<div><p>Functional fluorescence imaging has been widely applied to analyze spatio-temporal patterns of cellular dynamics in the brain and spinal cord. However, it is difficult to integrate spatial information obtained from imaging data in specific regions of interest across multiple samples, due to large variability in the size, shape and internal structure of samples. To solve this problem, we attempted to standardize transversely sectioned spinal cord images focusing on the laminar structure in the gray matter. We employed three standardization methods, the affine transformation (AT), the angle-dependent transformation (ADT) and the combination of these two methods (AT+ADT). The ADT is a novel non-linear transformation method developed in this study to adjust an individual image onto the template image in the polar coordinate system. We next compared the accuracy of these three standardization methods. We evaluated two indices, i.e., the spatial distribution of pixels that are not categorized to any layer and the error ratio by the leave-one-out cross validation method. In this study, we used neuron-specific marker (NeuN)-stained histological images of transversely sectioned cervical spinal cord slices (21 images obtained from 4 rats) to create the standard atlas and also to serve for benchmark tests. We found that the AT+ADT outperformed other two methods, though the accuracy of each method varied depending on the layer. This novel image standardization technique would be applicable to optical recording such as voltage-sensitive dye imaging, and will enable statistical evaluations of neural activation across multiple samples.</p></div
