The overall goal of these clock scan protocols is to allow the fast characterization of the average pixel intensity within, along the border of, and/or on the outside of selected regions of interest. This method can help answer key questions and teach on image analysts about average pixel intensity within specific regions of interest. The main advantages of the clock scan technique here are that it can be used to quickly characterize the average pixel intensity from orbit in single or multiple regions of interest.
For standard clock scan analysis, under the File menu, in the appropriate ImageJ program, select Open to open an image file of interest. For example, here, a tissue section with neurons labeled for a protein of interest will be analyzed. Select an appropriate drawing tool, and outline the region of interest.
Select Plugins and Clock Scan from the File menu to open the standard clock scan protocol, popup option window. The region of interest manager window, with the outline automatically added, will also open. In the Plugin option window, use the scroll bars to change the x and y coordinates at the center of the region of interest, as appropriate.
Depending on how much of the background region outside of the object should be covered by the scanning, use the scan limit scroll bar to adjust the scan limits. Select the real radius, subtract background, polar transform, and/or plot with standard deviation check boxes as experimentally appropriate. Then, click OK to run the plugin.
Once the analysis is complete, use the list command to access the numerical data for further analysis. For clock scan analysis of multiple regions, open an image file containing multiple regions of interest. For example, here, a panel of frames captured within the same tissue section, as just demonstrated, will be analyzed.
Click Analyze, then Tools, then Region of Interest Manager and use the appropriate drawing tool to outline each region of interest. The segmented line tool is useful for selecting asymmetrical regions of interest, and for excluding background labeling. Click Add in the Region of Interest Manager window to add each region of interest to the manager as it is selected.
in the Plugins menu, select Multi Clock Scan to open the protocol options popup window and reset the scan limit, as necessary. Check the plot with standard deviation and/or subtract background boxes, as experimentally appropriate. Then, click OK to run the protocol, which will generate two output plot windows.
For clock scan analysis of an image stack, open an image stack of interest. Here, an image stack of an isolated dorsal root ganglion, labeled with a calcium-sensitive fluorescent that was imaged before and after intracellular electrode stimulation, will be analyzed. Outline the region of interest within the image stack.
Add the selected image stack to the region of interest manager, as just demonstrated, and select Multi Clock Scan to open the protocol options popup window. Reset the scan limit as necessary, and check the plot with standard deviation, and/or subtract background boxes, as experimentally appropriate. Then, click OK to run the protocol, generating two output plot windows.
In this representative analysis of a tissue section of fluorescently labeled neurons, a region of interest was drawn through the bright signal for the protein of interest, and a standard clock scan analysis was performed to collect radial pixel intensity scans from the center of the region of interest to the scan limit outlined. The radial scans were normalized to the region of interest radius in the direction of the scan. An average, to produce an integral radio pixel intensity profile, revealing that most of the protein of interest was measured at the plasma membrane of the cell.
As plot with standard deviation was selected for this analysis, the vertical standard deviation lines indicated the between pixel intensities of the individual radial scan profiles at a given distance from the scan origin. In this analysis, multiple regions of interest within the neuron tissue sample were scanned, as just demonstrated, generating an integral pixel intensity profile for each scanned object in the sequence that they were added to the region of interest manager. And, an average pixel intensity profile for each selected region of interest.
These standard deviation bars, therefore, represent the variability between integral scans of individual objects, rather than individual radial scans. Here, an image stack analysis of an isolated dorsal root ganglion neuron captured at different times, before and after stimulation, via intracellular electrode, was performed. At the end of the analysis, an integral pixel intensity profile of the individual instances of the object of interest on different images within the stack, and an average pixel intensity profile for all of the instances of the object were displayed.
Further, the standard deviation analysis of a clock scanned image stack demonstrates the data variability between the integral scans throughout the stack of images. After watching this video, you should have a good understanding of how to measure pixel intensities using clock scan plugins. Our purpose in developing these plugins was to make these types of image analysis freely available for every investigator.
We believe that the clock scan protocol will be used for the many areas of research, including but not limited to, biological, chemical, and physical imaging application. As we continue to develop and operate these clock scan plugins, we will appreciate any feedback and any suggestions from interested parties on how to improve these tools.
