F-CUBIC: a rapid optical clearing method optimized by quantitative evaluation

In recent decades, various powerful optical clearing methods have emerged to facilitate deep-tissue imaging. However, a rapid and safe protocol for millimeter-thick specimens is still desired. In this study, we propose a simple and economical chemical screening method that uses porcine small intestine tissue as the testing sample to quantify the clearing speed of different optical clearing reagents. By screening with this method, we developed a fast and versatile clearing protocol, termed F-CUBIC (adding formamide to CUBIC). F-CUBIC allows easy clearing of millimeter-thick tissues within 2-20 min by one-step immersion at room temperature. It introduces negligible tissue distortion and shows high compatibility with various fluorescent labeling techniques. Based on endoscopic human colon specimens, we successfully demonstrated the potential of F-CUBIC for nondestructive three-dimensional (3D) biopsy in combination with two-photon microscopy. This study would substantially benefit rapid 3D tissue mapping in biomedical research and clinics, such as instant histopathological examinations.