Volumetric Imaging and Analysis of Tissues for Clinical Research through De Novo Formation of Citrate-Based Fluorophores

Two-dimensional (2D) slide-based histopathology is the current paradigm in diagnosis and prognosis. However, loss of volumetric information accompanied by slide preparation provides limited information about the spatial progression of diseases that may lead to an incorrect diagnosis. Recent technological advances, especially tissue-clearing techniques, have enabled three-dimensional (3D) imaging of pathological samples to overcome these limitations. However, affinity-based labeling strategies such as small molecule dyes and fluorescently-labeled antibodies show limited compatibility with tissue-clearing techniques. Moreover, the nonspecific binding of small fluorescent probes and slow penetration of antibodies into large-scale tissues due to their high molecular weight hinder the uniform staining of biological samples and may produce misleading results. Until now, effective labeling and imaging techniques for thick tissues have not been achieved despite the demand for accurate volumetric analyses of pathological samples.<br/>Here, we present an imaging technique, 3DNFC (“3D tissue imaging through <i>de Novo</i> Formation of Citrate-based fluorophores”), for 3D reconstruction of thick tissues through the integration of tissue clearing techniques and citrate-based fluorogenic reaction. 3DNFC provides the anatomical morphology of tissues by generating fluorophores on non-fluorescent amino-terminal cysteine and visualizes the 3D structure of the tissues. Furthermore, 3DNFC can unbiasedly analyze various features from pathological tissues in their natural context based on the 3D reconstructed images. We believe that 3DNFC overcomes many limitations of conventional histopathology and may be a suitable 3D analog for the microscopic examination of pathological samples.