Name: Focal Ca2+ Transient Detection in Smooth Muscle (Video) | JoVE Journal
Uploaded: 2009-06-29T00:00:00.000+00:00
Duration: 17 min 41 s
Description: Learn high-resolution calcium imaging in smooth muscle using confocal microscopy and isolated organ preparations. This protocol enables detailed analysis of neurotransmitter effects on calcium signaling events.

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5 minutes

The Wellcome Trust provided financial support (VIP Award to J.S.Y. and 074128 Research Fellowship to K.L.B.). Medical Research Council Studentship to R.J.A.
Leica_SP2_Stacker, an algorithm used to import TIFFs for &#x2018;reconstruction&#x2019; into a series, is based on Leica_tiff_sequence, a plugin for reading Leica SP2 TIFFs in ImageJ, developed by Tony Collins and used with his permission.
(<a href="http://www.uhnres.utoronto.ca/facilities/wcif/software/Plugins/LeicaTIFF.html">http://www.uhnres.utoronto.ca/facilities/wcif/software/Plugins/LeicaTIFF.html</a>)StackReg and TurboReg, algorithms used to correct for movement of tissue, are based on Th venaz and colleagues (1998)9.
Excel_writer.jar was written by Kurt De Vos (http://rsb.info.nih.gov/ij/plugins/excel-writer.html).
JNCT0.08JoVE.ijm, the particle detection algorithm, is based on an algorithm written by K.L. Brain and detailed previously5.

Part 1: Preparation of the Ca2+ indicator (Oregon Green 488 BAPTA-1 AM)
<ol>
<li>Oregon Green 488 BAPTA-1 AM comes in 500 &#x03BC;l vials as a small amount (50 &#x03BC;g) of powder. Add 40 &#x03BC;l Pluronic F-127 solution to the powder, shaking gently for 30 s, then add 460 &#x03BC;l PSS, and shake gently for 30 s. The final solution, 10 &#x03BC;M Oregon Green 488 BAPTA-1 AM, provides 4 x 125 &#x03BC;l aliquots. Avoid exposure to light and store at -20 C.</li>
</ol>
Part 2: Ca2+ loading
<ol>
<li>Remove the Ca2+ indicator (Oregon Green 488 BAPTA-1 AM) from the (-20 C) freezer and allow to thaw at room temperature.</li>
<li>Take 1 ml of PSS in a test-tube and place in water-bath. Bubble with 95% O2 / 5% CO2 at a rate of around 1 bubble appearing per second. The bubbler needs to be secured into the test-tube, e.g. with a bung or with Parafilm.</li>
<li>Dissect the tissue from the animal and, in a Sylgard-lined Petri dish, carefully remove connective tissue. Irrespective of smooth muscle organ type, PSS should be replaced every 10 mins and tissue should be washed well (e.g. by replacing the PSS three times) following dissection. For the vas deferens: consider cutting the organ length-ways to produce a sheet of tissue. For the urinary bladder: open longitudinally from the bladder neck (posterior) to the top of the dome (anterior). Prepare four strips, 6 &#x2013; 8 mm long and 1 &#x2013; 2 mm wide, along the dorsal surface of the detrusor, cutting in the direction of the dominant muscle bundles.</li>
<li>Place dissected tissue in the &#x2018;pre-warmed and bubbled&#x2019; PSS.</li>
<li>Add 125 &#x03BC;l of 10 &#x03BC;M Oregon Green 488 BAPTA-1 AM to the test-tube and give a little shake, by hand, to mix.</li>
<li>Resume bubbling and leave, covered with foil. The time is takes for sufficient tissue to take up the Ca2+ indicator varies with the size of tissue and thickness of the smooth muscle layer. For example: 70 mins (mouse detrusor, rat anococcygeus) to 120 mins (rat detrusor, mouse vas deferens).</li>
</ol>
Part 3: Preparing the &#x2018;Ca2+ loaded&#x2019; tissue for microscopy
Careful pinning of the tissue is necessary in order to minimize spontaneous movement of the tissue (a property of imaging of smooth muscle cells from relatively intact tissue) and to facilitate the location of a suitable site for imaging (as a flat piece of tissue may be surveyed in two dimensions, rather than three).
<ol>
<li>Rinse the tissue in bubbled PSS for at least 5 mins.</li>
<li>Mount in Sylgard-lined preparation dish, stretching the tissue (slightly) to form a flat sheet. Avoid using long &#x2018;pins&#x2019; that may scratch the objective; use instead short lengths of 25-50 &#x03BC;m diameter silver wire as &#x2018;pins&#x2019; and insert at an angle.</li>
<li>Position the preparation dish on the microscope stage, being careful to ensure that the PSS does not escape from the Sylgard-lined area of the dish (as this may lead to a large area being covered in PSS, when the superfusion begins).</li>
<li>Switch on the pump in order to superfuse the preparation with PSS. A rate of 100 ml per hour is often employed.</li>
<li>Switch on the heater unit.</li>
<li>Place the inflow and outflow tubes and temperature probe on the preparation dish (affixing each to the metal strip by the magnet on its base). The height of the tip of the outflow tube will determine the depth of the PSS. Be careful to ensure that the outflow is actually removing PSS (as it sometimes does not initially).</li>
<li>Set the temperature on the heater unit to reach the desired temperature, e.g. 33-35 C.</li>
<li>Allow the tissue to equilibrate for at least 10 mins.</li>
</ol>
Part 4: Selection of site to image
Exposure to excitation illumination will photobleach the indicator, so avoid using for more than a few seconds at a time.
<ol>
<li>With a low power objective (10x or 20x) use epifluorescence, exciting with a blue light, to view the smooth muscle and identify a suitable region to monitor. Try to pick a site based on: movement (which should be minimal) and the number of smooth muscle cells in the field. Bright &#x2018;structures&#x2019; may cause a high number of &#x2018;false positives&#x2019; in the image detection algorithm, so avoid sites with large numbers of (e.g.) interspersed epithelial cells or fibroblasts.</li>
<li>Switch to a higher power objective (40x).</li>
<li>Rotate the stage or the optical axis so the smooth muscle cell (s) lies horizontal (i.e. parallel to the rapid scan axis).</li>
</ol>
Part 5: Confocal microscopy
The details of how the confocal microscope is &#x2018;set up&#x2019; are specific to each microscope type, but the key considerations are: speed (minimum of 5 Hz is required for most Ca2+ transients); resolution and size of field (both of which are traded-off with speed). Some of the following protocol is specific to a Leica SP2 upright confocal microscope. A typical protocol is: 100 frame series, acquired at 5 Hz (256 x 256 pixels; approx. 100 x 100 &#x03BC;m) or 13.5 Hz (256 x 64 pixels; approx. 100 x 25 &#x03BC;m). The scanning head set to move bi-directionally (to maximize acquisition speed) and acquire at 1000 Hz per line.
<ol>
<li>Close the pinhole to one &#x2018;airy disk&#x2019;.</li>
<li>Set the laser (488 nm) power between 25 and 35% on the AOTF; this value is a trade-off between brightness and photobleaching. (The latter being a particular problem during long recordings.)</li>
<li>Acquire six series per site, and four - six sites per &#x2018;treatment&#x2019;. It&#x2019;s common to aim to monitor the same six sites pre-drug (i.e. &#x2018;control&#x2019;) and post-drug, although significant photobleaching may cause the experimenter to deviate from this.</li>
<li>It is essential with Ca2+ imaging that &#x2018;time controls&#x2019; are performed.</li>
</ol>
Part 6: Preparation for analysis
<ol>
<li>Download and install Image J from: http://rsb.info.nih.gov/ij/download.html. Download the platform-specific version, and then update to the latest version by downloading the latest imageJ.jar file from http://rsb.info.nih.gov/ij/upgrade/ij.jar, replacing the old file. Apple Mac users: if it runs slowly, make sure you&#x2019;re using the latest version of the java virtual machine (JVM) available from Apple (http://www.apple.com/macosx/features/java/).</li>
<li>Download Excel_writer.jar from: http://rsb.info.nih.gov/ij/plugins/excel-writer.html and install into the Plugins [directory] > jar directory.</li>
<li>Copy the following files into the Plugins directory: Leica_SP2_Stacker.class; JNCT0.08Release.ijm. Install each of these scripts using the Plugins [Menu] > Macros > Install&#x2026; function of ImageJ.</li>
<li>We use a Leica SP2 confocal microscope, the software (Leica LCS) for which generates each series as a single viewable &#x2018;movie&#x2019; within the software, but saves as individual frames. To reconstruct frames into series: (i) Make sure that all the &#x2018;frames&#x2019; to be reconstructed are in a single folder (e.g. &#x2018;DataFolder > Tiffs&#x2019;). (ii) Select Plugins [Menu] > Macros > Leica_SP2_Stacker. Select the root directory (e.g. &#x2018;DataFolder&#x2019;). Select the desired folder from the drop-down list (e.g. &#x2018;Tiffs/&#x2019;). Select an output directory or generate a new one (e.g. &#x2018;DataFolder > Stacks&#x2019;). The script will then reconstruct series from individual frames.</li>
</ol>
Part 7: Correcting for movement
Although movement of the imaged site may be minimized with good pinning and evenly-stretched tissue, some smooth muscle organs exhibit spontaneous contractions that cannot be abolished by careful pinning alone. Here we describe the use of a freely available algorithm that aligns or matches frames within a series.
<ol>
<li>Download &#x2018;StackReg&#x2019; (http://bigwww.epfl.ch/thevenaz/stackreg/) and &#x2018;TurboReg&#x2019; (http://bigwww.epfl.ch/thevenaz/turboreg/).</li>
<li>Install each into the Plugins directory using the Plugins > Macros > Install&#x2026;</li>
<li>Load a stack to be processed (File > Open&#x2026;) and then move to a frame that clearly shows the field of interest. Other frames will be aligned to this reference frame.</li>
<li>Plugins > Stacks > StackReg. Try each of the different &#x2018;Transformations&#x2019; (i.e. Translation, Rigid Body, Scaled Rotation, Affine) to determine what is most effective. (Further details: http://bigwww.epfl.ch/thevenaz/stackreg/)</li>
</ol>
Part 8: Particle detection algorithm
For each &#x2018;site&#x2019; imaged, factors will vary that affect the detection of Ca2+ transients. By measuring certain properties of each site (&#x2018;particle parameters&#x2019; - detailed below), the process of detection is facilitated. Thus for each site, one calculates &#x2018;threshold for subtracted&#x2019;, &#x2018;threshold for ratio&#x2019; and &#x2018;cell edge threshold&#x2019;.
<ol>
<li>Plugins [Menu] > Macros > Install&#x2026; and select &#x2018;JNCT0.08Release.ijm&#x2019;.</li>
<li>Plugins [Menu] > Macros > load stack (shortcut: l). Select a series and calculate particle parameters:<ol style="list-style:none;">
<li>a. Threshold for subtracted (= background)</li>
</ol></li>
<li>Select a rectangular ROI over what you believe to be &#x2018;background&#x2019;.</li>
<li>Measure (shortcut: m). Note down MEAN.<ol style="list-style:none;">
<li>b. Calculating threshold for ratio</li>
</ol></li>
<li>Select a rectangular ROI around an optically flat area of the cell(s).</li>
<li>Measure (shortcut: m). Note down MEAN and STANDARD DEVIATION.</li>
<li>Repeat a further two times OR you can draw three boxes at once by pressing &#x2018;shift&#x2019;.</li>
<li>Calculate and note down threshold for ratio = (1+SD/mean) x 32, averaging values from the three replicates / sites.<ol style="list-style:none">
<li>c. Cell edge threshold</li>
</ol></li>
<li>Image > Stacks > Z project... Choose projection type: &#x2018;Average intensity&#x2019;</li>
<li>Image > Adjust > Threshold.</li>
<li>Select black and white.</li>
<li>Define &#x2018;under&#x2019; (upper slider) limit and note down the corresponding value (to the right of the slider). Only events occurring in the black regions will be counted.</li>
<li>Click &#x2018;reset&#x2019;.</li>
<li>Analyse stacks (shortcut: 2)</li>
<li>Select just one series at this stage, i.e. the &#x2018;first file&#x2019; and &#x2018;last file&#x2019; should be the same.</li>
<li>Enter values of &#x2018;threshold for subtracted&#x2019;, &#x2018;threshold for ratio&#x2019; and &#x2018;cell edge threshold&#x2019;. Leave other settings at their default values.</li>
<li>The algorithm will produce a dual-pane window in which the processed series is shown on the left and the original on the right. Carefully go through this to assess the number of false positives and the occasions on which events, visible by eye, have not been detected. To get a more accurate detection of Ca2+ transients it may be necessary to adjust slightly some of the &#x2018;particle parameters&#x2019;. While the process may seem a little &#x2018;hit and miss&#x2019; on the first attempt, one quickly gets a feel as to what parameters are key.</li>
<li>For each series, the algorithm produces an Excel file that details characteristics of the detected event (such as amplitude, area and position). &#x2018;False positives&#x2019; - identified by reviewing the image files that the algorithm outputs (e.g. step 8.17) - may be removed, by hand, from this Excel file.</li>
</ol>

<ol><li>Gregory, J. Indicators for Ca2+, Mg2+, Zn2+ and other metal ions. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHaugland%2C+RP+%22Handbook+of+fluorescent+probes+and+research+products%22">Handbook of fluorescent probes and research products</a>. , Molecular Probes. Eugene, Oregon. 771-826 (2002).</li>
<li>Boland, B., Himpens, B., Gillis, J. M., Casteels, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationship+between+force+and+Ca2%2B+in+anococcygeal+and+vas+deferens+smooth+muscle+cells+of+the+mouse%5BTitle%5D)+AND+%22Pflugers+Arch%22%5BJournal%5D)">Relationship between force and Ca2+ in anococcygeal and vas deferens smooth muscle cells of the mouse.</a> Pflugers Arch. 421, 43-51 (1992).</li>
<li>Hashitani, H., Fukuta, H., Takano, H., Klemm, M. F., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Origin+and+propagation+of+spontaneous+excitation+in+smooth+muscle+of+the+guinea-pig+urinary+bladder%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Origin and propagation of spontaneous excitation in smooth muscle of the guinea-pig urinary bladder.</a> J Physiol. 150, 273-286 (2001).</li>
<li>Heppner, T. J., Bonev, A. D., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elementary+purinergic+Ca2%2B+transients+evoked+by+nerve+stimulation+in+rat+urinary+bladder+smooth+muscle%5BTitle%5D)+AND+%22J.+Physiol%22%5BJournal%5D)">Elementary purinergic Ca2+ transients evoked by nerve stimulation in rat urinary bladder smooth muscle.</a> J. Physiol. 564, 201-212 (2005).</li>
<li>Brain, K. L., Jackson, V. M., Trout, S. J., Cunnane, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intermittent+ATP+release+from+nerve+terminals+elicits+focal+smooth+muscle+Ca2%2B+transients+in+mouse+vas+deferens%5BTitle%5D)+AND+%22J.+Physiol%22%5BJournal%5D)">Intermittent ATP release from nerve terminals elicits focal smooth muscle Ca2+ transients in mouse vas deferens.</a> J. Physiol. 541, 849-862 (2002).</li>
<li>Fleichman, M., Schneider, T., Fetscher, C., Michel, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signal+transduction+underlying+carbachol-induced+contraction+of+rat+urinary+bladder+II.+Protein+kinases%5BTitle%5D)+AND+%22J.+Pharmacol.+Exp.+Ther%22%5BJournal%5D)">Signal transduction underlying carbachol-induced contraction of rat urinary bladder II. Protein kinases.</a> J. Pharmacol. Exp. Ther. 308, 54-58 (2004).</li>
<li>Young, J. S., Meng, E., Cunnane, T. C., Brain, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spontaneous+purinergic+neurotransmission+in+the+mouse+urinary+bladder%5BTitle%5D)+AND+%22J.+Physiol%22%5BJournal%5D)">Spontaneous purinergic neurotransmission in the mouse urinary bladder.</a> J. Physiol. 586, 5743-5755 (2008).</li>
<li>Nelson, M. T., Cheng, H., Rubart, M., Santana, L. F., Bonev, A. D., Knot, H. J., Lederer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relaxation+of+arterial+smooth+muscle+by+calcium+sparks%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Relaxation of arterial smooth muscle by calcium sparks.</a> Science. 270, 633-637 (1995).</li>
<li>Thévenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+pyramid+approach+to+subpixel+registration+based+on+intensity%5BTitle%5D)+AND+%22IEEE+Trans.+Image+Process%22%5BJournal%5D)">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans. Image Process. 7, 27-41 (1998).</li>
</ol>

Efforts were made to minimize the number of animals used and their suffering; all experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986 and European Communities Council Directive 86/09/EEC.

Choice of fluorescent Ca2+ indicator
Oregon Green 488 BAPTA-1 AM was chosen as the Ca2+ indicator because it (i) is bright compared to other indicators (e.g. Fluo-4 and Calcium Green)1; (ii) is sensitive to physiological changes in Ca2+ concentration with a Kd of similar magnitude1 to the intracellular Ca2+ concentration [Ca2+]i (e.g. of vas deferens2); (iii) loads many smooth muscle cells, enabling smooth muscle cell coupling to be monitored. However, Oregon Green 488 BAPTA-1 AM is a non-ratioable Ca2+ indicator, and as such cannot easily be used to determine the absolute [Ca2+]i. Furthermore, it may also buffer Ca2+ if present at high concentrations and becomes saturated with high amplitude changes in [Ca2+]i.
Inhibition of spontaneous contractility
The spontaneous contractility of smooth muscle organs may be reduced pharmacologically, such as by blocking the movement of Ca2+ through L-type Ca2+ channels (e.g. by: nifedipine3; diltiazem4). Note that these drugs will greatly reduce the amplitude of whole-cell Ca2+ flashes / transients.
Contraction of the vas deferens results from a combination of primarily purinergic and noradrenergic neurotransmission. Focal Ca2+ transients in this tissue are, however, insensitive to noradrenergic receptor blockade (e.g. &#x03B1;1 adrenoceptor, by prazosin5) so movement may be reduced without affecting focal Ca2+ transients.
Alternatively, contraction may be prevented &#x2018;downstream&#x2019; through the inhibition of myosin light chain kinase e.g. by wortmannin6.
Conclusions
Monitoring Ca2+ fluorescence at submicron resolution is a powerful technique to study the post-junctional effects of neurotransmitter release5,7 and release of Ca2+ from internal stores (e.g. &#x2018;sparks&#x2019;8). The analysis of Ca2+ transients using the methodology outlined here is cost-effective compared to commercially-available image analysis packages and allows tailor-made analysis through custom-written Java plugins for ImageJ.

<table><tbody><tr><td>Oregon Green 488 BAPTA-1 AM</td><td>Invitrogen</td><td>O6807</td><td>10 x 50 &amp;#x03BC;g</td></tr><tr><td>Pluronic F-127 solution</td><td>Invitrogen</td><td>P3000MP</td><td>20% solution in DMSO</td></tr><tr><td>Leica SP2 upright confocal microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Air table (&amp;#8216;vibration isolated workstation&amp;rsquo;)</td><td>Newport Corp.</td><td>M-VW-3046-OPT-012140</td><td></td></tr><tr><td>Sylgard 184 elastomer</td><td>Dow Corning</td><td></td><td></td></tr></tbody></table>

focal ca2+ transient detection in smooth muscle

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

First, a calcium loading solution is prepared, warmed, and bubbled. The urinary bladder is dissected out, opened, pinned out flat, and a strip of detrusor is taken. The detrusor strip is added to the loading solution, incubated and rinsed, pinned out flat.The calcium floor four within the tissue is excited by laser light and the emitted light captured resulting. Movies consisting of individual frames are analyzed by comparing successive frames. The background is subtracted.The cell edge threshold determines where events can be found and a ratio between successive brains is taken. If the remaining events match predefined criteria, they are highlighted and labeled. Hello, my name is John Young from the laboratories of Keith Brain and Tom Kinane at the Department of Pharmacology, university of Oxford.I'm Rob Amos, also from the Brain and Canne Labs and my name's Keith Brain. Today, John and Rob will show you a calcium imaging technique in smooth muscle. We use this in our laboratory to study the release of neurotransmitter from autonomic nerves and its effect on smooth muscle.So let's get started. The calcium indicator organ green, 4 88 BAFTA 1:00 AM comes in 500 microliter vials containing 50 micrograms of powder. To prepare the calcium indicator at 40 microliters of Onic F1 27 solution to the powder and shake gently for 30 seconds after that at 460 microliters of physiological saline solution PSS, and shake gently for another 30 seconds.The final solution, 10 micro molars of organ green. 4 88 BAFTA 1:00 AM provides four aliquot of 125 microliters each. Avoid exposure to light and store the solution at negative 20 degrees Celsius prior to dissecting the tissue of interest.Remove the calcium indicator from the freezer and thaw at room temperature while the calcium indicator is thine. Place one milliliter of PSS in a test tube and a water bath at 33 degrees Celsius. Secure a bubbler into the test tube with a bung, or you could also use perfil and then bubble with a 95%oxygen to 5%carbon dioxide mixture at a rate of around one bubble appearing per second.Dissect the smooth muscle organ from the animal. In this case, it's the urinary bladder from a mouse. Place the organ in about 50 millimeters of PSS to wash it after the tissue has been washed carefully remove the ciero adipose tissue and connective tissue.Dissect open according to the smooth muscle laurean type for the urinary bladder. Open longitudinally from the bladder neck, the coddle end to the top of the dome. The cranial end Prepare four strips, six to eight millimeters long and one to two millimeters wide along the dorsal surface of the resser cutting in the direction of the dominant muscle bundles.Place the dissected tissue and the pre-war and bubbled PSS to load the calcium indicator at 125 microliters of 10 micromolar organ green, 4 88 B to 1:00 AM to the tissue in the test tube and give it a little shake by hand to mix. Return the tube to the water bath. Resume bubbling and cover the water bath with a sheet of foil.The time it takes for sufficient tissue to take up the calcium indicator varies with the size of the tissue and the thickness of the smooth muscle layer. For example, for the mouse detrusor and rat oxs, it's 70 minutes. Whereas for the rat detrusor and mouse VAs deens, we incubate for 120 minutes.Once the tissue has been loaded with the calcium indicator, it can be prepared for microscopy. First, rinse the tissue in bubbled PSS for 10 minutes. Next, mount the tissue in a S guard lined preparation dish.Stretching it slightly to form a flat sheet. Pin the tissue using short lengths. For example, two millimeter of 25 to 50 micrometer diameter silver wire and insert the pins at an angle.Avoid using pins that are any longer than a few millimeters in length as they may scratch the objective. Careful pinning of the tissue is important from minimizing spontaneous of the tissue and to facilitate the location of a suitable site for imaging. After the tissue has been pinned, position the preparation dish on the microscope stage.Be careful that the PSS does not escape from the sil guard lined area of the dish. As this may lead to a large area being covered in PSS. When the superfusion begins for demonstration purposes, the following steps will be done in the light.Please note that these experiments are normally performed in darkness. Switch on the pump in order to super fuse the preparation. With PSS, A rate of 100 milliliters per hour is often used.Switch on the heater unit, place the inflow and outflow tubes and temperature probe on the preparation dish of fixing each to the metal strip by the magnet on its base. The height of the tip of the outflow tube will determine the depth of the PSS. Be careful to ensure that the outflow is actually removing PSS as it sometimes does not.Initially set the temperature on the heater unit to reach the desire temperature of between 33 and 35 degrees Celsius. Allow the tissue to equilibrate for at least 10 minutes with a low power objective. 10 or 20 times.Use epi fluorescence or exciting with a blue light to view the smooth muscle and identify a suitable region to monitor. Since exposure to excitation, illumination will photobleach the indicator. Avoid using for more than a few seconds at a time.Avoid sight with bright structures because they may cause a high number of false positives in the image detection algorithm. Try to pick a site with minimal movement and a large number of smooth muscle cells in the field. Once you have selected the site to image switch to a higher power objective such as 40 x.Rotate the stage or the optical axis so that the smooth muscle cell or cells lie horizontal. That is parallel to the rapid scan axis. The smooth muscle cells are now ready for imaging by confocal microscopy.In this protocol, we use a Leica SP two upright confocal microscope. Close the pinhole to one airy disc. Set the laser power between 25 and 35%on the custo optic tuneable filter.This value is a trade off between brightness and photo bleaching. To prepare for data analysis, download and install image J.Download the platform specific version and then update to the latest version by downloading the latest ImageJ jar file to replace the old file. Next, download the Excel writer jar file and install into the plugins jar directory.Copy these files into the plugins directory. Install each of these scripts using the plugins macros. Install function of image J.The software for the Leica SP two confocal microscope that we use called Leica.LCS saves individual frames of each series as a separate file. To reconstruct the series. Make sure that all the frames to be reconstructed are in a single folder.For example, data folder tiffs. Then select the Leica SP two stacker macro from the plugins macros menu. Select the root directory in this case data folder.Select the desired folder from the dropdown list. In this case, tiffs select an output directory or generate a new one. For example, data folder stacks.The script will then reconstruct series from individual frames to correct from movement. We use a freely available algorithm that aligns or matches frames within a series downloads stack, rig and turbo reg. Copy the files for each into the stacks folder.Load a stack to be processed and then move to a frame that clearly shows the field of interest. Other frames will be aligned to this reference frame. After that, go to plugins stacks.Stack Reg. Try each of the different transformations to determine what is most effective for each site. Imaged factors that affect the detection of calcium transients will vary by measuring certain properties, particle parameters of each site.The sensitivity and specificity of detection are increased. Thus, for each site, we will calculate threshold for subtracted threshold for ratio and cell edge threshold. To begin, go to plugins, macros, install and select jn CTO dot oh eight release dot IJM JM.Then go to plugins macros load stack.Select a series and calculate the following particle parameters. To calculate the threshold for subtracted, select a rectangular region of interest over what you believe to be background measure. Note down mean To calculate the threshold for ratio, select a rectangular region of interest around an optically flat area of the smooth muscle cell or cells.Measure note down mean and standard deviation. Repeat the process two more times, or you can draw three boxes at once by pressing shift. Calculate add note down the threshold for ratio.Calculate as the standard deviation divided by the mean add one, and then multiply the result by 32. Averaging values from the three replicates, the factor of 32 is necessary to account for the ratioing algorithm where no change from the proceeding frame is mapped to an integer value of 32. This gives a good dynamic range to detect increases and decreases in the ratio.For an eight bit image, select image adjust threshold. Select black and white. Define under the upper slide limit and note down the corresponding value.This is to the right of the slider. Only events occurring in the black regions will be counted. Click reset.Now that the threshold for subtracted threshold for ratio and sell edge threshold have been calculated, we can analyze the image stacks. Determine the location of the stacks to be analyzed. Select just one series at this stage.That is the first file, and last file should be the same. Then select a location for output of data files and stacks. For example, data folder output.Enter values of threshold for subtracted the threshold for ratio and sell edge threshold. Leave other settings at their default values. The algorithm will produce a dual pane window in which the processed series is shown on the left and the original on the right.Open this file from the output folder and carefully go through this to assess the number of false positives and the occasions on which events visible by eye have not been detected. For each series, the algorithm produces an Excel file that details characteristics of the detected event such as amplitude area, and position false positives identified by reviewing the image files that the algorithm outputs may be removed by hand from this Excel file. Although you should have strict criteria for doing so.This is a movie of calcium transience in smooth muscle. In this example, the movie is comprised of 100 individual frames acquired at five hertz. We'd acquire many of these movies per experiment when investigating, for example, the effect of a particular drug on the frequency of calcium transient.Here we see numerous spontaneous calcium transient in the smooth muscle cells. The outcome of the particle detection algorithm is shown here. For each series, the algorithm produces an Excel file that details characteristics of the detected event such as amplitude area and position.So we've demonstrated a method for imaging calcium trance in smooth muscle organs such as the urinary bladder, and we've demonstrated a way in which we analyze those calcium transients using an algorithm that we've produced in the lab. The important things to remember are to prepare the tissue well for the calcium imaging. So in the case of the tissue that we demonstrated today, that's removing connective tissue and and removing any fat that may obscure the field.It's also important to pin the tissue in a flat sheet. Well, good luck with your experiments and thank you for watching.

focal-ca2-transient-detection-in-smooth-muscle

Research

JoVE Journal

Biology

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy.   The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell.  Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Measuring Fast Calcium Fluxes in Cardiomyocytes

Vascular cell functiondepends on activity of intracellular messengers.  Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

This paper describes a protocol that assesses the changes of myofilament Ca2+ sensitivity during contraction in isolated cardiac myocytes from rat heart. Together with cardiac electrophysiology, systolic/diastolic cytosol Ca2+ levels and contraction/relaxation, this measurement is imperative in underpinning the mechanisms mediating cardiac excitation-contraction coupling in healthy and diseased hearts.

Assessment of Myofilament Ca2+ Sensitivity Underlying Cardiac Excitation-contraction Coupling

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

The present protocol describes preparing and utilizing mouse precision-cut lung slices to assess the airway and intrapulmonary arterial smooth muscle contractility in a nearly in vivo milieu.

Utilizing the Precision-Cut Lung Slice to Study the Contractile Regulation of Airway and Intrapulmonary Arterial Smooth Muscle

Functional site-directed fluorometry is a method to study protein domain motions in real time. Modification of this technique for its application in native cells now allows the detection and tracking of single voltage-sensor motions from voltage-gated Ca2+ channels in murine isolated skeletal muscle fibers.

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca2+) concentrations in cells using single-cell Ca2+ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca2+ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Learn high-resolution calcium imaging in smooth muscle using confocal microscopy and isolated organ preparations. This protocol enables detailed analysis of neurotransmitter effects on calcium signaling events.

Focal Ca²⁺ Transient Detection in Smooth Muscle

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