3D imaging with enhanced transparency, signal-to-background ratios, and antigen detection using HyPer-3D

We developed HyPer-3D (hydrogen peroxide-NaN₃-DMSO), a chemical treatment that addresses persistent challenges in 3D imaging of cleared tissues. Imaging across stem cell, zebrafish, and murine model systems demonstrated that HyPer-3D quenches tissue autofluorescence, increases signal-to-background ratios (SBRs) by 30×, potentiates optical clearing capacity by 6×, increases detection of poorly recognized antigens by 4.5×, and enables a 5× increase in fluorescent reporter detection, which are conversely often diminished in 3D imaging. These improvements enhanced multiplex interrogation of highly autofluorescent and fibrous organs, including previously undocumented nephron segments, cardiac conduction system elements, and diffuse spermatogonial stem cells. Optimization for human tissues, which remain among the most difficult to 3D imaging due to autofluorescence and structural density far exceeding those of commonly used animal tissues, yielded a 5× increase in SBRs and enabled multiplex imaging with cellular resolution. Collectively, HyPer-3D provides a robust approach for high-contrast 3D imaging of basic and clinical specimens.