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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.