We thank Amanda Rychel for assistance in developing the time lapse protocol and Lynn Pillitteri for constructing POLAR::POLAR-eGFP. We are also grateful to ABRC for providing the pm-rb construct. This protocol was developed through a support from the PRESTO award from Japan Science Technology and Agency. Research on POLAR was also supported by the University of Washington Royalty Research Fund (RRF-4098) and the National Science Foundation (MCB-0855659). KMP is an NSF Graduate Research Fellow (DGE-0718124), and KUT is an HHMI-GBMF investigator.

1. Seed Sterilization
<ol>
	<li>Prepare seed sterilization solution: 33% household bleach, 0.1% Triton X-100.</li>
	<li>Place seeds carrying desired fluorescent reporter construct(s) and genotype(s) in 1.7 ml tube and apply 1 ml of sterilization solution. Incubate on nutator for 15 min.</li>
	<li>In a sterile hood, use a pipettor to remove sterilization solution from tube, leaving seeds behind. Rinse with 1 ml sterile water. Repeat four times.</li>
	<li>Incubate at 4 &deg;C for two days or more.</li>
</ol>
2. Preparation of Chamber Slide Media
<ol>
	<li>Mix 20 ml of 0.5% solution of Bacto Agar (not pure agarose) in water in a 200 ml flask, and microwave until dissolved. Be cautious when boiling the solution.</li>
	<li>Cool the solution to around 60 &deg;C.</li>
	<li>Collect 1 ml of agar solution with a pipettor and slowly apply to the cover glass inside the chamber slide (Lab-Tek II Chambered #1.5 German Coverglass System). Solution which is too hot or applied too quickly may melt glue or crack glass.</li>
	<li>Immediately add a second ml of agar solution. Agar media should now completely cover the bottom of the slide chamber. This minimal media is sufficient to support development in a plant cotyledon, which contains stored nutrients, for four to five days by maintaining moisture and allowing gas diffusion.</li>
	<li>Cool slide on benchtop with chamber covered. If flask is covered tightly, solution may be saved and reheated for future use.</li>
</ol>
3. Seed Dissection
<ol>
	<li>Remove tube of sterile seed from 4 &deg;C storage.</li>
	<li>Place a paper tissue on the stage of a dissecting microscope and moisten with water. Adjust tissue to create a smooth surface. Tissue is used to stabilize seeds for dissection.</li>
	<li>Pipet 20-30 seeds from the tube to the tissue, being careful to place them in the dissecting scope's field of view.</li>
	<li>Using sharp forceps (Roboz, #5 biology tip), carefully remove the seed coat from the seedling inside. Both outer and inner integuments must be removed.</li>
	<li>When imaging the cotyledon, it is beneficial to remove the hypocotyl and radicle. Cotyledons contain sufficient nutrients to develop for several days under this protocol. If intact, the hypocotyl will straighten and lengthen dramatically, moving the cotyledon out of the time lapse field of view. Use a scalpel to slice the cotyledons free of the hypocotyl.</li>
	<li>Hold dissected cotyledons immersed in water, in a microtube or similar.</li>
	<li>Repeat 3.4-3.6 until the desired number of cotyledons is reached. 15-20 successful dissections are recommended before mounting.</li>
</ol>
4. Mounting Cotyledons in the Chamber Slide
<ol>
	<li>Using a small (18 mm2) cover slip, cut through the agar media and lift the entire layer up from the chamber slide's glass base.</li>
	<li>Pipet dissected cotyledons with a minimum of water from holding tube into the chamber slide, under the lifted agar layer.</li>
	<li>Gently lower the agar onto the cotyledons. If excess water is present, soak it away with a tissue, being careful not to disturb the cotyledons. Specimens should no longer move easily when mount is complete.</li>
	<li>Place cover on chamber slide and move slide to microscope stage.</li>
</ol>
5. Time Lapse Imaging
<ol>
	<li>Set up inverted confocal microscope to image the desired fluorophore(s). LSM700 parameters used: for GFP, excitation at 488 nm and collection with a band pass filter at 440-530 nm; for RFP, excitation at 555 nm and collection with a band pass filter at 570-610 nm.</li>
	<li>Inspect mounted specimens for damage. Program the locations of intact, properly mounted cotyledons into microscope software. In Zen 2009: Focus on a cotyledon with 20x objective and move objective to center of chamber slide. Under Acquisition Mode, change Zoom to 0.5 and Frame Size to 48x48 pixels. Select Positions checkbox and Scan overview image... button under Positions, then increase Horizontal and Vertical tile numbers to 30-35. When scan is complete, click the Positions button under the Dimensions tab and place crosshairs at each cotyledon to observe.</li>
	<li>Set up z series encompassing the cotyledon epidermis of all samples.In Zen 2009: Click the Z-Stack checkbox. Select each position under Position List and click the Move to button. Focus on the uppermost plane of the cotyledon epidermis and click the Set First button under Z-Stack. Focus to the lowest position at which epidermis is usefully visible and click the Set Last button. Click the button next to Optimal, which shows the best z-slice thickness, to set. Check each cotyledon to confirm that these settings encompass all samples; the z parameters cannot be set for individual positions in Zen 2009.</li>
	<li>Set up time series at desired resolution and length. Intervals of 15-30 min for three days are effective for protein dynamics in epidermal cell division. This interval can be changed as necessary. In Zen 2009: Click the Time Series checkbox. Under Time Series, set Cycles for the number of images desired using the slider or typing in the text field. Set Interval to 30 and use the pull-down menu to select min.</li>
	<li>Begin xyzt multi-position scan. Note that the number of positions must be limited by the scan specifications; if total scan time is greater than the desired interval, the time points will not be correct. A maximum of six simultaneous positions are possible when imaging GFP and RFP at 59 z-slices in a 30-min interval using our system. In Zen 2009: Change Frame Size to desired resolution and click Start Experiment.</li>
</ol>
6. Video Editing
<ol>
	<li>From the confocal data file, make a maximum intensity z-projection of each time/position combination and export as image files in a lossless format such as TIFF. File names should be in one series per position (e.g., POLAR-GFP_pos1_0001, POLAR-GFP_pos1_0002, etc., not POLAR-GFP_0001_pos1) for software to combine them into a movie. In Zen 2009: Use Copy:Subset under the Processing tab to split positions into separate files, then Maximum Intensity Projection. Finally, export as TIFF (File:Export, Full resolution image window - series).</li>
	<li>Use FIJI7,8 to align successive images by running the bUnwarpJ macro9(Plugins:Registration:bUnwarpJ). Change Registration Mode to Mono. All Advanced Options can use default values. For long time lapse sequences, download and install the macro "Affine + Consistent Elastic 2D Image Registration," which will apply bUnwarpJ to a series of images automatically, and open your data using File:Import:Image Sequence to use it. Uncheck MOPS landmarks extraction and run.</li>
	<li>Use FIJI/ImageJ or Quicktime Pro to open the sequence of aligned images and save in the desired video format (AVI is a good choice).</li>
</ol>

<ol>
	<li>Sulston, J. E., Schierenberg, E., White, J. G., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Embryonic+Cell+Lineage+of+the+Nematode+Caenorhabditis+elegans.">The Embryonic Cell Lineage of the Nematode Caenorhabditis elegans.</a> Developmental Biology. 100, 64 (1983).</li><li>Foe, V., Odell, G., Edgar, B. A., Bate, M., Martinez Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3+Mitosis+and+Morphogenesis:+Point+and+Counterpoint.">Chapter 3 Mitosis and Morphogenesis: Point and Counterpoint.</a> Development of Drosophila melanogaster. , (1993).</li><li>Vincent, C. A., Carpenter, R., Coen, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Lineage+Patterns+and+Homeotic+Gene+Activity+During+Antirrhinum+Flower+Development.">Cell Lineage Patterns and Homeotic Gene Activity During Antirrhinum Flower Development.</a> Current Biology. 5, 1449 (1995).</li><li>Beemster, G. T. S., De Vusser, K., De Tavernier, E., De Bock, K., Inz&#233;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Variation in Growth Rate between Arabidopsis Ecotypes Is Correlated with Cell Division and A-Type Cyclin-Dependent Kinase Activity. Plant Physiology. 129 (2), 854 (2002).</li><li>Grandjean, O., Vernoux, T., Laufs, P., Belcram, K., Mizukami, Y., Traas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Analysis+of+Cell+Division,+Cell+Growth,+and+Differentiation+at+the+Shoot+Apical+Meristem+in+Arabidopsis.">In Vivo Analysis of Cell Division, Cell Growth, and Differentiation at the Shoot Apical Meristem in Arabidopsis.</a> Plant Cell. 16 (1), 74 (2004).</li><li>Pillitteri, L. J., Peterson, K. M., Horst, R. J., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Profiling+of+Stomatal+Meristemoids+Reveals+New+Component+of+Asymmetric+Cell+Division+and+Commonalities+among+Stem+Cell+Populations+in+Arabidopsis.">Molecular Profiling of Stomatal Meristemoids Reveals New Component of Asymmetric Cell Division and Commonalities among Stem Cell Populations in Arabidopsis.</a> Plant Cell. 23 (9), 3260 (2011).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11 (7), 36 (2004).</li><li>Arganda-Carreras, I., Sorzano, S. &. #. 2. 2. 5. ;. n. c. h. e. z., Marabini, C. O., Carazo, R., de Solorzano, J. M. O. r. t. i. z. -., C, J., Kybic, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consistent+and+Elastic+Registration+of+Histological+Sections+Using+Vector-Spline+Regularization.">Consistent and Elastic Registration of Histological Sections Using Vector-Spline Regularization.</a> Computer Vision Approaches to Medical Image Analysis, ser. Lecture Notes in Computer Science. 4241, 85-95 (2006).</li><li>Peterson, K. M., Rychel, A. L., Torii, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Out+of+the+Mouths+of+Plants:+The+Molecular+Basis+of+the+Evolution+and+Diversity+of+Stomatal+Development.">Out of the Mouths of Plants: The Molecular Basis of the Evolution and Diversity of Stomatal Development.</a> Plant Cell. 22 (2), 296 (2010).</li><li>Czymmek, K. J., Fogg, M., Powell, D. H., Sweigard, J., Park, S. -. Y., Kang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Time-lapse+Documentation+Using+Confocal+and+Multi-Photon+Microscopy+Reveals+the+Mechanisms+of+Invasion+into+the+Arabidopsis+Root+Vascular+System+by+Fusarium+oxysporum.">In vivo Time-lapse Documentation Using Confocal and Multi-Photon Microscopy Reveals the Mechanisms of Invasion into the Arabidopsis Root Vascular System by Fusarium oxysporum.</a> Fungal Genetics and Biology. 44 (10), 1011 (2007).</li><li>Campilho, A., Garcia, B., Toorn, H. V., Wijk, H. 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No conflicts of interest declared.

This time-lapse confocal technique allows longitudinal studies of fluorescently tagged protein expression and localization in individual cells of the Arabidopsis cotyledon epidermis, which in the case of POLAR and other dynamically changing proteins is critical to a proper understanding of their function. Previously, sustained time lapse imaging has been used to examine Arabidopsis root fungal infection11 and meristem growth5,12, but adding the cotyledon epidermis expands the vers...

<table border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bacto Agar</td>
 <td>BD</td>
 <td>214010</td>
 <td></td>
 </tr>
 <tr>
 <td>One-chamber slide</td>
 <td>Nunc (Thermo Scientific)</td>
 <td>155360</td>
 <td>Or two-chamber (155379)</td>
 </tr>
 <tr>
 <td>Laser scanning confocal microscope</td>
 <td>Zeiss</td>
 <td>LSM700</td>
 <td>Zen 2009 software</td>
 </tr>
 <tr>
 <td>20x objective lens</td>
 <td>Zeiss</td>
 <td>420650-9901</td>
 <td>NA 0.8, Plan-APOCHROMAT</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Benz (National)</td>
 <td>431TBL</td>
 <td>Illuminates from below</td>
 </tr>
 <tr>
 <td>#5 forceps, biology tip</td>
 <td>Roboz Surgical Instrument</td>
 <td>RS-4978</td>
 <td>Very fine tips are critical</td>
 </tr>
 </tbody>
</table>

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.

A set of informative time points collected with this method is shown in Figure 3. Cell membranes are labeled with RFP (pm-rb) and GFP is fused to POLAR protein under its native promoter (POLAR::POLAR-GFP)6 At the 30-min time scale, we see cell divisions along with the changes in protein localization preceding them. Asymmetric cell divisions in the stomatal lineage form stem-cell-like stomatal precursors called meristemoids, which retain the ability to undergo further asymmetric divisi...

Watch this Scientific Journal Video about Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination at JoVE.com

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.

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 ...

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13.7K Views.  University of Washington. 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.

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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

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 ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Using Flatbed Scanners to Collect High-resolution Time-lapsed Images of the Arabidopsis Root Gravitropic Response

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories can face is the development and attainment of these methods. Observation of phenotypes in a process of interest is a typical objective of research labs studying gene function and this is often achieved through image capture. A particular process that is amenable to observation using imaging approaches is the corrective growth of a seedling root that has been displaced from alignment with the gravity vector. Imaging platforms used to measure the root gravitropic response can be expensive, relatively low in throughput, and/or labor intensive. These issues have been addressed by developing a high-throughput image capture method using inexpensive, yet high-resolution, flatbed scanners. Using this method, images can be captured every few minutes at 4,800 dpi. The current setup enables collection of 216 individual responses per day. The image data collected is of ample quality for image analysis applications.

This protocol describes a process for rapid collection of images of Arabidopsis seedlings responding to a gravity stimulus using commercially-available flatbed scanners. The method allows for inexpensive, high-volume capture of high-resolution images amenable for downstream analysis algorithms.

Research efforts in biology increasingly require use of methodologies that enable high-volume collection of high-resolution data. A challenge laboratories ...

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 ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

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 ...