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

The overall goal of this procedure is to measure calcium or nitric oxide in a freshly isolated vessel using two photon microscopy. This is accomplished first by mechanical isolation and preparation of the vessel. Next, the vessel is incubated in the calcium labeling dye or an O labeling dye with onic acid.

Then the vessel is fixed to a silicone coated dish with a grid and pins to provide a stable view during two photon imaging. Finally, two photon imaging is performed and the results are quantified. This video shows elevation of intracellular calcium signaling in endothelial cells of isolated blood vessels in response to changes in extracellular calcium concentration.

Ultimately, this method will allow investigators to monitor calcium or nitric oxide changes in endothelial cells or smooth muscle cells of a freshly isolated blood vessel. The main advantage of this technique over existing methods is that it can be provided better signal resolution and precise measurements of the intracellular processes in vascular tissues. Here, this technology was applied in combination with classical, not geometric dice, to measure calcium transient and ntic oxide production in individual cells on an isolated hollow orca.

We believe that this technique will help forward our knowledge about the mechanism regulated intracellular calcium and oxide levels within the different vascular cell types. Two photon excitation Microscopy imaging of living cells, especially in intact tissues such as blood vessels, provides the advantages of high resolution signal and low out of focus background fluorescence. The narrow localization of the two photon excitation illuminates a single focal volume in the tissue, greatly reduces out of focus absorption and decreases light scattering, allowing us to image fluoro four dynamics in highly autofluorescent tissues containing collagen and elastin fibers.

After isolating a rat aorta, according to the text protocol under a dissecting microscope, use fine tipped forceps and micro scissors to dissect the fat and connective tissue from the aorta. Once it is clean, cut the organ longitudinally and transfer it to physiological salt solution for later use. To prepare the loading cocktail at seven microliters of ready-made one millimolar flu oh 4:00 AM dye and DMSO solution to 450 microliters of PSS containing no calcium.

Then add 40 microliters of non DMSO based 10%onic acid solution to the loading cocktail to help disperse the acet methyl lesters and improve loading of the calcium dye. Place the dissected aortas into 500 microliter tubes containing the loading cocktail. Cover the tubes with foil and place on a rotating shaker at room temperature for one hour.

After following the text protocol to pin the aortic tissue to the surface of a silicone coated dish, place the dish beneath the nose piece of an upright two photon microscope and install and position a 25 x water immersion objective lens Above the specimen, install a corresponding emission filter into the microscope port. Using the software, activate the two photon infrared laser and tune the laser excitation to 820 nanometers under transmitted light. Using course objective adjustment, locate the aorta and focus the objective on the endothelial surface.

Switch the microscope into two photon laser scanning mode, ensuring that the excitation laser is mode locked. The non DS scanned detectors are engaged with about five to 10%laser power. Start live scanning.

And finally, adjust the objective to bring the endothelial or smooth muscle cells into focus. Collect sequential images according to the experimental protocol Text. Save the images in a format that includes the metadata to carry out image processing and calculation download and install the Fiji image processing package, which is released under the general public license.

Using the software, open an image file. Then when prompted, split the transmitted light and fluorescent channels to use only the fluorescent channels for the data analysis within the Fiji program. Click on the analyze tab and scroll down to the tools option.

Then click on the ROI manager tab, and a new window will appear.Next. Under the analyze tab, click on set measurements and select the specific measurements of interest. For the calcium transient measurements.

Use the mean gray value option. Then with the circular trace tool, begin to trace the regions of interest and on the ROI manager click add for every ROI chosen. Once all the cells of interest have been traced under the more tab in the ROI manager, select multi mesure.

Finally, either save the measurements directly or copy and paste them into a spreadsheet and carry out calculations according to the text protocol. Using the protocol described in this video, the endothelial cells of the aorta are easy to locate due to their shape and strong fluorescent signal. Smooth muscle cells will also be visible in the same field with endothelial cells, which will lead to a better understanding of how the two cell types jointly yet independently respond to different agonists and stimuli to demonstrate the utility of this method.

Measurements of the response to acetylcholine or a CHA potent calcium dependent vasodilator of the endothelium were determined as shown here after the addition of a CH endothelial cell fluorescence increases compared to cells in PSS alone indicating a rapid increase in intracellular calcium content that slowly returns to baseline. This protocol is also suitable for the detection of endothelial nitric oxide production. In this experiment, aortic endothelial cells were loaded with DAF FM D acetate.

Dye ENO Production increased in a calcium dependent manner in response to a CH stimulation, and this response was blocked with the endothelial OCH synthase inhibitor L name. This method can help answer key questions in the area of cardiovascular physiology. Use of experimental procedure described here, together with multiple available genetically modified animal models, provides new perspectives for the FAR FSA application of two photo microscopy in basic and translational research.

Use of this approach might provide you an in-depth understanding and mechanistic insight into specific intracellular signaling pathways within the blood vessel cells. After watching this video, you will learn how to apply this technique for real time monitoring of the calcium and nitric oxide levels within the individual cells of freshly isolated blood vessels.