Three-dimensional Multicolor Subcellular Imaging by Fast Serial Sectioning Tomography for Centimeter-scale Specimens

Abstract

AbstractRapid multicolor three-dimensional (3D) imaging for centimeter-scale specimens with subcellular resolution remains a challenging but captivating scientific pursuit. Here, we present a fast, automated, cost-effective, and versatile multicolor 3D imaging method with ultraviolet (UV) surface excitation and vibratomy-assisted sectioning, termed translational rapid ultraviolet-excited sectioning tomography (TRUST). TRUST enables exogenous molecular-specific fluorescence and endogenous content-rich autofluorescence imaging simultaneously with the help of a UV light-emitting diode and a color camera. Commonly applied tissue preparation procedures (e.g., staining or clearing) are laborious, time-consuming, and may induce detrimental effects on processed samples. In TRUST, formalin-fixed specimens are stained with real-time double labeling layer by layer along with serial widefield optical illumination with raster scanning and mechanical sectioning to improve the staining speed and reveal rich biological information. All vital organs in mice have been imaged by TRUST to demonstrate its fast, robust, and high-content multicolor 3D imaging ability. Moreover, its potential for developmental biology has also been validated by imaging entire mouse embryos (taking ∼2 days for imaging the embryo at the embryonic day of 15). TRUST offers a way for multicontrast and multicolor whole-organ 3D imaging with high resolution and high speed while relieving researchers from heavy sample preparation workload.