The capacity to acquire large amounts of data from calcium imaging experiments has drastically improved, especially with tissue imaging experiments. Our protocols describe appropriate analysis techniques to quantify and describe these data sets. Our protocols generate a range of spatial, temporal, and intensity values that are intrinsic to calcium signaling in entire tissues and this can be lost in more rudimentary-based analysis.
One hour before the imaging transfer a small intestine harvested from a mouse with interstitial cells of Cajal or ICC that specifically express a genetically-encoded calcium indicator to the stage of a spinning disk confocal microscope. And continuously perfuse the tissue with 37 degrees Celsius Krebs-Ringer bicarbonate or KRB solution. Using a 60 to 100 X magnification locate the stellate-shaped myenteric plexus ICC or ICC-MY between the circular and longitudinal smooth muscle layers of the small intestine.
Then locate the spindle-shaped deep muscular plexus ICC or ICC-DMP in a single plane between the circular smooth muscle layer and the submucosal plexus and save the movies as a stack of TIFF images. To create spatiotemporal maps or STMs of the ICC-DMP calcium activity open the image stack in ImageJ, and click Image, Type and 32-bit to modify the image quality. To create a line scan right-click on the line selection tool and select segmented line.
Using single left clicks draw a line along the mid access of an individual ICC-DMP, double-clicking when the entire cell has been outlined. Select Image, Stacks and Reslice. A pop-up window will appear.
Select Rotate 90 degrees to orient the line scan from left to right so that it can be calibrated and read against time on the x-axis with space on the y-axis and click OK.The spatiotemporal map will appear. To accurately measure the amplitude of the calcium signals from the map, select rectangular selection and draw a region of interest around the area within the STM that displays the most uniform and least intense area of fluorescence. Click Analyze and Histogram to obtain a mean value of the intensity within the selected region of interest and click Edit, Selection, Select All to select the entire STM.
Next, click Process, Math and Divide and enter the mean value of the intensity with the selected STM region of interest in the pop-up box. The STM will turn black. Correct this by clicking Image, Adjust, Brightness/Contrast, and Auto in the pop-up window.
The line scan will now be calibrated for the amplitude with the intensity of the fluorescence expressed as the fluorescence divided by the zero fluorescence. The STM will display the number of frames in the TIFF stack on the x-axis and the number of pixels representing the length of the cell on the y-axis. To quantify the temporal and spatial data from the calcium signals click Image and Properties and enter the appropriate values to fully calibrate the STM for space and time in the pop-up window.
To analyze the individual calcium events click Straight Line and click and drag on the STM to draw a straight horizontal line through the center of a calcium event parallel to the x-axis. Then click Analyze and Plot Profile. A box will appear showing the plot profile of the calcium event.
For particle-based analysis of the ICC-MY calcium signaling data, open the movie file in Volumetry and right-click to select STK Filter, Differentiate. Right-click again to input a value to differentiate. Press Enter and left-click to initiate the differentiation.
To create particles, use the middle mouse button to scroll through the movie to select a 20 to 40 frame section that includes a quiescent period, followed by the occurrence of a calcium transient cluster and 20 to 40 frames that follow right after the cluster. When all of the frames have been selected right-click in the movie window to access STK OPS Ramp DS particle info and click to run a particle analysis routine that progressively ramps the threshold from the maximum to the minimum intensity. To ensure a successful particle analysis, ensure that you incorporate as much pre-calcium transient cluster non-activity, and as much post-calcium transient cluster non-activity as you can.
This will help to boost the signal-to-noise ratio. The analysis will be displayed graphically in the plot window as plot of noise and in the traces window as three color traces with the green trace representing the number of particles, the red trace representing the average particle size, and the blue trace representing the absolute intensity threshold. To histogram balance the plot of particle noise press H on the keyboard.
Then press the right bracket key to cycle through color schemes until the background is white with the color traces on top. Press F on the keyboard one time to bring up a measuring tool. Then press F a second time to mark a single vertical line in the scroll bar of the traces window on the plot at the intersection where the colored plots begin to shift to the right-hand side.
Within the traces window use the middle mouse button to scroll from the inflection point of the red trace to the marked white vertical line. After noting the displayed Y average, click the middle mouse button again inside the traces window to deselect the blue trace. In the movie window click C to bring up a color wheel, and D to adjust the threshold.
Valid particles will now be shown in red, and those that saturate will be white. Use the left mouse button to scroll until the white areas are removed and use the middle mouse button to adjust the yellow numerical value in the color wheel to the Y average value to assign everything in red as an active calcium transient particle at the determined threshold point. To save the data as a coordinate-based particle file right-click to access STK 3D, save particle zero, and left-click to save the file.
To create particle activity heat maps open the saved particle file and right-click in the movie window to access particle STKops, StatMap Flag and enter flag assignments to analyze. Click to apply the analysis and a heat map showing the total particles for the entire length of recording with different colors representing the percent occurrence throughout the recording. Right-click to save the heat map as a TIFF file.
Then right-click in the movie window to access particle measure, particle stats STK equals, and enter a flag assignment for the analysis. A series of traces will be generated in the trace window. STM analysis provides the ability to monitor and record the spatial characteristics of calcium signaling, such as the spatial spread and propagation velocity.
This information can be amassed to provide a rather complete view of calcium signaling behaviors within cells in their native environments. Using particle analysis, quantitative information about calcium signaling can be calculated by measuring the particle area and the particle count, which together can be used to determine the spatial ranges of calcium signal activation within a specified field of view. Particle analysis also allows the in-depth quantification of subcellular calcium signaling through examination of the location and firing probabilities of calcium firing sites.
By allocating the particles into different flags based on their temporal characteristics, the initiating particles can be accurately mapped. And a wealth of hard data acquired on the number of initiation sites, the size of the site in pixels and micrometers, probability of each initiation site firing either once or multiple times during each calcium transient cluster, and the percentage of firing sites that fire during each calcium transient cluster cycle can be obtained. Consistency is the key to success in this protocol.
All data sets and all movies must be treated exactly the same to ensure reliable, repeatable results. This is especially true when dealing with particle files. Our protocols allow a deep characterization of calcium signaling in situ, and a range of statistical methods, when used appropriately, can assess differences between a range of spatial and temporal parameters.
These analysis techniques in our protocol have allowed researchers to ask and answer previously unaddressable questions relating to intestinal pacemaking, and intestinal innervation, as well as the neuronal control of the lower urinary tract.
