Translational rapid ultraviolet-excited sectioning tomography for whole-organ multicolor imaging with real-time molecular staining

Abstract

Rapid multicolor three-dimensional (3D) imaging for centimeter-scale specimens with subcellular resolution remains a challenging but captivating scientific pursuit. Here, we present a fast, cost-effective, and robust multicolor whole-organ 3D imaging method assisted with ultraviolet (UV) surface excitation and vibratomy-assisted sectioning, termed translational rapid ultraviolet-excited sectioning tomography (TRUST). With an inexpensive UV light-emitting diode (UV-LED) and a color camera, TRUST achieves widefield exogenous molecular-specific fluorescence and endogenous content-rich autofluorescence imaging simultaneously while preserving low system complexity and system cost. Formalin-fixed specimens are stained layer by layer along with serial mechanical sectioning to achieve automated 3D imaging with high staining uniformity and time efficiency. 3D models of all vital organs in wild-type C57BL/6 mice with the 3D structure of their internal components (e.g., vessel network, glomeruli, and nerve tracts) can be reconstructed after imaging with TRUST to demonstrate its fast, robust, and high-content multicolor 3D imaging capability. Moreover, its potential for developmental biology has also been validated by imaging entire mouse embryos (~2 days for the embryo at the embryonic day of 15). TRUST offers a fast and cost-effective approach for high-resolution whole-organ multicolor 3D imaging while relieving researchers from the heavy sample preparation workload.

Automated whole-organ histological imaging assisted with ultraviolet-excited sectioning tomography and deep learning

“…During the imaging process with the TRUST system, we applied two fluorogenic dyes (4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) together for double labeling to achieve high color contrast and reveal rich biological information 16 . Once the uppermost layer of the organ has been labeled, the whole section will be raster-scanned by the TRUST imaging system, which is based on the ultraviolet surface excitation for block-face imaging 17 .…”

“…1, marked with a green dashed box, termed HistoTRUST). HistoTRUST is based on our recently developed high-speed block-face imaging technique named translational rapid ultraviolet-excited sectioning tomography (TRUST), which can acquire serial histological images with a subcellular resolution of whole organs with ultraviolet illumination and vibratome-assisted sectioning 16 . The obtained images will be further transformed into virtual H&E stained histological images by our developed deep-learning neural network.…”

Deep Learning Enables Rapid Whole-Organ Histological Imaging with Ultraviolet-Excited Sectioning Tomography

“…During the imaging process with the TRUST system, we applied two fluorogenic dyes (4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) together for double labeling to achieve high color contrast and reveal rich biological information. 16 Once the uppermost layer of the organ has been labeled, the whole section will be rasterscanned by the TRUST imaging system, which is based on the ultraviolet surface excitation for block-face imaging. 17 Then, the imaged surface layer will be sliced off by a vibratome to expose the adjacent layer of the tissue for the next round of staining and image scanning.…”

“…HistoTRUST is based on our recently developed high-speed block-face imaging technique named translational rapid ultraviolet-excited sectioning tomography (TRUST), which can acquire serial histological images with a subcellular resolution of whole organs with ultraviolet illumination and vibratome-assisted sectioning. 16 The obtained images will be further transformed into virtual H&E stained histological images by our developed deep learning neural network. With HistoTRUST, the imaging process for acquiring 3D virtual H&E stained histological images of all six organs (e.g., brain, heart, liver, lung, kidney, and spleen) takes 3 days in maximum without any human involvement.…”

Multi-scale tissue fluorescence mapping with fibre optic ultraviolet excitation and generative modelling

“…DUV-excited fluorescence microscopy, also known as MUSE, has been celebrated for its simplicity and effectiveness in concept -the combination of oblique DUV illumination and widefield detection. However, prior published illustrations of real-world implementations [14,15,35,[56][57][58] showed inevitably large and complex setups, with the notable exception of Pocket MUSE, which is generally incompatible with large surgical or mesoscale samples [16]. Also notable in the MUSE literature was the conspicuous omission of imaging capabilities at >40X magnification, a mainstay of biomedical inspection and unusually difficult in MUSE due to the classically awkward implementation of oblique illumination.…”