The authors thank David Larson for developing the original protocol for imaging the live Drosophila pupal eye. We thank our colleagues Richard Carthew, Shoichiro Tsukita and Matthew Freeman, who developed the transgenic flies that we combined to generate our live-imaging fly lines. Brittany Baldwin-Hunter gave helpful comments on the manuscript. This work was supported by start-up funds awarded to Ruth Johnson by Wesleyan University.

Figure 3 shows a summary of the experimental procedure.
1. Tissue Preparation
<ol>
	<li>To determine the consequences of modified expression of a gene of interest, set up the following cross in duplicate and maintain at 25 &#176;C: UAS-transgene (males)X GMR-GAL4; UAS-&#945;-cat:GFP, ubi-DE-Cad:GFP ; + / SM5-TM6b (virgin females) NOTE: To obtain control tissue cross UAS-lacZ males to GMR-GAL4; UAS-&#945;-cat:GFP, ubi-DE-Cad:GFP females. &#946;-Galactosidase generates no defects in cell behavior in the retina.</li>
	<li>To enhance efficacy of RNAi transgenes, cross UAS-RNAi males to GMR-GAL4, UAS-Dcr-2; UAS-&#945;-cat:GFP, ubi-DE-Cad:GFP; + / SM5-TM6b virgin females. 
	NOTE: Occasional defects in cell behavior in retinae expressing ectopic Dcr-2 have been observed (data not shown). Vigilance is recommended if using this approach.</li>
	<li>Select white pre-pupae of appropriate genotypes and place in 1.5 ml microcentrifuge tubes. If the pupae express Dcr-2, select either male or female pupae: expression of UAS-Dcr-2, an insert on the X chromosome, is stronger in males. Pupae should be sexed at 0 hr APF before pigmentation of the pupal case &#8211; the gonads of males are visible as large translucent globes on the lateral sides of the pupa (approximately one-third from the posterior).</li>
	<li>Incubate microcentrifuge tubes containing pupae, in a clean plastic tip-box at 25 &#176;C. To prevent desiccation of pupae place a 10 cm2 piece of tissue, soaked in distilled water, into the tip-box. 
	NOTE: if the humidity in the tip-box becomes too high the pupal case may become soggy and difficult to dissect in subsequent steps.</li>
	<li>Incubate for appropriate time. To capture cell behaviors associated with patterning the eye, prepare pupae for imaging at around 17 hr APF, 20 hr APF or 24 hr APF. See Representative Results for further discussion on when specific cell behaviors are best observed.</li>
	<li>Place a piece of fresh double-sided tape onto a black sylgard dissection dish. Position a pupa, dorsal side up, with the head of the pupa adhered to the double-sided tape (Figure 1A).Try not to adhere the thoracic and abdominal regions of the pupa to the double-sided tape. With forceps carefully lift and remove the operculum to expose the pupal head. Tear along the side of the pupal case to expose the area around one eye.</li>
	<li>Gently remove the pupa from the adhesive tape. If the pupa remains adherent, apply a small volume of distilled water to the junction between the pupal case and tape to weaken adhesion.</li>
</ol>
2. Mounting
<ol>
	<li>Construct a small 15 mm2 frame from blotting paper and punch a hole in the middlethat is 5.5 mm in diameter (Figure 1B). Immerse in distilled water and place in the center of a microscope slide. Using a 30 cc syringe, squeeze out a uniform ring of petroleum jelly to surround the blotting paper frame. Ensure that the petroleum jelly ring is marginally higher than the width of the pupa and that the diameter of the ring is marginally less than the width of a cover slip.</li>
	<li>Add a small bead of petroleum jelly directly onto the slide, in the hole punched into the blotting paper(Figure 1C).Carefully arrange the pupa, on its side, supported by the petroleum jelly bead (Figure 1D).</li>
	<li>Place a coverslip on top of the petroleum jelly ring so that it contacts the epithelium above the eye neuroepithelium (Figure 1D). Gently compress the preparation to seal the coverslip against the petroleum jelly and generate a small flat contact surface between the pupal eye region and the coverslip.</li>
</ol>
3. Fluorescence Imaging
<ol>
	<li>Image the pupal preparation using a fluorescent microscope. Every 7 min capture serial sections through the apical domain of the eye neuroepithelium, in the region of the adherens junctions. 
	NOTE: a) Appropriate serial sections are usually 4-5 per&#160;z-stack&#160;comprised of 0.2 &#956;m z-steps. b) Irregularities in the topology of the epithelium, including mild bumps and folds, may make it challenging to acquire in-focus images of large regions of tissue. While imaging, one may find part of an area of interest to be in-focus, and a neighboring region to be out-of-focus. Including a small droplet of distilled water between the pupal eye and coverslip can eliminate some acute tissue folds but not the mildly uneven topology characteristic of the early stages of pupal eye morphogenesis. In these instances oversample the tissue by extending the z-stack until in-focus slices are acquired for the entire field. c) Capturing serial sections every 7 min should enable live-imaging for 3 to 4 hr without a significant reduction in the intensity of GFP fluorescence that may compromise image quality. Shorter time-intervals may reduce the total time of imaging.</li>
	<li>Continue imaging for 3 to 4 hr. Do not use an automatic focus and time function that is available on many fluorescent microscopes since pupal growth and pumping of the hemolymph moves the position of the retina and vigilant re-focusing is required every 14-21 min. NOTE that imaging beyond 4 hours may slow or stall morphogenesis of the eye.</li>
	<li>Use appropriate deconvolution software to reduce background and enhance contrast of the serial section images. For each z-stack file, perform the following in LAS AF software (see Table of Materials): Navigate to the Tools panel, select 3D Deconvolution and click Apply.</li>
	<li>Generate a maximum projection (MP) image for each deconvoluted stack file: Navigate to the Tools panel, select 3D Projection and click Apply. 
	NOTE: The Maximum Projection algorithm may fail to generate a uniformly in-focus image for tissue that has been oversampled. A technique for sharpening out-of-focus regions is outlined in step 5.2.</li>
</ol>
4. Post-imaging Pupal Rescue and Phenotype Verification
<ol>
	<li>To address whether artifacts were introduced or the pupa harmed during imaging, carefully remove the pupa from the imaging rig: using forceps remove the coverslip and transfer the pupa to a clean vial of food. Store at room temperature or 25 &#176;C until the adult fly emerges.</li>
	<li>Carefully examine the phenotype of the adult eye and compare to the eyes of adult flies emerging from the original fly cross.</li>
</ol>
5. Image Processing
<ol>
	<li>Automatic Image Alignment: 
	NOTE: The live Drosophila tissue will shift and grow throughout the imaging process. Consequently, the centers of images gathered at successive time points may not correspond to the same point in the Drosophila tissue. In addition the entire retinal disc rotates about 30&#176; between ~21 and 23 hr APF. To highlight individual cell behaviors and reduce distractions caused by organismal growth,align each MP image.While this can be performed manually,the image editing software used here (see Table of Materials) has built-in algorithms that can expedite the process.
	<ol>
		<li>From the File tab of the main menu, open Scripts and select Load Files into Stack.</li>
		<li>Import the MP image files into the Load Layers panel and select OK. Make sure that the Attempt to Automatically Align Source Images option is not selected. 
		NOTE: If this option is selected, the software may use an alignment algorithm that distorts the image data.</li>
		<li>Once all MP files have loaded into the Layers pane, make sure that all images are in chronological order such that the earliest time point is at the top of the layer stack. If the layers are not in the correct order, drag and drop them until they are ordered correctly.</li>
		<li>Select Auto-Align Layers from the Edit tab of the main menu. Choose Reposition and click OK.</li>
		<li>If the Auto-Align algorithm fails to orient the frames to a relevant focal point, make minor adjustments using the Move tool.</li>
	</ol>
	</li>
	<li>Composite Sharpening: 
	NOTE: Out-of-focus regions of an initial MP image can be sharpened by &#8216;cropping&#8217; the corresponding deconvoluted stack in LAS AF software. Cropping a stack file allows one to manually restrict the interval of a z-stack that is subjected to the Maximum Projection algorithm.
	<ol>
		<li>Locate the Crop function in the Tools panel (under the Process tab). Manually restrict the initial and final slices such that they span only the adhesion belt within the initially out-of-focus region. Click Apply to generate a new stack file that is optimized for this region.</li>
		<li>Generate a new Maximum Projection for the locally optimized stack and export as a TIFF file.</li>
		<li>In the image editing software (see Table of Materials), open Scripts (located in the File tab of the main menu) and select Load Files into Stack.</li>
		<li>Import the initial MP image file and locally optimized MP file for a particular time point into the Load Layers panel and select OK. Make sure that the Attempt to Automatically Align Source Images option is not selected.</li>
		<li>Select both layers in the Layers pane and then choose Auto-Blend Layers (located in the Edit tab of the main menu) to automatically mask the appropriate regions of the two images, yielding a uniformly in-focus retinal field. Save this composite image as a TIFF file.</li>
		<li>Repeat steps 5.2.1 through 5.2.5 for all suboptimal MP images.</li>
	</ol>
	</li>
	<li>Further Adjustments: Rotate, crop, and Adjust the Levels of the Movie Layers:
	<ol>
		<li>With all of the layers selected, use the Transform tool (Edit &#62; Free Transform) to rotate the images so that the dorsal-ventral axis of the retina is aligned to the y-axis of the movie frame.</li>
		<li>If necessary, use the Crop tool to reduce the field of view to a desired size and shape.</li>
		<li>Use level adjustment (of individual frames) to help enhance contrast between the cell membrane and cell body.</li>
		<li>For stylistic purposes and to emphasize specific cell behaviors, introduce false color to highlight points of interest in each frame.</li>
	</ol>
	</li>
	<li>Animation: Convert the Images into a Movie:
	<ol>
		<li>Open the Animation pane from the Windows tab of the main menu. Select Make Frames from Layers from the menu located in the upper right hand corner of the Animation pane.</li>
		<li>Note that the layers are added to the animation pane in sequential order beginning from the bottom of the stack. To reverse the order of the frames, first select all of the frames and then select Reverse Frames from the Animation pane menu.</li>
		<li>Select a frame delay time for each frame using the dropdown menu below the frame thumbnail. 
		NOTE: The frame delay for Movies 1 to 4 presented in this paper is 0.8 sec.</li>
		<li>From the File tab of the main menu, open Export and select Render Video. Choose QuickTime Export under File Options and select a desired video format. Under Render Options set Frame Rate to Custom, enter a frame rate of 15, and click Render.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

The description of wild-type development (above) forms the basis for comparisons of patterning events in RNAi or overexpression genotypes. Comparisons of live tissue development are invaluable when determining precisely which cell behaviors are regulated by a protein of interest. Further, and not described here, live cell imaging enables one to make qualitative and/or quantitative descriptions of the role of a gene of interest in the events that pattern the eye. By 30-32 hr APF most pigment cells have acquired stable pos...

The compound Drosophila eye is characterized by the stereotyped arrangement of its ~750 ommatidia separated by a honeycomb-lattice of accessory pigment cells 4,5. These pigment cells are patterned by a coordinated combination of events: local cell movements, cell growth, changes in cell shape, and apoptosis. Live visualization of this epithelium allows one to study the molecular mechanisms underpinning these events in a physiologically relevant and unperturbed three-dimensional context.
In contrast to previous protocols 6,7, the technique outlined here incorporates an efficient method for stabilizing extraneous tissue movement that cannot be uncoupled from the imaging process. This method enhances studies of cell behavior in the developing Drosophila pupal eye epithelium &#8211; a tissue that grows, rotates, and shifts over the course of imaging. In addition the motion-stabilization technique described here will be useful for studying cells in other contexts where extraneous movement occurs.
To visualize cell boundaries in the Drosophila retinal field, transgenic fly lines were generated that express ubi-DE-Cadherin:GFP as well as UAS-&#945;-catenin:GFP under control of the eye-specific driver GMR-GAL4 1-3. The use of two GFP-tagged membrane markers allows for the visualization of cell boundaries at lower intensity light. This minimizes tissue damage and photobleaching on repeated exposure to high-energy wavelengths, enabling increased frame rate and movie duration. To enhance efficacy of RNAi transgenes, UAS-Dcr-2 was also incorporated into a second fly line 8.
In a third update from previous protocols, a simpler imaging rig that is easily assembled in most laboratories is described. This apparatus obviates the requirement to have a specialized imaging rig generated by a university&#8217;s &#8216;machine shop&#8217; or similar service. This imaging rig is similar to that used to image other pupal tissues 9,10.
Presented here is a simple live-cell imaging protocol that can be used to directly assess the morphogenetic events that contribute to eye patterning from ~17 to 42 hr after puparium formation (hr APF). Specifically, this protocol enables one to determine the consequences of modifying gene expression during pupal development.

<table><tbody><tr><td>GMR-GAL4; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP ; + / SM5-TM6b</td><td>Available on request from authors</td><td></td><td></td></tr><tr><td>UAS-dcr-2; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP; + / SM5-TM6b&amp;nbsp;</td><td>Available on request from authors</td><td></td><td></td></tr><tr><td>Scotch double stick tape</td><td>3M</td><td>665</td><td></td></tr><tr><td>Black Sylgard dissection dish</td><td></td><td></td><td>Fill glass petri dish to rim with Sylgard (colored black with finely-ground charcoal powder) or Sylgard 170; leave at room temperature for 24-48 hr to polymerize</td></tr><tr><td>Sylgard</td><td>Dow Corning</td><td>SYLG184</td><td></td></tr><tr><td>Sylgard (Black)</td><td>Dow Corning</td><td>SYLG170</td><td></td></tr><tr><td>Glass petri dish</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>25 x 75 x 1 mm glass microscope slide</td><td>Fisher Scientific</td><td>12-550-413</td><td></td></tr><tr><td>22 x 40 mm glass coverslip</td><td>VWR</td><td>48393-172</td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>91150-20</td><td></td></tr><tr><td>Whatman 3mm Chromatography Paper</td><td>Fisher Scientific</td><td>05-713-336</td><td></td></tr><tr><td>Vaseline</td><td>Fisher Scientific</td><td>19-086-291</td><td>Or purchase at local pharmacy.</td></tr><tr><td>30 ml syringe</td><td>Fisher Scientific</td><td>S7510-30</td><td></td></tr><tr><td>Leica TCS SP5 DM microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>LAS AF Version 2.6.0.7266 microscope software</td><td>Leica Microsystems</td><td></td><td></td></tr></tbody></table>

live-imaging of the drosophila pupal eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the pupal eye is a successful strategy to observe cell behaviors that contribute to patterning of the neuroepithelium. The role of specific proteins can be readily assessed by expressing transgenes that modify protein levels during eye development. To do this GMR-GAL4 is used to drive transgene expression behind the morphogenetic furrow. This offers the advantage of not perturbing earlier events that establish the eye field during the first two larval instars. In addition many RNAi transgenes req...

Watch this Scientific Journal Video about Live-imaging of the Drosophila Pupal Eye at JoVE.com

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Research

JoVE Journal

Developmental Biology

9.4K Views.  Wesleyan University. This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Video: Live-imaging of the Drosophila Pupal Eye

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Neuroscience

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Biology

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

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 ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

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.

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Extended Live Imaging of Drosophila Pupal Leg Morphogenesis Without Puparium

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which fate-determined cells form three-dimensional organismal shapes remains unclear. Recent advances in confocal microscopy have facilitated efforts to observe cell dynamics during development through live imaging. The Drosophila melanogaster pupa is ideal for live imaging due to its immobility, transparent pupal cuticle, and the availability of fluorescent reporter lines. A primary challenge for imaging is the puparium, the cuticle surrounding the pupa, which obstructs optical imaging. While previous methods involved either partial or complete removal of the puparium, maintaining pupal viability for extended periods after this procedure has remained challenging. Here, a simple method is presented for days-long live imaging of the Drosophila leg during the pupal stage, involving complete puparium removal. The method includes removing the puparium from a pupa adhered to double-sided tape, followed by assembling a small chamber on a glass-bottom dish to enclose the pupa and a drop of water. This setup is straightforward, reliable, and supports extended pupal survival by preventing desiccation. Long-term live imaging of the Drosophila pupa has significantly contributed to capturing how the adult leg undergoes dramatic three-dimensional structural changes over 2-3 days. These changes include the transient formation of an intriguing structure (the Parthenon-like structure) by epithelial cells, rapid tissue narrowing, joint formation, and bristle elongation. This method is applicable to the observation of various tissues and can potentially be combined with other techniques, such as optical gene induction, to advance the understanding of cell dynamics during the final shape formation of tissues in the pupal stage.

Long-Term Live Imaging of Drosophila Pupal Leg Development After Puparium Removal

Here, a protocol is presented for long-term live imaging of the Drosophila leg during the pupal stage, involving complete removal of the puparium. This protocol can also be applied to other tissues.

Over the past decades, significant progress has been made in understanding the mechanisms of cell fate determination. However, the process by which ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Live-imaging of the Drosophila Pupal Eye

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