This work was supported by grants from the Japan Society for the Promotion of Science (17H05007 and 18H05491) to MT, the National Science Foundation (IOS1557899 and MCB2016177) and the National Aeronautics and Space Administration (NNX14AT25G and 80NSSC19K0126) to SG.

1. Plant material preparation
<ol>
	<li>In a 1.5 mL microtube, surface sterilize the seeds of Arabidopsis thaliana (Col-0 accession) plant expressing either GCaMP3 or CHIB-iGluSnFR by shaking with 20% (v/v) NaClO for 3 min and then wash 5 times with sterile distilled water. 
	NOTE: The transgenic lines of Arabidopsis expressing GCaMP3 or CHIB-iGluSnFR have been described previously6.</li>
	<li>In a sterile hood, sow 13 surface-sterilized seeds on a 10 cm square plastic Petri dish filled with 30 mL sterile (autoclaved) Murashige and Skoog (MS) medium [1x MS salts, 1% (w/v) sucrose, 0.01% (w/v) myoinositol, 0.05% (w/v) MES&#160;and 0.5% (w/v) gellan gum; pH 5.7 adjusted with 1N KOH]. Replace the lid and wrap with surgical tape.</li>
	<li>After incubation in dark at 4 &#176;C for 2 days, place the plates horizontally at 22 &#176;C in a growth chamber under continuous light (90-100 &#181;mol m-2 s-1) for approximately 2 weeks before use. After 2 weeks, count the number of Arabidopsis leaves from oldest to youngest44 (Figure 1). Wound responses preferentially move from the damaged leaf (n) to leaves numbered n &#177; 3 and n &#177; 56. 
	NOTE: In this protocol, the Petri dish will be opened for imaging the wound and glutamate effects under a fluorescence microscope. Therefore, subsequent steps in this experiment should be conducted under temperature- and humidity-controlled room conditions. This is because Ca2+ signals are also elicited by changes in these environmental conditions. It is also known that the blue light, emitted from the microscope during recording for excitation of the biosensor protein&#39;s fluorescence, may elicit an increase of cytosolic Ca2+ concentration45 and so the plant should be acclimated to the blue light irradiation for several minutes before beginning the experiment.</li>
</ol>
2. Chemical preparation
<ol>
	<li>Dissolve L-Glutamate in a liquid growth medium [1/2x MS salts, 1% (w/v) sucrose&#160;and 0.05% (w/v) MES; pH 5.1 adjusted with 1N KOH] to make a 100 mM working solution. 
	NOTE: Avoid use of salts of glutamate such as sodium glutamate to prevent potential cation-related effects on Ca2+ dynamics.</li>
</ol>
3. Microscope setting and conducting real-time imaging
<ol>
	<li>Turn on the motorized fluorescence stereomicroscope equipped with a 1x objective lens (NA = 0.156) and a sCMOS camera (Figure 2) and configure the device settings to irradiate with a 470/40 nm excitation light and acquire an emission light passing through a 535/50 nm filter. 
	NOTE: Any GFP-sensitive fluorescence microscope can be used to detect GCaMP3 and iGluSnFR signals in&#160;real-time, but a low power objective lens and highly sensitive camera with a wide sCMOS chip are recommended to acquire signals from the entire plant. The low power objective allows for imaging of an entire Arabidopsis plant&#39;s response and use of a highly sensitive camera permits the fast data acquisition needed to capture the rapid time course of the wound-triggered Ca2+ wave. For the fluorescence microscope used in this study, the maximum values of the field of view and temporal resolution are 3 cm x 3 cm and 30 frames per second (fps), respectively.</li>
	<li>Remove the lid and place the dish under the objective lens.</li>
	<li>Check the fluorescence signal from the plant and then wait for approximately 30 min in the dark until plants are adapted to the new environmental conditions. This adaptation step is required because changes in humidity elicit [Ca2+]cyt elevation in plants that can interfere with any wound-related events.</li>
	<li>Adjust the focus and magnification to see the whole plant in the field of view. In the current protocol, a 0.63x magnification was used.</li>
	<li>Before starting real-time imaging, set up the acquisition parameters to detect the fluorescence signals using microscope imaging software. The settings for imaging in the current protocol are: exposure and interval times set to 1.8 s and 2 s (i.e., 0.5 fps), respectively. Set recording time to 11 min.</li>
	<li>Image for 5 min prior to starting the experiment to acclimate the plant to the blue light irradiation from the microscope, then start recording by clicking on Run Now, or the equivalent command in the microscope software being used. To determine the average baseline fluorescence, record at least 10 frames (i.e., at least 20 s in the current protocol) before wounding or glutamate application (see Section 4).
	<ol>
		<li>For real-time imaging of wound-induced [Ca2+]cyt and [Glu]apo changes, cut the petiole or the middle region of leaf L1 with scissors (Figure 3 and Figure 4).</li>
		<li>For real-time imaging of glutamate-triggered [Ca2+]cyt changes, cut the edge (approximately 1 mm from the tip) of leaf 1 across the main vein with scissors. After at least 20 min recovery period, apply 10 &#181;L of 100 mM glutamate to the leaf&#39;s cut surface (Figure 5). 
		NOTE: This pre-cutting was necessary to allow glutamate access to the leaf apoplast in order to trigger responses. In addition, applying a drop of distilled water to the cut surface of leaf L1 was found to be critical to prevent the samples from desiccating during the recovery before applying glutamate.</li>
	</ol>
	</li>
	<li>After finishing the 11 min recording, save the data.</li>
</ol>
4. Data analysis
<ol>
	<li>For fluorescence intensity analysis over time, define a region of interest (ROI) at the place where fluorescence intensity is to be analyzed (Figure 6 and Figure 7). For the velocity calculation of the Ca2+ wave, define 2 ROIs (ROI1 and ROI2) for analysis. In the imaging software, click on Time Measurement | Define | Circle. Measure the distance between ROI1 and ROI2 by clicking on Annotations and Measurement | Length | Simple Line (Figure 6).</li>
	<li>Measure the raw fluorescence values (F) in each ROI over time by clicking on Measure. Export raw data to a&#160;spreadsheet software to convert the fluorescence signal into numbers at each time point by clicking on All to Excel | Export.</li>
	<li>Determine the baseline fluorescence value, which is defined as F0, by calculating the average of F over the first 10 frames (i.e., prior to treatment) in the recorded data.</li>
	<li>Normalize the F data (by calculating &#916;F/F) using the equation &#916;F/F = (F&#8722;F0)/F0, where &#916;F is the time-dependent change in fluorescence.</li>
	<li>For Ca2+ &#160;wave velocity wave analysis, define a significant signal rise point above the pre-stimulated values as representing detection of a Ca2+ increase in each ROI (t1 and t2) using the criterion of a rise to 2&#215; the standard deviation (2x SD) that is calculated from the F0 data using statistical software. 95% of the F0 data falls within 2x SD from the mean, indicating that a rise in signal above this level by chance is &#8804;5%. Calculate the time difference of the Ca2+ increase between ROI1 and ROI2 [t2- t1 time-lag (&#916;t)] and measure the distance between ROI1 and ROI2, then determine the velocities of any Ca2+ wave.</li>
</ol>

The authors do not have any conflicts of interest.

Systemic signaling is important for plants to respond to localized external environmental stimuli and then to maintain their homeostasis at a whole plant level. Although they are not equipped with an advanced nervous system like animals, they employ rapid communication both within and between organs based on factors such as mobile electrical (and possibly hydraulic) signals and propagating waves of ROS and Ca2+&#160;46,47. The protocol described above ...

Plants cannot escape from biotic stresses, e.g., insects feeding on them, so they have evolved sophisticated stress sensing and signal transduction systems to detect and then protect themselves from challenges such as herbivory1. Upon wounding or herbivore attack, plants initiate rapid defense responses including accumulation of the phytohormone jasmonic acid (JA) not only at the wounded site but also in undamaged distal organs2. This JA then both triggers defense responses in the directly damaged tissues and preemptively induces defenses in the undamaged parts of the plant. In Arabidopsis, the accumulation of JA induced by wounding was detected in distal, intact leaves within just a few minutes of damage elsewhere in the plant suggesting that a rapid and long-distance signal is being transmitted from the wounded leaf3. Several candidates, such as Ca2+, reactive oxygen species (ROS), and electrical signals, have been proposed to serve as these long-distance wound signals in plants4,5.
Ca2+ is one of the most versatile and ubiquitous second messenger elements in eukaryotic organisms. In plants, caterpillar chewing and mechanical wounding cause drastic increases in the cytosolic Ca2+ concentration ([Ca2+]cyt) both in the wounded leaf and in unwounded distant leaves6,7. This systemic Ca2+ signal is received by intracellular Ca2+-sensing proteins, which lead&#160;to the activation of downstream defense signaling pathways, including JA biosynthesis8,9. Despite numerous such reports supporting the importance of Ca2+ signals in plant wound responses, information on the spatial and temporal characteristics of Ca2+ signals induced by wounding is limited.
Real-time imaging using genetically encoded Ca2+ indicators is a powerful tool to monitor and quantify the spatial and temporal dynamics of Ca2+ signals. To date, versions of such sensors have been developed that enable the visualization of Ca2+ signals at the level of a single cell, to tissues, organs and even whole plants10. The first genetically encoded biosensor for Ca2+ used in plants was the bioluminescent protein aequorin derived from the jellyfish Aequorea victoria11. Although this chemiluminescent protein has been used to detect Ca2+ changes in response to various stresses in plants12,13,14,15,16,17,18, it is not well-suited for real-time imaging due to the extremely low luminescent signal it produces. F&#246;rster Resonance Energy Transfer (FRET)-based Ca2+ indicators, such as the Yellow cameleons, have also been successfully used to investigate the dynamics of a range of Ca2+ signaling events in plants19,20,21,22,23,24. These sensors are compatible with imaging approaches and most commonly are composed of the Ca2+ binding protein calmodulin (CaM) and a CaM-binding peptide (M13) from a myosin light chain kinase, all fused between two fluorophore proteins, generally a Cyan Fluorescent Protein (CFP) and a Yellow Fluorescent Protein variant (YFP)10. Ca2+ binding to CaM promotes the interaction between CaM and M13 leading to a conformational change of the sensor. This change promotes energy transfer between the CFP and YFP, which increases the fluorescence intensity of the YFP while decreasing the fluorescence emission from the CFP. Monitoring this shift from CFP to YFP fluorescence then provides a measure of the increase in Ca2+ level. In addition to these FRET sensors, single fluorescent protein (FP)-based Ca2+ biosensors, such as GCaMP and R-GECO, are also compatible with plant imaging approaches and are widely used to study [Ca2+]cyt changes due to their high sensitivity and ease of use25,26,27,28,29,30. GCaMPs contain a single circularly permutated (cp) GFP, again fused to CaM and the M13 peptide. The Ca2+-dependent interaction between CaM and M13 causes a conformational change in the sensor that promotes a shift in the protonation state of the cpGFP, enhancing its fluorescent signal. Thus, as Ca2+ levels rise, the cpGFP signal increases.
To investigate the dynamics of Ca2+ signals generated in response to mechanical wounding or herbivore feeding, we have used transgenic Arabidopsis thaliana plants expressing a GCaMP variant, GCaMP3, and a wide-field fluorescence microscope6. This approach has succeeded in visualizing rapid transmission of a long-distance Ca2+ signal from the wound site on a leaf to the whole plant. Thus, an increase in [Ca2+]cyt was immediately detected at the wound site but this Ca2+ signal was then propagated to the neighboring leaves through the vasculature within a few minutes of wounding. Furthermore, we found that the transmission of this rapid systemic wound signal is abolished in Arabidopsis plants with mutations in two glutamate receptor-like genes, Glutamate Receptor Like (GLR), GLR3.3, and GLR3.66. The GLRs appear to function as amino-acid gated Ca2+ channels involved in diverse physiological processes, including wound response3, pollen tube growth31, root development32, cold response33, and innate immunity34. Despite this well-understood, broad physiological function of the GLRs, information on their functional properties, such as their ligand specificity, ion selectivity, and subcellular localization, are limited35. However, recent studies reported that GLR3.3 and GLR3.6 are localized in the phloem and xylem, respectively. Plant GLRs have similarities to ionotropic glutamate receptors (iGluRs)36 in mammals, which are activated by amino acids, such as glutamate, glycine, and D-serine in the mammalian nervous system37. Indeed, we demonstrated that the application of 100 mM glutamate, but not other amino acids, at the wound site induces a rapid, long-distance Ca2+ signal in Arabidopsis, indicating that extracellular glutamate likely acts as a wound signal in plants6. This response is abolished in the glr3.3/glr3.6 mutant suggesting that glutamate may be acting through one or both of these receptor-like channels and indeed, AtGLR3.6 was recently shown to be gated by these levels of glutamate38.
In plants, in addition to its role as a structural amino acid, glutamate has also been proposed as a key developmental regulator39; however, its spatial and temporal dynamics are poorly understood. Just as for Ca2+, several genetically encoded indicators for glutamate have been developed to monitor the dynamics of this amino acid in living cells40,41. iGluSnFR is a GFP-based single-FP glutamate biosensor composed of cpGFP and a glutamate binding protein (GltI) from Escherichia coli42,43. The conformational change of iGluSnFR, that is induced by glutamate binding to GltI, results in an enhanced GFP fluorescence emission. To investigate whether extracellular glutamate acts as a signaling molecule in plant wound response, we connected the iGluSnFR sequence with the basic chitinase signal peptide secretion sequence (CHIB-iGluSnFR) to localize this biosensor in the apoplastic space6. This approach enabled imaging of any changes in the apoplastic glutamate concentration ([Glu]apo) using transgenic Arabidopsis plants expressing this sensor. We detected rapid increases in the iGluSnFR signal at the wounding site. This data supports the idea that glutamate leaks out of the damaged cells/tissues to the apoplast upon wounding and acts as a damage signal activating the GLRs and leading to the long-distance Ca2+ signal in plants6.
Here, we describe a plant-wide real-time imaging method using genetically encoded biosensors to monitor and analyze the dynamics of long-distance Ca2+ and extracellular glutamate signals in response to wounding6. The availability of wide-field fluorescence microscopy and transgenic plants expressing genetically encoded biosensors provides a powerful, yet easily implemented approach to detect rapidly transmitted long-distance signals, such as Ca2+ waves.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Arabidopsis expressing GCaMP3</td>
			<td>Saitama University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arabidopsis expressing CHIB-iGluSnFR</td>
			<td>Saitama University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamate</td>
			<td>FUJIFILM Wako</td>
			<td>072-00501</td>
			<td>Dissolved in a liquid growth medium [1/2x MS salts, 1% (w/v) sucrose, and 0.05% (w/v) MES; pH 5.1 adjusted with 1N KOH].</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog (MS) medium</td>
			<td>FUJIFILM Wako</td>
			<td>392-00591</td>
			<td>composition: 1x MS salts, 1% (w/v) sucrose, 0.01% (w/v) myoinositol, 0.05% (w/v) MES, and 0.5% (w/v) gellan gum; pH 5.7 adjusted with 1N KOH.</td>
		</tr>
		<tr>
			<td>Nikon SMZ25 stereomicroscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements AR analysis</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1x objective lens (P2-SHR PLAN APO)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sCMOS camera (ORCA-Flash4.0 V2)</td>
			<td>Hamamatsu Photonics</td>
			<td>C11440-22CU</td>
			<td></td>
		</tr>
		<tr>
			<td>Square plastic Petri dish</td>
			<td>Simport</td>
			<td>D210-16</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Signal propagation of [Ca2+]cyt and [Glu]apo in response to wounding is presented in Figure 3, Figure 4, Movie S1, and Movie S2. Cutting the petiole of the leaf 1 in plants expressing GCaMP3&#160;(at 0 s) led to a significant increase in [Ca2+]cyt that was rapidly induced locally through the vasculature (at 40 s) (Figure ...

Watch this Scientific Journal Video about Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis at JoVE.com

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.

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. ...

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4.7K Views.  Saitama University. 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.

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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

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.

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.

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

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Local Field Fluorescence Microscopy: Imaging Cellular Signals in Intact Hearts

In the heart, molecular signaling studies are usually performed in isolated myocytes. However, many pathological situations such as ischemia and arrhythmias can only be fully understood at the whole organ level. Here, we present the spectroscopic technique of local field fluorescence microscopy (LFFM) that allows the measurement of cellular signals in the intact heart. The technique is based on a combination of a Langendorff perfused heart and optical fibers to record fluorescent signals. LFFM has various applications in the field of cardiovascular physiology to study the heart under normal and pathological conditions. Multiple cardiac variables can be monitored using different fluorescent indicators. These include cytosolic [Ca2+], intra-sarcoplasmic reticulum [Ca2+] and membrane potentials. The exogenous fluorescent probes are excited and the emitted fluorescence detected with three different arrangements of LFFM epifluorescence techniques presented in this paper. The central differences among these techniques are the type of light source used for excitation and on the way the excitation light is modulated. The pulsed LFFM (PLFFM) uses laser light pulses while continuous wave LFFM (CLFFM) uses continuous laser light for excitation. Finally, light-emitting diodes (LEDs) were used as a third light source. This non-coherent arrangement is called pulsed LED fluorescence microscopy (PLEDFM).

In the heart, molecular events coordinate the electrical and contractile function of the organ. A set of local field fluorescence microscopy techniques presented here enables the recording of cellular variables in intact hearts. Identifying mechanisms defining the cardiac function is critical in understanding how the heart works under pathological situations.