We thank Dr. William Cashdollar, and the Northwestern Mutual Foundation Imaging Center at the Blood Research Institute of Wisconsin for help with the imaging studies. We also thank Dr. Daria Ilatovskaya for critical reading of this manuscript and helpful discussion. This study was supported by National Institutes of Health Grants HL108880 (to A.S.) and DP2-OD008396 (to A.M.G.).

The experimental procedures described below were approved by the Institutional Animal Care and Use Committee (IACUC) at the Medical College of Wisconsin and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
1. Isolation of the Rat Aorta
<ol>
	<li>Anesthetize rats with isoflurane (5% induction, 1.5 to 2.5% maintenance) or another IACUC approved method.</li>
	<li>Place the rat in a supine position. In order to expose the abdominal organs, make a small ventral midline incision through the skin and the subcutaneous abdominal tissue. Using gauze pads gently deflect the intestines to expose the aorta and vena cava as shown in Figure 1.</li>
	<li>Briefly, blunt dissect the aorta from the connective tissue and from the vena cava. Using suture, tie off the aorta as close as possible to the kidney. Dissect about 1 - 2 cm of aorta and place it in a 15 ml&#160;conical tube containing normal Physiological Salt Solution (PSS; in mM: 140 NaCl, 1.2 MgCl2, 2 CaCl2, 4.5 KCl, 6 Glucose, 10 HEPES) on ice.</li>
	<li>Once the aorta is isolated, euthanize the animal according to approved IACUC protocols. At the completion of all non-survival procedures euthanize deeply anaesthetized animals by thoracotomy inducing pneumothorax to ensure the humane demise of the animal.3</li>
	<li>Using a dissection microscope, fine-tipped forceps, and microscissors dissect the fat and connective tissue off of the aorta. Once the aorta is clean, cut the aorta longitudinally and place it in PSS on ice for later.</li>
</ol>
2.&#160;Dye Loading and Incubation
<ol>
	<li>Pipet 7 &#181;l of 1 mM Fluo-4 AM dye, which is supplied in DMSO-based solution, into individual 500 &#181;l tubes that have been covered with aluminum foil. Store in the freezer at -20 &#730;C to preserve the dye properties over time. Note: these aliquoted solutions should be stable for at least six months.</li>
	<li>Before use, allow the dye solutions to warm up to RT before opening. Prepare working solutions immediately before use. Do not store the diluted reagent for later use.</li>
	<li>Add 7 &#956;l of ready-made 1 mM Fluo-4 AM dye dissolved in DMSO solution to 450 ul of PSS containing no calcium. Add 40 &#956;l of non DMSO based 10% Pluronic Acid solution to the loading cocktail to help disperse the acetoxymethyl (AM) esters and improve loading of the calcium dye.</li>
	<li>Place the dissected aortas into the 500 &#181;l tubes containing the loading cocktail (as described in 2.3) for 1 h. Cover all tubes with foil and place on a rotating shaker at RT.</li>
	<li>To monitor NO production in the vasculature, use DAF-FM diacetate dye. Incubate the aorta in a solution containing 450 &#181;l PSS, 40 &#181;l pluronic acid and 5 &#181;l of 10 mM DAF-FM diacetate. Similar to the calcium incubation protocol (described in 2.3), place the aorta in an 500 &#181;l tube containing the NO-dye solution covered in foil and place on a rotating shaker for 30 min at 4 &#730;C followed by next 30 min at RT.</li>
	<li>When NO production is measured, use the endothelial NO synthase blocker, L-NAME, to block NO production as a control as shown in Figure 4B. For that procedure, add to cocktail (as described at 2.5) 100 nM L-NAME for the last 30 min of incubation (at RT).</li>
</ol>
3. Laser Scanning Two photon Microscopy Protocol
<ol>
	<li>After the 1 h incubation, wash the aorta 2 - 3 times in clean PSS to remove extracellular dye.</li>
	<li>Transfer the aorta to a silicone-coated dish containing either normal PSS or Ca2+-free buffer (depends on the experimental protocol). Pin the aorta down onto the silicone coating with the adventitia in contact with the silicone (i.e., endothelial lumen exposed to the dish opening) using &#181;-shaped pins. Alternatively, use a Slice Anchor grid or glue to fix the tissue. 
	Note: To reduce stress and possible mechanical artifacts, transfer and fix aorta in Ca2+-free solution and wait 5 - 10 min before starting the experiment.</li>
	<li>Connect a peristaltic pump or a syringe to the silicone-coated plate for automatic or manual application of drugs or for changing the extracellular solution.</li>
	<li>Place the dish beneath the nose piece of the upright two-photon microscope, and install and position a 25&#215; (N.A. 1.05 and working distance 2 mm) water-immersion objective lens above the specimen. Note: If this specific lens is not available, use an objective specifically manufactured for two photon imaging, because it can increase signal resolution dramatically compared with a regular objective.</li>
	<li>Activate the two photon laser using the software (the laser begins to pump and turn on). Tune the laser excitation to 820 nm.</li>
	<li>Using epifluorescence, locate the aorta and focus the objective on the endothelial surface using coarse adjustment.</li>
	<li>Switch the microscope into two photon laser scanning mode, ensuring that the excitation laser is mode locked, the non-descanned detectors are engaged and the emission filters appropriate to the expected emission spectra are installed. Note: Here, the FV10-MRL/R&#160;(495 to 540 nM) were used.</li>
	<li>Start live scanning using approximately 5% laser power and finely focus the objective in order to visualize the endothelial cells. 
	Note: The endothelial cells will exhibit a strong fluorescent signal when incubated in normal PSS. This will be noticeably weaker when the vessels are incubated in calcium-free buffer. The endothelial cells will be present on the top of the opened aorta and the smooth muscle cells can be seen just below the endothelial cells (see Figure 2). Because blood vessels are neither smooth nor even, there will be places where both smooth muscle cells and endothelial cells are present.</li>
	<li>Once the cells are in focus, program the microscope software to collect sequential images in fast scan mode (raster scan, 512 x 512 pixel window with a frame collection frequency of 1.1 s). In parallel with Fluo-4 AM fluorescence, collect transmitted light images in order to detect changes in vessel contraction and better visualize the experimental conditions.</li>
	<li>After collection of baseline signal images, change the Ca2+ ion concentration in the external solution or slowly apply pharmacological stimulus agents using the peristaltic pump while imaging. Observe the fluorescent signal increase within the loaded cells. 
	Note: Endothelial cells respond to changes in flow, thus it is recommended to use low laminar applications and vehicle testing before application of pharmacological activators of Ca2+ or NO signaling. To recalculate intracellular calcium to nM concentration values in vessels loaded with Fluo-4 (dissociation constant for Fluo-4 is 345 nM), fluorescence intensity could be recorded at baseline and after addition of ionomycin and MnCl2.4</li>
</ol>
4. Image Processing and Calculations
<ol>
	<li>Download and install Fiji image processing package.</li>
	<li>Open the image file in Fiji. Upon importing the file, split the channels (transmitted light and fluorescent signal) when prompted. Use only the fluorescent signal for the data analysis.</li>
	<li>Within the Fiji program, click on the analyze tab and scroll down to the tools option. Under the tools option, find the tab that says ROI manager and click on it; a new window will appear.</li>
	<li>After this, click on set measurements under the analyze tab. Select the specific measurements of interest. For the calcium transient measurements, use the mean gray value option.</li>
	<li>With the circular trace tool, begin to trace the regions of interest (ROI) and click &#8220;add&#8221; on the ROI manager screen for every ROI. Once all cells of interest have been traced, in the ROI manager window, select Multi Measure under the &#8220;more&#8221; tab. Remember to either save the measurements directly or copy and paste all of these measurements into a spreadsheet.</li>
	<li>Open *.txt file or Excel spreadsheet from Fiji and transfer the numbers to a new Origin worksheet. Note: Alternatively, use any program like Sigma Plot or Prism for further analysis.</li>
	<li>Within the Origin program, use Plot &#8594; Line+Symbol menu to observe an overview of all transient responses. Deselect the unresponsive cells.</li>
	<li>Recalculate trace number to time scale (X axis), use Column &#8594; Set Column Values and multiply the trace number column to the image collection frequency (in our case 1.1s).</li>
	<li>Use Analysis &#8594; Statistics on Rows for all selected recordings to calculate Mean (Y axis) and Standard Error trace. Plot final Graph for current group of cells Plot &#8594; Line + Symbol.</li>
	<li>The mean calcium transient calculation is described as followed.
	<ol>
		<li>For total calcium release within the Origin program, click on Graph, then use menu Analysis &#8594; Calculus &#8594; Integrate. Total integral of area under curve appears in Result Log.</li>
		<li>For transient kinetics,&#160;click on Graph, then use menu: Analysis &#8594;&#160;Non-linear Curve Fit &#8594;&#160;Select Data Set (choose X axis range from maximum to end of transient response). Start Fitting&#160;&#8594;&#160;Select Function &#8594;&#160;Exponential Decay 1 &#8594;&#160;Done. 
		Note: Exponential fitting appears in Graph window and Result Log. Obtained data (values) represent total amount of calcium released, and kinetics of the channels mediated the transient response.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Experimental Overview. To better understand the contribution of calcium and NO to vascular physiology, a novel method was developed for measuring [Ca2+]i and NO within smooth muscle and endothelial cells of isolated intact aortas. Together, this protocol consists of these critical steps: 1) Mechanical isolation and preparation (not enzymatic digestion) of the vessel. It is important to keep the tissue healthy and intact as much as possible to obtain optimal physiological recordings. 2) Incu...

Calcium is a fundamental second messenger within vascular cells such as endothelial and smooth muscle cells. It is the primary stimulus for vascular contraction and plays a major role in vascular dilation, including its effects through NO generation within the endothelium. Due to limitations of imaging technologies, it has been virtually impossible to observe calcium handling within the intact vessel. The development of two photon imaging systems and the creation of new calcium or NO labeling dyes, makes it possible to image at a sufficient depth and resolution to begin to understand calcium dynamics and NO production within the vasculature.
Two photon microscopy has recently been applied in&#160;tissue, organs and even whole animal studies because of its superior ability to deeply penetrate tissues with low background fluorescence and high signal sensitivity.1,2 The narrow spectrum of two photon excitation at the illumination focal point and the use of non-descanned detectors are the reasons why two photon microscopy is superior to traditional confocal microscopy. Confocal microscopy cannot produce high-quality images at the necessary tissue depth due to the auto-fluorescence and the scattering of out-of-focus light into the confocal pinhole. Consequently, we have developed a method using a two photon microscope to measure [Ca2+]i signaling and NO production in intact, individual blood vessel cells with high resolution and a low signal-to-noise ratio.&#160;

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DAF-FM</td>
			<td>Life Technologies</td>
			<td>D-23842</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo 4 AM</td>
			<td>Life Technologies</td>
			<td>F14217</td>
			<td>500&#181;l in DMSO</td>
		</tr>
		<tr>
			<td>Pluronic F-68 solution</td>
			<td>Sigma-Aldrich</td>
			<td>P5556</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Name</td>
			<td>Tocris</td>
			<td>0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus upright microscope</td>
			<td>Olympus</td>
			<td>Fluoview FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MaiTai HP DeepSee-OL&#160;</td>
			<td>Spectra Physics</td>
			<td></td>
			<td>Ti:sapphire laser 690nm &#8212; 1040nm</td>
		</tr>
		<tr>
			<td>Filters</td>
			<td>Olympus</td>
			<td>FV10-MRL/R&#160;</td>
			<td>495 to 540 nm&#160;</td>
		</tr>
		<tr>
			<td>25&#215; water-immersion objective lens</td>
			<td>Olympus</td>
			<td>XLPL25XWMP</td>
			<td>N.A. 1.05 and working distance 2 mm</td>
		</tr>
		<tr>
			<td>Slice Anchor grid&#160;</td>
			<td>Warner Instrument&#160;</td>
			<td>SHD-27LH</td>
			<td></td>
		</tr>
		<tr>
			<td>Other basic reagents&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

two-photon imaging of intracellular ca2+ handling and nitric oxide production in endothelial and smooth muscle cells of an isolated rat aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

In order to accurately assess the contribution of calcium to vascular physiology (vasodilation and vasoconstriction), a protocol was designed to load calcium dyes into both endothelial cells and smooth muscle cells in isolated intact aortae. The general experimental set up depicted in Figure 1, shows the basic strategy for isolation and preparation of the vessel before imaging. Briefly, after isolation of the aorta from the rat, it should be cleaned of fat and connective tissue and slit longitudinally. T...

Watch this Scientific Journal Video about Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta at JoVE.com

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

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

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

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11.2K Views.  Medical College of Wisconsin. 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. 