Name: larvaspa, a method for mounting drosophila larva for long-term time-lapse imaging
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Description: Watch this Scientific Journal Video about LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging at JoVE.com

We thank Lingfeng Tang for establishing an earlier version of the LarvaSPA method; Glenn Swan at Cornell Olin Hall Machine shop for making earlier prototypes of the imaging chamber; Philipp Isermann for constructing metal frames and providing suggestions on making PDMS cuboids; Cornell BRC Imaging facility for access to microscopes (funded by NIH grant S10OD018516); Maria Sapar for critical reading of the manuscript. This work was supported by a Cornell Fellowship awarded to H.J.; a Cornell start-up fund and NIH grants (R01NS099125 and R21OD023824) awarded to C.H. H.J. and C.H. conceived the project and designed the experiments. H.J. conducted the experiments. H.J and C.H. wrote the manuscript.

1. Making the imaging chamber
<ol>
	<li>The metal frame can be constructed from an aluminum block in a typical machine shop. The specifications of the frame are illustrated in Figure 1A.</li>
	<li>To construct the imaging chamber, seal the bottom of the metal frame using a long coverslip (22 mm x 50 mm) and UV glue (Figure 1A). Cure the UV glue using a hand-held UV lamp.</li>
</ol>
2. Making PDMS cuboids<...

<ol>
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Here we describe LarvaSPA, a versatile method of mounting live Drosophila larvae for long-term time-lapse imaging. This method does not require recovering or remounting larvae, enabling uninterrupted imaging. It is therefore ideal for tracking biological processes that take hours to complete, such as dendrite degeneration and regeneration. This method can be also used for imaging intracellular calcium dynamics and subcellular events such as microtubule growth. As the larval body wall is stable during the imaging...

Time-lapse live imaging is a powerful method for studying dynamic cellular processes. The spatial and temporal information provided by time-lapse movies can reveal important details for answering cell biology questions. The Drosophila larva has been a popular in vivo model for investigations using live imaging because its transparent body wall allows for noninvasive imaging of internal structures1,2. In addition, numerous genetic tools are available in Drosophila to fluorescently label anatomical structures and macromolecules3. However, long-term time-lapse imaging of Drosophila larvae is challenging. Unlike stationary early embryos or pupae, Drosophila larvae move constantly, necessitating immobilization for live imaging. Effective ways of immobilizing live Drosophila larvae include mounting in halocarbon oil with chloroform4, anesthetizing using isoflurane or Dichlorvos solution5, and compressing between the coverslip and the microscope slide6. Although some of these methods have been used for microscopy, none of them is effective for long-term live imaging. Other methods were developed for imaging body wall neurons in crawling larvae using conventional confocal microscopy or light-sheet microscopy7,8,9. However, these methods are not ideal for monitoring cellular dynamics due to the movement of the larvae.
New methods have been developed to achieve long-term time-lapse imaging of Drosophila larvae. Using a polydimethylsiloxane (PDMS) &#34;larva chip&#34;, Drosophila larvae can be effectively immobilized through vacuum-generated suction in a specialized microchamber without anesthetization. However, this method does not offer high temporal resolution for cell biology studies and it has strict limitations on animal size10. Another method using an anesthetization device achieved live imaging of Drosophila larvae at multiple time points and has been applied to study neuromuscular junctions11,12,13,14,15,16. However, this method also does not allow for continuous imaging for longer than 30 min and requires using desflurane repeatedly, which can inhibit neural activity and affect the biological process studied17,18. Recently, a new method that combines microfluidic device and cryoanesthesia has been used to immobilize larvae of various sizes for short periods of time (minutes)19. However, this method requires specialized devices such as a cooling system and longer periods of immobilization require repeated cooling of the larvae.
Here we present a versatile method of immobilizing Drosophila larvae that is compatible with uninterrupted time-lapse imaging for longer than 10 h. This method, which we call &#34;Larva Stabilization by Partial Attachment&#34; (LarvaSPA), involves adhering the larval cuticle to a coverslip for imaging in a custom-built imaging chamber. This protocol describes how to make the imaging chamber and how to mount larvae at a variety of developmental stages. In the LarvaSPA method, the desired body segments are affixed to the coverslip using a UV-reactive glue. A PDMS cuboid additionally applies pressure to the larvae, preventing escape. The air and moisture in the imaging chamber ensure the survival of the partially immobilized larvae during imaging. Advantages of LarvaSPA over other techniques include the following: (1) It is the first method that allows for continuous live imaging of intact Drosophila larvae for hours with high temporal and spatial resolution; (2) The method has fewer limitations on larval size; (3) The imaging chamber and PDMS cuboids can be manufactured at a minimal cost and are reusable.
In addition to describing the larval mounting method, we provide several examples of its application for studying dendrite development and dendrite degeneration of Drosophila dendritic arborization (da) neurons.

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			<td>6061 Aluminum bars</td>
			<td>McMaster-Carr</td>
			<td>9246K421</td>
			<td></td>
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		<tr>
			<td>3M double-sided tape</td>
			<td>Ted Pella, Inc.</td>
			<td>16093</td>
			<td></td>
		</tr>
		<tr>
			<td>3M Scotch Packaging tape</td>
			<td>3M</td>
			<td></td>
			<td>1.88&#34;W x 22.2 Yards</td>
		</tr>
		<tr>
			<td>DUMONT #3 Forceps</td>
			<td>Fisher Scientific</td>
			<td>50-241-34</td>
			<td></td>
		</tr>
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			<td>Glass coverslip</td>
			<td>Azer Scientific</td>
			<td>1152250</td>
			<td></td>
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			<td>Isoflurane</td>
			<td>Midwest Veterinary Supply</td>
			<td>193.33161.3</td>
			<td></td>
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			<td>Leica Confocal Microscope</td>
			<td>Leica</td>
			<td>SP8 equipped with a resonant scanner</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens paper</td>
			<td>Berkshire</td>
			<td>LN90.0406.24</td>
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			<td>Petri dishes (medium)</td>
			<td>VWR</td>
			<td>25373-085</td>
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			<td>Petri dishes (small)</td>
			<td>VWR</td>
			<td>10799-192</td>
			<td></td>
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			<td>Razor blade</td>
			<td>Ted Pella, Inc.</td>
			<td>121-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular petri dish</td>
			<td>VWR</td>
			<td>25384-322</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD 184 kit (PBMS kit)</td>
			<td>Electron Microscopy Sciences</td>
			<td>24236-10</td>
			<td></td>
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			<td>Transferring pipette</td>
			<td>Thermo Fisher Scientific</td>
			<td>1371126</td>
			<td></td>
		</tr>
		<tr>
			<td>UV glue</td>
			<td>Norland products</td>
			<td>#6106, NOA 61</td>
			<td>Refractive Index 1.56</td>
		</tr>
		<tr>
			<td>UV lamp (Workstar 2003)</td>
			<td>Maxxeon</td>
			<td>MXN02003</td>
			<td></td>
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		<tr>
			<td>Vacuum desiccator</td>
			<td>Electron Microscopy Sciences</td>
			<td>71232</td>
			<td></td>
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			<td>Wipes</td>
			<td>Kimberly-Clark</td>
			<td>Kimwipes</td>
			<td></td>
		</tr>
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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.

The larva imaging chamber is constructed by gluing a custom-made metal frame and two coverslips together. The design of the metal frame is specified in Figure 1A. Drosophila larvae inside the chamber are adhered to the top coverslip with the aid of UV glue and PDMS cuboids. The groove on the PDMS cuboid and the double-sided tape the cuboid is attached to create the space to hold the larvae (Figure 1B,C). The PDMS also applies gentle pressure to ...

Watch this Scientific Journal Video about LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging at JoVE.com

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.

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 ...

LarvaSPA is the first method that allows for continuous live imaging of intact drosophila larvae for more than 10 hours with high temporal and spatial resolution. This method is used for revealing dynamic cellular processes of larvae peripheral sensory neurons and for studying many other cellular processes that happen near the larva body wall. This method is easy to use and has less limitation on larva size.Six to nine larvae can be mounted in the imaging chamber at the same time, which makes the experiment possible. The imaging chamber at the PDMS cuboids can be manufactured at a minimal cost and are reusable. This method is particularly useful for studying mechanisms of dendrite development and dendrite degeneration using drosophila larva dendritic authorization neurons.Being able to image the same neuron for hours can reveal how long term global changes of dendritic patterns arise from short term behaviors at the individual branch level. Using PDMS cuboids that match the sizes of larvae will increase the success rate of immobilizing larvae. Please find more useful tapes in our troubleshooting section.After constructing an aluminum block from a machine shop, seal the bottom of the metal frame using a long cover slip with UV glue. Cure the UV clue using a handheld UV lamp for 30 seconds. Attach layers of packaging tape to the inner surface of a rectangular Petri dish or round cell culture plate.Use one layer for second in-star larvae, two layers for early third in-star larvae, or three layers for late third in-star larvae. Cut the tape into trips of specific width with a razor blade, 1.5 millimeters for the one layer tape and two millimeters for the two layer or three layer tape. Leave at least a five millimeter space between the two strips.Remove the tape layers covering the space. Remove dust from the inner surface of the plate using sticky tape. After that, the mold is ready for use.To prepare the PDMS mix, mix seven grams PDMS base in 0.7 grams of curing agent thoroughly in a small container. Place the container in a vacuum desiccator for at least 15 minutes to remove air from the mixture. Slowly pour about 5.5 grams PDMS mixture onto the mold to reach a one to two millimeter thickness.Place the PDMS mixture in the vacuum desiccator again for at least 15 minutes to remove remaining air bubbles from the mixture. Break the last few bubbles with a pipette tip. Cure the PDMS on a flat surface in a heat incubator at 65 degrees Celsius for two hours.Next, use a razor blade to loosen the cured PDMS along the edge of the mold and detach it from the mold. Store the PDMS between two pieces of large sticky tape at room temperature. For early and late third in-star larvae, cut the PDMS into eight by two by one millimeter cuboids by positioning the groove created by the tape strip at the center of the long side of the cuboid.For second in-star larvae, cut the cuboid to eight by one by one millimeter. To prepare the top cover slip for mounting, choose six PDMS cuboids with grooves matching the sizes of the larvae. Remove dust from the surface of the PDMS with sticky tape.Attach four pieces of double sided tape at the size of 12 by five millimeters on a long cover slip of 22 by 50 millimeters for fixing PDMS cuboids later. Adjust the spaces between the two pieces of double sided tape to the same as the width of the PDMS groove. Apply a small drop of UV glue into the groove of each PDMS cuboid and add six small drops of UV glue into the space between the double sided tapes on the cover slip.To prepare the larvae for mounting, using a pair of forceps, clean the larvae in water to remove food from the body surface. Place the clean larvae on a small piece of moistened tissue paper in a small Petri dish without a lid and place the small Petri dish into a large Petri dish containing a piece of dry tissue paper. In a chemical hood, use a plastic transferring pipette to apply 160 to 240 microliters of isoflurane onto the dry tissue paper and close the lid of the large Petri dish.Wait two to three minutes while monitoring the larvae. Once their mouth hooks stop moving, take out the larvae from the large Petri dish. Place the immobilized larvae onto the UV glue between the double sided tapes on the cover slip with the dorsal cuticle facing the cover slip.Cover each larva with a PDMS block and fit the trunk of the larva into the groove of the PDMS. Leave the head and the tail of the larva outside the PDMS groove. Avoid blocking the spiracles of the larva by the glue.Press down on the ends of the PDMS block onto the double sided tape without applying force on the groove. Gently pull on the tail of the larva to flatten the cuticle under the PDMS. Wear safety glasses and use a handheld UV lamp to cure the UV glue for four minutes on the high setting.Flip the cover slip upside down and use the UV lamp to cure the UV glue for another four minutes. Place a small piece of lens paper with the size of 15 by 30 millimeters at the bottom of the imaging chamber. Moisten the paper with 20 to 30 microliters of water.Place the cover slip on the chamber so that the larvae are facing the inside of the chamber. Use the UV glue to adhere both ends of the cover slip to the metal surface. The dorsal side of the larvae is ready for imaging under a confocal microscope.After imaging, remove the oil on the top cover slip using lens paper. Detach the top cover slip from the metal frame by cutting into the space between the cover slip and the metal frame with a razor blade. The imaging chamber is ready for reuse.Detach the PDMS cuboids from the top cover slip with forceps. Roll the PDMS cuboids on sticky tape to remove glue residue and dust. The PDMS cuboids are ready for reuse.In this protocol, drosophila larvae were immobilized for longer than 10 hours for continuous imaging. This figure shows six late third in-star larvae mounted in the chamber. The trunks of the larvae were fixed while their heads and tails were free to move.Here, we demonstrate the application of LarvaSPA in studying neuronal dendrite dynamics and dendrite degeneration using class four DA neurons as a model. To successfully immobilize larvae using this method, it is critical to stop exposing the larvae to isoflurane once the mouth hooks stop moving and cure the UV glue before larvae fully wake up. To study dendrite degeneration and regeneration, laser injury can be performed when larvae are immobilized in the imaging chamber.LarvaSPA has helped us to understand how dynamic growing dendrites fail to innervate epidermal cells like in hepatic cell proteoglycans and how phosphatidylserine is exposed on degenerating dendrites after laser injury. Protect eyes with safety glasses while using the UV lamp. Knocking out larvae using isoflurane should be performed in a fume hood.

larvaspa-method-for-mounting-drosophila-larva-for-long-term-time

Research

JoVE Journal

Biology

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...