AbstractCerebral microhemorrhages (CMHs) are associated with cerebrovascular disease, cognitive impairment, and normal aging. One method to study CMHs is to analyze histological sections (5–40 μm) stained with Prussian blue. Currently, users manually and subjectively identify and quantify Prussian blue-stained regions of interest, which is prone to inter-individual variability and can lead to significant delays in data analysis. To improve this labor-intensive process, we developed and compared three digital pathology approaches to identify and quantify CMHs from Prussian blue-stained brain sections: (1) ratiometric analysis of RGB pixel values, (2) phasor analysis of RGB images, and (3) deep learning using a mask region-based convolutional neural network. We applied these approaches to a preclinical mouse model of inflammation-induced CMHs. One-hundred CMHs were imaged using a 20 × objective and RGB color camera. To determine the ground truth, four users independently annotated Prussian blue-labeled CMHs. The deep learning and ratiometric approaches performed better than the phasor analysis approach compared to the ground truth. The deep learning approach had the most precision of the three methods. The ratiometric approach has the most versatility and maintained accuracy, albeit with less precision. Our data suggest that implementing these methods to analyze CMH images can drastically increase the processing speed while maintaining precision and accuracy.
Using Laurdan and Spectral Phasor Analysis to Study Erythrocytes Membrane Solubilization
