This work was supported by the division of intramural research at the National Institutes of Health/NHLBI (1ZIAHL006126) and a Lenfant Biomedical Postdoctoral Fellowship awarded to DAL.

1. Preparation of Materials Required to Explant Third Instar Larval Brains
<ol>
	<li>Prepare tools required for microdissection.
	<ol>
		<li>Forge two dissecting tools (a scalpel and a hook) by first placing a single dissecting pin into each pin holder. Secure the pins tightly by hand or with pliers (Figure 1C).</li>
		<li>Use a pair of old forceps to bend one dissecting pin to an approximately 120&#176; angle. Do this under a dissecting microscope to ensure that the top half of the pin lies flat when the pin holder is held in hand. This tool will be used as a scalpel to hold down or slice through tissue.</li>
		<li>Use a pair of old forceps to bend the second pin into a hook that will be used to hold and scrape away tissue.</li>
		<li>Cover each dissecting tool with a pipette tip to protect the tools from damage. With care, well-formed tools may be used for dissection indefinitely. Replace damaged or deformed pins as necessary.</li>
	</ol>
	</li>
	<li>Prepare dissecting medium.
	<ol>
		<li>In a tissue culture hood, dilute the antibiotic-antimycotic supplement into the Schneider&#8217;s culturing medium. Swirl the media to incorporate the supplement.</li>
		<li>Prepare several 5 ml aliquots of the supplemented media. Store all media at 4 &#176;C until ready for use.</li>
		<li>Warm a 5 ml aliquot of supplemented medium to room temperature. Draw the medium into a sterile 5 ml syringe and screw on a sterile 0.2 &#181;m syringe filter.</li>
	</ol>
	</li>
	<li>Select larvae for dissection. Use a dissecting probe to select crawling third instar larvae as the brain will be easiest to dissect. Optional: deposit larvae on a grape agar plate. 
	Note: Do not use overly crowded vials or bottles with &#8220;wet food,&#8221; as these tend to force second or first instar larvae to crawl out prematurely. 
	Note: Some genetic backgrounds will necessitate using younger larvae. This does not affect the protocol, but does make the dissection more difficult.</li>
</ol>
2. Dissection of the Central Nervous System
<ol>
	<li>On a single glass slide, create two 50 ml pools of warm dissecting media separated by about 3 cm. One pool will be used for gross dissection and other used for fine dissection (Figure 1C).</li>
	<li>Transfer several larvae to one of the media drops.</li>
	<li>Move the coverslip with larvae to a dissecting microscope. Zoom into the pool containing the larvae such that the anterior and posterior ends of the larvae are discernible.</li>
	<li>Use a pairs of forceps to grasp the mid-region of a single larva. Take a second pair of forceps in the other hand and grasp the larva close to the same region. Pull the two pairs of forceps apart to gently tear the larva in half. Repeat the above two steps for all larvae on the coverslip.</li>
	<li>Dispose of the posterior halves of the larval body by transferring them to a tissue.</li>
	<li>Zoom into a larva so that the anterior mouthhooks are clearly visible. Use both pairs of forceps to create a small incision, or notch, on opposite sides of the larval mouthhooks between the third thoracic and first anterior denticle bands.</li>
	<li>Grasp the mouthhooks with the forceps in the non-dominant hand. Use the forceps in the other hand to gently peel away cuticle in an anterior-to-posterior direction. Continue to slowly peel away cuticle until the central nervous system is exposed.</li>
	<li>Isolate the central nervous system from the rest of the larva by tearing tissues with the forceps. Use caution not to disturb the central nervous system. 
	Critical step: Do not tug on the central nervous system! Discard damaged samples with a torn ventral nerve cord (Figure 2A) or distorted optic lobes (Figures 2B and 2B&#8217;).</li>
	<li>Repeat the above two steps for all larvae on the coverslip. Transfer the healthy explanted tissue to the second (clean) pool of medium on the coverslip for the fine dissection stage.</li>
	<li>Use the dissecting pin tools to clear away the peripheral tissues (e.g., eye, wing, and leg discs) from the brain. Slide the scalpel between the wanted (brain) and the unwanted tissue, pinning the connective tissue to the coverglass. Use the hook to gently tease away the undesired tissue while simultaneously pressing the scalpel to the coverslip in saw-like movements.</li>
	<li>Critical step: Work slowly and deliberately to remove all discs from each brain. Use caution not to disturb the brain morphology. A healthy brain will have an intact ventral nerve cord and symmetric, round optic lobes (Figure 2C).</li>
</ol>
3. Mounting Explanted Brains for Live Imaging
<ol>
	<li>Invert a 50 mm gas-permeable culture dish with the clear film facing up (Figure 2D). Depress the syringe to deposit a small drop (about 30 &#181;l) of medium onto the center of the dish.</li>
	<li>Transfer the isolated brains, one at a time, to the drop of medium on the dish. Use the hook tool to scoop up a brain underneath the optic lobes. Alternatively, use forceps to grasp axons projecting from the ventral nerve cord.</li>
	<li>Collect 5-10 brains in the drop of medium. Submerge the brains by using the dissecting pin tools to push individual brains to the bottom of the drop of medium as many of them will be trapped at the meniscus (Figure 2E).</li>
	<li>Orient the brains using the dissecting pin tools to align brains depending on the NBs to be imaged (Figure 3A). To image the dorsal NBs, place the ventral nerve cord facing down (along the membrane). To image the anterio-ventral NBs, place the brains with the ventral nerve cord facing upward (away from membrane). Align the brains such that all the ventral nerve cords point in the same direction within the x-y plane.</li>
	<li>Use a syringe or plastic transfer pipette to place 4 halocarbon (HC) oil drops (about 30 &#181;l each) onto the gas permeable membrane. The volume of each drop of oil should be equivalent to each other and to the drop of medium. Space the drops of oil to correspond to the four corners of a coverslip centered around the drop of culturing medium. Once complete, the five drops (four drops of oil and one drop of medium in the center) will resemble the five facets on Western-style dice (Figure 3B).</li>
	<li>Lower a #1.5 22 mm coverslip on the assembly such that it hits all 5 drops at the same time. Maintaining an even pressure across the samples reduces the likelihood that they will be disrupted. Wait about 3 min as the oil and medium disperse under the weight of the coverslip. A cross-like shape of media will form (Figures 3C and 3D).</li>
	<li>Critical step: Lower the coverslip onto the surface of the brains by removing excess media using a tissue paper wick (Figure 3C). Do this while watching the sample under the dissecting scope. As media is removed, the coverslip will lower. Stop once the coverslip touches the brains. Do not over-wick as this will cause tissue distortion or brain explosions.</li>
	<li>If imaging dorsal NBs (Figure 3E), move to step 3.9. If imaging anterio-ventral NBs, the coverslip must be slightly moved (by gently nudging it with forceps) in the direction of the ventral nerve cord tips. This causes the brain to rotate, which brings the brain lobes in direct contact with the coverslip to facilitate imaging (Figure 3F).</li>
	<li>Seal the chamber by encircling the coverslip with small amounts of halocarbon oil (Figure 3D). Excess oil oozing from the coverslip should be minimized and blotted away with a tissue.</li>
</ol>
4. Live Imaging of Central Brain Neuroblasts
Note: For this work, a Nikon Eclipse Ti spinning-disk confocal microscope (inverted microscope) was used for live imaging; details of our setup have been previously published31. Alternatively, the sample can be imaged using an upright system.
<ol>
	<li>Add a drop of immersion oil to the coverslip just above the mounted brains. This will help center the objective directly above the sample.</li>
	<li>Critical step: Maintain brains at a constant temperature of 25 &#176;C with a stage incubator. Gently position the mounted sample into the stage incubator and cover the chamber with its lid.</li>
	<li>Locate central brain NBs using transmitted light by focusing on the large, round cells that reside in the central and medial areas of the optic lobes; do not use epi-fluorescence. This becomes easier with practice. It is very important to expose the sample to the least amount of light as possible. Do not waste time imaging brains that are damaged.</li>
	<li>Once in place, take quick exposures using the confocal to determine proper location and depth. Limit depth to the first 10-15 &#181;m. Adjust as needed, and take another test image.
	<ol>
		<li>Optional step: Collect a test movie of the ventral nerve cord to ensure there is no x-y drift. If drift is detected, wait 15-30 min for the sample to settle. If the drift does not settle in 30 min, prepare a new sample. Major drift can usually be traced back to the addition of excess oil, or the unequal size of oil drops during mounting.</li>
	</ol>
	</li>
	<li>To eliminate axial drift (z-drift) use a continuous automatic focusing device that uses an infrared LED. Alternatively, manually correct the focal plane.</li>
	<li>Once a NB of interest is identified, proceed with imaging regimen. As with all live cell imaging, the amount of laser light, exposure time, camera binning, objective magnification, time and/or z-step interval must all be empirically optimized for a particular experiment to answer the question at hand. The goal is to minimize light damage while still collecting useful data. See discussion for additional guidance.
	<ol>
		<li>Image the entire volume of the NB by collecting 12-14 images spaced by 1 &#181;m in the z-axis. Adjust the time resolution to capture single or multiple cell cycles of the same NB. As a general guideline, use 10-30 sec intervals for single cell cycle imaging and 1-3 min intervals for multiple cell cycles.</li>
	</ol>
	</li>
	<li>Critical step: Confirm that the sample is healthy by monitoring the duration of mitosis. If mitosis takes more than 15 min, move on to another brain. A healthy brain will show many NBs within the same field of view undergoing several rounds of mitosis. Note that each cell cycle is slightly longer than the previous one due to light damage.</li>
	<li>Once imaging is complete, disassemble the incubation chamber, and discard everything but the gas-permeable culturing dishes. Rinse the culture dish surface with 95% ethanol and wipe well with a tissue. Repeat two more times.</li>
</ol>
5. Preparation of Brains for Immunofluorescence
<ol>
	<li>Collect 20 or more explanted brains into a 1.5 ml tube containing approximately 0.2 ml of supplemented medium.</li>
	<li>Remove medium from the tube and rinse brains once with 0.5 ml PBSTx (Phosphate Buffered Saline, PBS, with 0.3% Triton X-100). Let brains settle to the bottom of the tube between all exchanges of liquid. The Triton X-100 detergent is used to permeabilize the tissue. Rinsing typically takes less than 30 sec; simply remove the PBSTx once brains have settled to the bottom of the tube.</li>
	<li>Replace the solution with 0.5 ml of 9% electron microscopy grade paraformaldehyde diluted in PBSTx. Incubate with nutation for 15 min at room temperature.</li>
	<li>Dispose of fixative according to hazardous waste regulations.</li>
	<li>Wash the samples with 0.5 ml PBSTx for 15 min. Repeat the wash step with fresh PBSTx two more times.</li>
	<li>Replace the solution with 0.5 ml PBT (PBS,1% Bovine Serum Albumin (BSA), and 0.1% Tween-20). Incubate with nutation for 1 hr at room temperature. This step blocks the nonspecific binding of antibody to the tissue.</li>
	<li>Replace the solution with 0.5 ml primary antibody diluted in PBT supplemented with 4% normal goat serum (NGS). Incubate with nutation overnight at 4 &#176;C.</li>
	<li>Discard or save the diluted primary antibody.</li>
	<li>Wash the samples with 0.5 ml PBT for 15 min. Repeat the wash step with fresh PBT two more times.</li>
	<li>Replace the solution with 0.5 ml of modified PBT (PBS, 2% BSA, 0.1% Tween-20, and 4% NGS). Incubate with nutation for 1 hr at room temperature.</li>
	<li>Replace the solution with 0.5 ml secondary antibody diluted into modified PBT. Incubate with nutation for 2 hr at room temperature.</li>
	<li>Replace the solution with 0.5 ml PBST (PBS with 0.1% Tween-20). Incubate with nutation for 15 min at room temperature. Repeat the wash step with fresh PBST two more times.</li>
</ol>
6. Mounting Samples for Immunofluorescence
<ol>
	<li>Carefully transfer the samples as a drop on a glass slide, restricting the drop towards the left of the center of slide.</li>
	<li>Add a large drop (about 2 cm in diameter) of mounting medium (for example, Aqua-Poly/Mount) to the center of the slide. Avoid the pool of PBST that is toward the left.</li>
	<li>Use forceps to transfer the brains from the pool of PBST to the pool of mounting medium. Adding mounting medium directly on top of the brains may disrupt their orientation and should be avoided.</li>
	<li>Under a light microscope, arrange the samples in the mounting medium with the side that will be imaged facing up.</li>
	<li>With the slide under the light microscope, slowly lower a #1.5 glass coverslip on top of the samples. Optional step: Dislodge minor air bubbles by lightly pushing on the coverslip.</li>
	<li>Allow the mounting medium to polymerize for several hours, or overnight, by lying slides flat, light-protected, at room temperature.</li>
	<li>Slides may be imaged the next day, or stored prior to imaging.</li>
</ol>

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<li>Prasad, M., Jang, A. C., Starz-Gaiano, M., Melani, M., Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+protocol+for+culturing+Drosophila+melanogaster+stage+9+egg+chambers+for+live+imaging%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">A protocol for culturing Drosophila melanogaster stage 9 egg chambers for live imaging.</a> Nat. Protoc. 2, 2467-2473 (2007).</li>
<li>Rusan, N. M., Peifer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+a+novel+centrosome+cycle+in+asymmetric+cell+division%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">A role for a novel centrosome cycle in asymmetric cell division.</a> J. Cell Biol. 177, 13-20 (2007).</li>
<li>Martinez-Campos, M., Basto, R., Baker, J., Kernan, M., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Drosophila+pericentrin-like+protein+is+essential+for+cilia%2Fflagella+function%2C+but+appears+to+be+dispensable+for+mitosis%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis.</a> J. Cell Biol. 165, 673-683 (2004).</li>
<li>Dix, C. I., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+Spd-2+recruits+PCM+to+the+sperm+centriole%2C+but+is+dispensable+for+centriole+duplication%5BTitle%5D)+AND+%22Curr.+Biol%22%5BJournal%5D)">Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication.</a> Curr. Biol. 17, 1759-1764 (2007).</li>
<li>Moutinho-Santos, T., Sampaio, P., Amorim, I., Costa, M., Sunkel, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+localisation+of+the+mitotic+POLO+kinase+shows+a+highly+dynamic+association+with+the+mitotic+apparatus+during+early+embryogenesis+in+Drosophila%5BTitle%5D)+AND+%22Biol.+Cell%22%5BJournal%5D)">In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila.</a> Biol. Cell. 91, 585-596 (1999).</li>
<li>Megraw, T. L., Kilaru, S., Turner, F. R., Kaufman, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+centrosome+is+a+dynamic+structure+that+ejects+PCM+flares%5BTitle%5D)+AND+%22J.+Cell+Sci%22%5BJournal%5D)">The centrosome is a dynamic structure that ejects PCM flares.</a> J. Cell Sci. 115, 4707-4718 (2002).</li>
</ol>

The authors have nothing to disclose.

Live cell imaging is an invaluable approach used to study the mechanisms that regulate ACD of stem cells, the differentiation of cell lineages, and the morphogenesis of complex tissues. Although research groups have historically employed a variety of techniques to visualize NB ACD, these approaches can be generally grouped into two categories that primarily differ with respect to whether the brain tissue is left intact or dissociated.
Imaging dissociated NBs has its advantages. For one, dissoc...

Stem cells maintain a balance of differentiation and self-renewal to generate cellular diversity during early development and replace damaged cells in adult tissues. Regulation of this homeostasis prevents both the loss and over-expansion of the stem cell population, which can result in deleterious tissue degeneration or tumorigenesis.
Neuroblasts (NBs) are the widely studied neural stem cells of the Drosophila central nervous system (Figure 1A) that first form during embryonic stages1,2. NBs undergo repeated rounds of asymmetric cell division (ACD) to produce two unequally fated cells: a self-renewing stem cell and a differentiating cell. ACD is guided by centrosomes, the non-membranous organelles that act as the microtubule-organizing centers of most cells3. During mitosis, NB centrosomes organize and orient the bipolar mitotic spindle along the apical-basal polarity axis. Upon cleavage of the dividing NB, apical fate determinants that specify the stem cell fate, and basal fate determinants that specify differentiation, are segregated into the unequal daughter cells.
In the larval central brain, two types of NBs may be distinguished by their number, position, transcription factor expression, and cell lineage (Figure 1A)4-6. Type I NBs are the most abundant, and about 90 of them populate the anterior and posterior sides of each optic lobe of the brain7. These NBs express the transcription factor Asense (Ase), and they characteristically divide into one self-renewing NB and one smaller ganglion mother cell (GMC; Figure 1B). Each GMC undergoes a single terminal division to generate two neurons or glia (Figure 1B). In contrast, the eight Type II NBs that populate the posterior side of each optic lobe lack Ase expression5. They undergo ACD to produce one self-renewing NB and one intermediate neural progenitor (INP).
The INP, in turn, divides asymmetrically three to five times. Each of these divisions results in regeneration of the INP and the production of a single GMC4. Collectively, the specific NB identity and the temporal order of GMC birth gives rise to the astounding neuronal diversity of the adult central nervous system.
Understanding the cell biology that underlies NB ACD has been vastly improved through the use of live cell imaging techniques. Published protocols used by researchers to image live NBs vary widely. Overall, however, these methods may be grouped into two general categories distinguished by whether the larval brain is left intact or mechanically dissociated. Both techniques have distinct advantages and disadvantages depending on the researcher&#8217;s application.
Early reports revealing live NB cell divisions involve some degree of manual dissociation of the larval brain. These protocols detail smearing8 or teasing apart9 the brain in order to grow short-term primary cultures of the NBs on glass coverslips. To improve imaging, the round NBs are usually flattened on the coverslips with either glass slides8 or agarose pads9. Although flattened cells have improved optics, these techniques often lead to NB mitotic defects, including regression of the cleavage furrow and the inability to divide more than one time. Therefore, protocols that involve both manual dissociation and physical distortion of the larval brain tissue are generally only suited to very short-term (i.e., one cell cycle or less) applications. Likewise, semi-squashing or completely flattening intact brains between glass slides and coverslips limits the time one may spend imaging a given specimen to an approximately 30 min period10. Despite this limitation, this approach has been used successfully11-14.
Recent efforts to image live dissociated NBs limit physical distortion of the isolated cells. These techniques are particularly useful for applications where it is necessary to sustain cell cultures for multiple cell division cycles. For example, dispersed cells from manually dissociated brains may be partially embedded in a clot made from the mixture of fibrinogen and thrombin15 for long-term imaging16. Alternatively, explanted brains may be first chemically dissociated with the enzyme collagenase, next manually disrupted by repeated passage through a pipet tip, and subsequently plated on poly-L-lysine-coated glass bottom dishes17,18. Coating glass dishes with either fibrinogen or poly-L-lysine promotes the attachment and slight spreading of round cells, bringing them as close to the coverslip as possible without major physical distortion. In addition, these methods involve culturing the NBs in medium that may be readily exchanged for pharmacological perturbation experiments18. Overall, directly plating dispersed NBs improves imaging optics without sacrificing long-term imaging capabilities. Recently, a protocol describing the long-term culturing of dissociated NBs has been detailed19.
However, this approach is not without its limitations. There are several caveats to imaging live dissociated NBs. In a field of dispersed cells, for example, it is difficult to identify specific NB lineages that are spatially organized in the intact brain1. Likewise, distinguishing the Type I versus Type II NBs within dissociated tissue is also challenging without the expression of lineage tracers or subtype-specific transgenes, such as worniu-GAL4, asense-GAL8020. Although these transgenes are quite useful for some applications, they do limit researchers to those transgenes expressed under the control of the UAS enhancer element21 and require more complex genetic schemes. Studies that concern the spatial or temporal development of NBs, therefore, require in vivo imaging in the context of an intact tissue.
Moreover, studies of dissociated embryonic NBs indicate that the physical contact of adjacent cells is critical for the interphase localization of key polarity determinants22,23. These studies show completely isolated NBs no longer maintain the invariant mitotic spindle axis. Instead, these cells display a more randomized spindle axis and wide angles of separation between successive GMC buds, perhaps due to the loss of apical centrosome positioning22. Although the relationship between the larval NBs and their neighboring cells has not been extensively studied, it is worth considering that the larval stem cell microenvironment may impinge upon cell polarity or other behaviors. To maintain the physiological context of larval NBs, we image ACD from whole brains.
Given the inherent limitations associated with imaging dissociated NBs, our lab and others have established protocols to image successive rounds of NB ACD from whole larval brains. Techniques to image intact brains often replace the rigid surface of glass on one side of the culture chamber with a flexible membrane to prevent tissue distortion or damage and allow for gas exchange. Some methods involve mounting explanted brains in a mixture of insect culturing medium, serum, and ascorbic acid with the fat bodies isolated from ten larvae24. The isolated fat bodies are added because they secrete a mitogen known to stimulate NB cell division25. This protocol preserves the integrity of the tissue for over 3&#160;hr&#160;and is, therefore, amenable to long-term imaging24,26-28. Recently, a protocol describing the long-term imaging of intact larval brains in the presence of ascorbic acid, serum, and fat bodies has been detailed29. Importantly, because the tissue is neither exposed nor immobilized, this approach is less useful for applications where culturing medium is exchanged (e.g., drug studies, immunodepletion, etc.).
Our lab has simplified the whole mount preparation of live larval brains for long-term imaging30,31. The main advantage to our protocol over others is its simplicity: our observations indicate neither serum, ascorbic acid, insulin, nor fat bodies are necessary additives to image successive rounds of NB ACD. Although additives, such as insulin, have been useful for the prolonged imaging of stem cell divisions32 and collective cell migration in Drosophila ovaries33, they appear to be dispensable for NB ACD, as our imaging medium consists of a simple mixture of standard insect cell culturing medium supplemented with antibiotic-antimycotic to prevent contamination. Through the use of reusable gas-permeable or glass bottom culture dishes, we have been able to sustain several rounds of successive NB ACDs. The same explanted samples can readily be used for immunofluorescence and other procedures with fixed tissue. In this protocol, we detail the dissection of intact larval brains, as well as the preparation of brains for both live and fixed analysis.

<table><tbody><tr><td>Lumox tissue culture dish 50 x 12 mm</td><td>Sarstedt</td><td>94.6077.410</td><td>A reusable culture dish with a gas-permeable and optically clear film base.</td></tr><tr><td>Schneider&#039;s S2 Medium</td><td>Invitrogen</td><td>21720-024</td><td></td></tr><tr><td>Gibco Antibiotic-Antimycotic (100x)</td><td>Invitrogen</td><td>15240-062</td><td>A supplement containing penicillin (10,000 U/ml), streptomycin (10,000 &amp;#956;g/ml), and amphotericin B (25 &amp;#956;g/ml) that is added to Schneider&#039;s S2 media to prevent bacterial and fungal contamination.&amp;nbsp;</td></tr><tr><td>Glass coverslips, #1.5 22 x 22 mm</td><td>Fisher Scientific</td><td>12-541-B</td><td></td></tr><tr><td>Halocarbon oil 700</td><td>Sigma-Aldrich</td><td>H8898</td><td>A high viscosity inert oil that is clear and colorless.</td></tr><tr><td>Pair of nickel-plated pin holders, 17 cm long</td><td>Fine Science Tools</td><td>26018-17</td><td></td></tr><tr><td>Pair of Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Dissecting needle/probe with plastic handle</td><td>Fisher Scientific</td><td>08-965-A</td><td></td></tr><tr><td>Syringe filter, 0.22 &amp;#956;m pore size</td><td>Fisher Scientific</td><td>09-719A</td><td></td></tr><tr><td>Syringe, 5 ml</td><td>BD Biosciences</td><td>309646</td><td></td></tr><tr><td>Plastic transfer pipette</td><td>Fisher Scientific</td><td>S30467-1</td><td></td></tr><tr><td>Paraformaldehyde, 32% EM-grade</td><td>Electron Microscopy Sciences</td><td>15714</td><td>CAUTION: Formaldehyde is toxic; it should be handled in a fume hood. Follow MSDS guidelines for safe handling.</td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole, dihydrochloride)</td><td>Invitrogen</td><td>D1306</td><td>A dye that labels DNA and is excited by UV light.</td></tr></tbody></table>

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 has elucidated a number of mechanisms that regulate NB ACD. Prior to image acquisition, it is necessary to determine the optimal imaging conditions. It is necessary to balance the frame capture rate with the duration of live cell imaging. For fine cellular analysis, for example, increased temporal and optical resolution may be required. When visualizing a single NB cell cycle (Figure 4A), the rate at which images are captured may be increased without introducing photodamage. Typically, singl...

Watch this Scientific Journal Video about Live Imaging of Drosophila Larval Neuroblasts at JoVE.com

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.

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 ...

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Neuroscience

14.9K Views.  National Institutes of Health. 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.

Video: Live Imaging of Drosophila Larval Neuroblasts

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 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.

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

Developmental Biology

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Bioengineering

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

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...

Imaging Live Brain Explants from Drosophila melanogaster Third Instar Larvae

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

Author Spotlight: Investigating Asymmetric Cell Division Dynamics: A Protocol for Live-Imaging of Drosophila Larval Brain Explants 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

Real-Time Imaging of Germline Stem Cells in Drosophila Ex Vivo Germaria Culture

Extended Live Imaging of Female Drosophila melanogaster Germline Stem Cell Niches

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to explore the dynamics of a myriad of developmental processes, such as cell division, stemness, differentiation, migration, apoptosis, autophagy, cellular adhesion, etc., over time. Recently, we have implemented an extended ex vivo culture and live imaging of the female Drosophila GSC niche. Using a Drosophila line harboring a GFP::Par-1 transgene as an example, this method allows the visualization of the GSCs&#39; asymmetric division within their niche and the description of the changes in the spectrosome morphology along the cell cycle. Here, we present a detailed protocol for the ex vivo culture of Drosophila germaria, enabling prolonged visualization of the female GSC niche. Importantly, this protocol is broadly applicable to live imaging GSCs with multiple fluorescently tagged proteins of interest that are available in stock centers and/or in the Drosophila research community.

Here, we provide a detailed protocol to perform live imaging of the asymmetric division of germline stem cells (GSCs) in the Drosophila ovarian niche. We use a transgenic line that ubiquitously expresses a green fluorescent protein (GFP) fusion of the spectrosome protein Par-1.

Live imaging methods allow the analysis of dynamic cellular processes in detail and in real-time. The Drosophila ovary represents an excellent model to ...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...

Live Imaging of Drosophila Larval Neuroblasts

Summary

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