A Spectral Principal Component Analysis-Based Framework for Composite Hard/Soft Tissue Fluorescence Image Investigation

Traditional thin sectioning microscopy of large bone and dental tissue samples using demineralization may disrupt structure morphologies and even damage soft tissues, thus compromising the histopathological investigation. Here, we developed a synergistic and original framework on thick sections based on wide-field multi-fluorescence imaging and spectral Principal Component Analysis (sPCA) as an alternative, fast, versatile, and reliable solution, suitable for highly mineralized tissue structure sustain and visualization. Periodontal 2-mm thick sections were stained with a solution containing five fluorescent dyes chosen for their ability to discriminate close tissues, and acquisitions were performed with a multi-zoom macroscope for blue, green, red, and NIR (near-infrared) emissions. Eigen-images derived from both standard scaler (Std) and Contrast Limited Adaptive Histogram Equalization (Clahe) pre-preprocessing significantly enhanced tissue contrasts, highly suitable for histopathological investigation with an in-depth detail for sub-tissue structure discrimination. Using this method, it is possible to preserve and delineate accurately the different anatomical/morphological features of the periodontium, a complex tooth-supporting multi-tissue. Indeed, we achieve characterization of gingiva, alveolar bone, cementum, and periodontal ligament tissues. The ease and adaptability of this approach make it an effective method for providing high-contrast features that are not usually available in standard staining histology. Beyond periodontal investigations, this first proof of concept of an sPCA solution for optical microscopy of complex structures, especially including mineralized tissues opens new perspectives to deal with other chronic diseases involving complex tissue and organ defects. Overall, such an imaging framework appears to be a novel and convenient strategy for optical microscopy investigation.