We are grateful to B. Stevenson for technical assistance and Dr Marc R. Knight for providing Aequorin transgenic line. This work was funded by the National Institutes of Health Grant R01 GM059138.

1. Aequorin Based Ca2+ Imaging Using the FAS System
<ol>
	<li>Prepare seedlings for luminescence imaging. Sterilize seeds of Arabidopsis plants expressing Aequorin with 10% bleach solution containing 0.01% Triton-100. Sow the sterile seeds on a square plate (10 x 10 cm square Petri dish with grid,) containing full strength MS (Murashige and Skoog Basal Salt Mixture), 1% sucrose, and 1.2% agar. Place plates vertically in a growth chamber after stratification at 4 &#176;C for 2&#160;days (Figure 1A).</li>
	<li>Transfer the seedlings onto a film. Place an adhesive film (Figure 1B) on the top of 7-10 day old seedlings growing on the plate. Gently push the film by hand to ensure that seedlings adhere to the film (Figure 1C). Peel the film gently so that the seedlings remain adhered to the film (Figure 1D and 1E)</li>
	<li>Incubate the seedlings with cofactor. Place the adhered seedlings onto the square plate (10 x 10 cm) containing 15 ml of 2 &#956;g/ml h-CTZ (coelenterazine) in water. Incubate the seedlings at room temperature for 4 hr to overnight (Figure 1F).</li>
	<li>Prepare for luminescence imaging. Take the film out of the h-CTZ solution and cut it down the middle, forming two pieces. Place each piece of film with seedlings face up in two different plates. Leave the plates in the dark for 5 min.</li>
	<li>Acquire luminescence images. In the dark, place the two plates next to each other on the stage of the luminescence imaging system (Figure 1G). Acquire images immediately upon adding 20 ml of stimuli solution to the plates simultaneously.</li>
	<li>Analyze luminescence images. Choose the same display range for all luminescence images. Crop the region of interest (ROI) and generate the images as JPEG files (Figure 2A). Alternatively, export the images as SPE format files and import them into the ImageJ image analysis software. Set measurements for calculation of the mean gray value of ROI. Select the same size of ROI area and measure the mean gray and present data as bar graphs (Figure 2B).</li>
</ol>
2. Live Cell Confocal Ca2+&#160;Imaging
<ol>
	<li>Prepare seedlings for confocal imaging. Sterilize seeds of Arabidopsis plants expressing case12 with 10% bleach solution containing 0.01% Triton. Sow the sterile seeds on a plate containing full- strength MS salts, 1% sucrose, and 1.2% agar. Place plates vertically in a growth chamber after stratification at 4 &#176;C for 2&#160;days.</li>
	<li>Setup Imaging Chambers
	<ol>
		<li>Assemble an imaging chamber using a slide and coverslip (Chamber A). Place a piece of water soaked cotton wool onto the middle of the slide. Then transfer one or two 5 day old seedlings from the plate onto the top of water soaked cotton wool. Stick small pieces of clay to each corner of a coverslip, and place coverslip on the top of seedlings to create a gap (chamber) between the coverslip and slide (Figure 3A, left panel). Connect one end of polyethylene tube (0.58 mm diameter) to a 1 ml syringe and place the other end of tube immediately adjacent to the chamber. Hold the tubing in place with tape (Figure 3A, right panel).</li>
		<li>Assemble an imaging chamber using a plexiglass chamber (Chamber B).
		<ol>
			<li>Spread a thin layer of silicone grease around two polyethylene tubes (0.58 mm diameter) and press the coated tubes into the channels on each side of the plexiglass chamber (Figure 3B left panel). Spread a thin layer of silicone grease onto the surface of the chamber that contains the tube grooves and press a coverslip into the grease to seal one side of the chamber opening (cut out).</li>
			<li>Transfer a five day old seedling from the plate into the chamber and place a piece of cotton wool soaked with water on the top of the seedling. Spread silicone grease onto the other surface of the chamber, and press another coverslip on top to seal. Connect the end of one of the tubes to a 1 ml syringe. Leave the end of the other tube open (Figure 3B right panel).</li>
		</ol>
		</li>
		<li>Prepare an imaging chamber using a chambered cover glass (Chamber C). Sterilize the chambered cover glass with 70% ethanol and leave it on the hood until dry. Add 0.6 ml or 0.2 ml of full strength MS medium containing 1% sucrose and 0.5% phytagel into each well of an 2-well chamber or an 8-well chamber (Figure 3C right panel), respectively. Sterilize seeds and sow the seeds directly in a chambered cover glass containing a thin layer of clear gel and let them grow vertically for 5 days (Figure 3C).</li>
	</ol>
	</li>
	<li>Acquire images using a confocal microscope. To apply stress stimuli, such as salt, cold or peroxide, slowly inject about 200 &#956;l of 150 mM NaCl, ice-cold water or 1 mM H2O2 solution into the chamber (Chamber A or B) just prior to acquiring images, or gently add 500 &#956;l or 100 &#956;l of stimulus solution to the well of a 2- or 8-well chamber, respectively. Capture images immediately after applying the stimulus solution using an inverted Nikon A1R confocal laser-scanning microscope with a 20X water immersion lens (numerical aperture 0.75). Collect a time series of images at 4 sec intervals with excitation and emission wavelengths of 488 nm and 500 - 550 nm, respectively, and at a pixel resolution of 512 x 512.</li>
	<li>Image Analysis 
	Using Nikon Elements, ROIs were drawn around each cell (or area) of interest. Total intensity within each ROI was measured over time using the Time Measurement dialog box (ImageJ could be used instead). Total intensity measurements were exported and processed in DataGraph. Ca2+ spike amplitude was defined as peak intensity minus resting intensity, duration as the time between initiation and completion of a spike, and period as the time interval between adjacent spike peaks of two spikes. t-test was used to compare the means.</li>
</ol>

<ol>
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Genomics. 39 (5), 225-235 (2012).</li><li>Ranf, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defense-related+calcium+signaling+mutants+uncovered+via+a+quantitative+high-throughput+screen+in+Arabidopsis+thaliana.">Defense-related calcium signaling mutants uncovered via a quantitative high-throughput screen in Arabidopsis thaliana.</a> Mol. Plant. 5 (1), 115-130 (2012).</li><li>Zhu, X., Feng, Y., Liang, G., Liu, N., Zhu, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aequorin-based+luminescence+imaging+reveals+stimulus-+and+tissue-specific+Ca2++dynamics+in+Arabidopsis+plants.">Aequorin-based luminescence imaging reveals stimulus- and tissue-specific Ca2+ dynamics in Arabidopsis plants.</a> Mol. Plant. 6 (2), 444-455 (2013).</li><li>Knight, M. R., Campbell, A. K., Smith, S. 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The authors have nothing to disclose.

We have demonstrated a FAS system for recording the spatial-temporal Ca2+ response of Arabidopsis seedlings. This FAS Ca2+ recording system provides a simple, sensitive and robust approach that could be adapted for measuring Ca2+ dynamics triggered by various stimuli in addition to the abiotic stress stimuli that are presented here. Using this system, we can easily compare tissue- or stimuli-specific spatial-temporal Ca2+ dynamics at the whole plant level. The high sensitivity ...

The plant cell responds to the environment via signaling that coordinates cell actions. An early cell signaling event in response to environmental stimuli is a transient Ca2+ increase. The pattern, or signature of a transient increase in free Ca2+ concentration is characterized by its amplitude, frequency, and duration. Distinct spatio-temporal Ca2+ signatures regulate different cellular activities1. Specific stimuli, such as heat, cold, salt, drought, light, or plant hormones, may fine-tune the spatio-temporal activity of membrane-localized Ca2+ channels and/or transporters, resulting in specific Ca2+ signatures. Although Ca2+ transporters have been well characterized, little is known about the molecular identities and functions of Ca2+ channels in plants1. Genetic screens for mutants with altered Ca2+ response to stress stimuli may be an effective approach for identifying the components that compose Ca2+ signatures. Recently several Aequorin based Ca2+ detection systems have been developed that facilitate genetic screens for Ca2+ signaling components in response to pathogen attack and abiotic stress2-4.
Aequorin was first used to detect Ca2+ signals in plants in the early 1990s5. Since then, Aequorin has been targeted to different cellular compartments, such as the cytoplasm5, nucleus6, chloroplasts7, tonoplast8, mitochondria9, and stroma10, as well as to different cell types in the root to monitor cell specific Ca2+ signals11. Aequorin based Ca2+ measurements reveal the spatial and temporal Ca2+ response of a population of cells to stress stimuli. However, in most cases, the Ca2+ responses of single cells are unsynchronized in the responding tissue4. Therefore, Aequorin Ca2+ recording does not necessarily report the Ca2+ signal in individual cells. In recent years, genetically encoded fluorescent protein (FP)-based Ca2+ indicators, such as yellow cameleon (YCs)12 and CASEs1213 have been used to study Ca2+ signaling with high subcellular resolution. YCs are fluorescence resonance energy transfer (FRET)-based Ca2+ indicators, containing CFP and YFP variants linked by the Ca2+-binding protein calmodulin and calmodulin-binding peptide M13. Calmodulin undergoes a conformational change as it binds to Ca2+, thereby brings CFP and YFP closer together, resulting in increased energy transfer (enhanced FRET). The FRET level over time, calculated roughly as the ratio of YFP to CFP signal intensities, reflects intracellular Ca2+ dynamics. Several YC versions have been used in plants. YC3.6 was targeted to the cytosol14,15, nucleus16, mitochondria17, and plasma membrane18, and YC4.6 and D4ER were targeted to the ER15,19, and D3cpv was targeted to the peroxisomes20. Transgenic plants expressing YCs allow the live-cell imaging of Ca2+ dynamics within different cellular compartments of different cell types. CASEs (presumably Calcium sensor) are single circularly permuted fluorescent proteins (cpFPs) harboring a calmodulin and calmodulin-binding peptide M13. Upon binding to Ca2+, CASEs undergo conformational changes, leading to an increase of fluorescence intensity. The correlation between the CASE&#8217;s fluorescence response and Ca2+ concentration allows intracellular Ca2+ dynamics to be measured quantitatively. The Case12 variant has 12 fold increased fluorescence in the Ca2+-saturated forms. N. benthiminana plants transiently expressing Case12 or Arabidopsis plants stably expressing Case 12 were used to study Ca2+ signaling in defense and abiotic stress4,21 . Asynchronous spatial and temporal Ca2+ oscillations in cells responding to pathogen attack, or to dehydration stress have been revealed with Case12 based Ca2+ imaging.
Here, we present detailed instructions for Aequorin based luminescence imaging of tissue- and stimuli specific Ca2+ dynamics in Arabidopsis seedlings, and for confocal imaging of cytosolic and nuclear Ca2+ dynamics in Arabidopsis root cells that express Case 12. Luminescence imaging of FAS could be adapted to analyze stress-induced Ca2+ dynamics in intact plants or tissues not described here, or to screen mutagenized Arabidopsis plant populations for mutants with altered stress induced Ca2+ signals. The live cell Ca2+ imaging setup could be adapted to analyze Ca2+ dynamics within different subcelluar compartments or in different cell types using other Ca2+ indicators.

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Name of Material/ Equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:175px;">Catalog Number</td>
			<td style="width:184px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X10 cm square petri dish&#160;</td>
			<td>VWR</td>
			<td style="width:175px;">60872-310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">adhesive film&#160;</td>
			<td style="width:172px;">VWR</td>
			<td>60941-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyethylene tubing</td>
			<td>PerkinElmer</td>
			<td style="width:175px;">9908265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL syringe&#160;</td>
			<td style="width:172px;">VWR</td>
			<td style="width:175px;">53548-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">silicone grease&#160;</td>
			<td style="width:172px;">Beckman</td>
			<td style="width:175px;">335148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-well chambered cover glass&#160;</td>
			<td>Nalge Nunc international</td>
			<td>155379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">8-well chambered cover glass</td>
			<td>Nalge Nunc international</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">luminescence imaging system&#160;</td>
			<td>Princeton Instruments</td>
			<td style="width:175px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">inverted confocal laser-scanning microscope&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:184px;">Nikon A1R&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Imaging software</td>
			<td>Nikon Instruments Inc.</td>
			<td style="width:175px;">N/A</td>
			<td>Nikon Elements&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">ImageJ</td>
			<td style="width:172px;">NIH</td>
			<td style="width:175px;">N/A</td>
			<td>Public domain, Java-based image processing program</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DataGraph</td>
			<td>Visual Data Tools Inc</td>
			<td style="width:175px;">N/A</td>
			<td>DataGraph 3.1.1 is the newest version</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coelenterazine</td>
			<td>NanoLight Technolgies</td>
			<td style="width:175px;">#301B NF-BCTZ-FB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">All purpose Bleach</td>
			<td style="width:172px;">Any local store</td>
			<td style="width:175px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Triton X-100</td>
			<td style="width:172px;">Fisher</td>
			<td style="width:175px;">BP151500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">MS salt</td>
			<td style="width:172px;">Phytotechnology Labs</td>
			<td style="width:175px;">M524-50L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">sucrose</td>
			<td style="width:172px;">VWR</td>
			<td style="width:175px;">BDH8029-12KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Agar</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:175px;">A1296-5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Phytagel</td>
			<td style="width:172px;">Sigma</td>
			<td style="width:175px;">P8169-1KG</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spatial and temporal ca2+ signals in arabidopsis plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Mannitol, NaCl and H2O2 were used as proxies for dehydration, salt and oxidative stress stimuli, respectively. To check if the heavy metal ion Cu2+ synergizes with any of these three stress stimuli, we compared the Ca2+ response to each stimuli in the presence or absence of Cu2+. As shown in Figure 2, FAS luminescence imaging revealed that Arabidoposis seedlings responded differently to dehydration, salt and oxidative stress. For the concentration of...

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Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

measuring-spatial-and-temporal-ca2-signals-in-arabidopsis-plants

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Biology

12.0K Views.  Purdue University. Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Video: Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Modeling Biological Membranes with Circuit Boards and Measuring Electrical Signals in Axons: Student Laboratory Exercises

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology used in electrophysiology. The same equipment is used in the membrane model as on live preparations. Some properties of an isolated nerve cord are investigated: nerve action potentials, recruitment of neurons, and responsiveness of the nerve cord to environmental factors.

This is a demonstration of how biological membranes can be understood using electrical models. We also demonstrate procedures for recording action potentials from the ventral nerve cord of the crayfish for student orientated laboratories.

This is a demonstration of how electrical models can be used to characterize biological membranes. This exercise also introduces biophysical terminology ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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 ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...