with the metal ring and (D) close the chamber. (E) Now the chamber is ready to be mounted on the microscope.
<img src="/files/ftp_upload/2249/2249fig2.jpg" alt="Figure 2" /> Figure 2 Body-wall muscles in Drosophila larvae. Muscles and NMJs were visualized by expression of a CD8-GFP-Sh fusion protein8. The muscles are shown as observed when focusing at the ventral side through the cuticle into the larva. In (A-C) the most superficial muscle, 27, is shown, in (K-L) the most interior muscles, 6 and 7, are shown. Scale Bar: 100 &mu;m, &Delta;Z between slices is 2 &mu;m.
<img src="/files/ftp_upload/2249/2249fig3.jpg" alt="Figure 3" /> Figure 3 Identity of body-wall muscles in Drosophila larvae. The identity of muscles in L3 Drosophila larvae, segment A3, is shown. Muscles and NMJs were visualized by expression of a CD8-GFP-Sh fusion protein8. The muscles are displayed as observed when focusing at the ventral side through the cuticle into the larvae. In A-C the most superficial muscle, 27, is shown, in K-L the most interior muscles, 6 and 7, are shown. Scale Bar: 100 &mu;m, &Delta;Z between slices is 2 &mu;m.
<img src="/files/ftp_upload/2249/2249fig4.jpg" alt="Figure 4" /> Figure 4 Identity of neuromuscular junctions of Drosophila larvae. (A-D) NMJs were visualized by expression of a DGluRIIA-mRFP fusion protein1. The NMJs are shown as observed when focusing at the ventral side through the cuticle into the larva. In (A-B) the most superficial NMJ, 27, is shown, in C-D the most interior NMJs, 6 and 7, are shown. Scale Bar: 100 &mu;m, &Delta;Z between slices is 5 &mu;m. (E) and (F) the identity of the superficial (E) and more interior (F) NMJs is given for reference.

<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>

No conflicts of interest declared.

The presented method was initially developed to study glutamatergic synapses on the body wall muscles of Drosophila melanogaster larvae. The Drosophila neuromuscular junction (NMJ) is characterized by a stereotypical cyto-architecture of muscles and neurons and is thus ideally suited for in vivo imaging. However, the described anesthetization protocol is not limited to imaging the NMJ; the transparency of Drosophila larvae facilitates the adaptation of the described protocol to study the development of organs, migration of cells, transport of axonal cargo and sub-cellular reorganization within cells.

Reagents:
<ul>
<li> Desflurane (Suprane, Baxter, Unterschlei&szlig;heim, Germany)</li>
<li> Halocarbon oil (e.g. Voltalef H10S oil / Atofina, Puteaux, France)</li>
<li> Fly culture medium</li>
</ul>
Equipment:
<ul>
<li> Inverted confocal microscope</li>
<li> Binocular microscope (e.g. Stemi 2000, Carl Zeiss, Jena, Germany)</li>
<li> Small paintbrush as used to sort flies</li>
<li> Incubator</li>
<li> Custom built imaging chamber (For mechanical drawings see Ref. 3)</li>
<li> Vaporizer/anesthetization device (For details see Ref. 3)</li>
</ul>

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.

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

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 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 ...

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

Research

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Biology

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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