“Clock-scan” protocol for image analysis

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Comparative analysis of extra- and intracellular distributions of protein markers in immunohistochemical and immunofluorescent studies relies on techniques of image analysis. Line or region of interest pixel intensity scans are methods routinely used. However, although having good spatial resolution, linear pixel intensity scans fail to produce integral image of the cellular distribution of the label. On the other hand, the regions of interest scans have good integrative capacity but low spatial resolution. In this work, we describe a "clock-scan" protocol that, when applied to convex objects (such as neuronal cell bodies and the majority of cells in culture), combines advantages and circumnavigates limitations of the above-mentioned techniques. The protocol 1) collects multiple radial pixel intensity profiles scanned from the cell center to the periphery, 2) scales these profiles according to the cell radius measured in the direction of the scan, and finally, 3) averages these individual profiles into one integral radial pixel intensity profile. Because of scaling, the mean pixel intensity profiles produced by the clock-scan protocol depend on neither the cell size nor, within reasonable limits, the cell shape. This allows direct comparison or, if required, averaging or subtraction of profiles of different cells. We have successfully tested the clock-scan protocol in experiments with immunostained dorsal root ganglion neurons. In addition, the protocol seems to be equally applicable for studies in a variety of other preparations.