Revolutionizing Cancer Detection: Enhancing Diagnosis with a Novel 3D Histopathological Approach

Despite these advancements, existing 3D pathology methods are time-consuming, requiring weeks to months for completion, which is impractical for clinical applications. This study introduces a novel 3D staining and clearing protocol combined with automated robotic systems and 3D imaging techniques to elevate cancer detection within a feasible timeframe. A total of 14 samples were processed. Our method facilitates the 3D reconstruction of entire biological samples in an efficient, reliable, and accurate manner, encompassing steps for clearing, staining, and imaging. The protocol is adaptable for reverse clearing, enabling the sample to be rehydrated and used for secondary processes with standard 2D clearing and staining. The iDISCO protocol, employed for the clearing step, is effectively paired with IMAI’s mechanical antibody injection and 3D imaging using a light-sheet microscope (MesoSPIM). Analysis of the images is conducted using ImageJ (FIJI software), with a wide variety of lens zoom settings in the MesoSPIM enabling an in-depth understanding of the sample's morphology and detailed exploration of microscopic structures. This Master’s thesis presents a ground-breaking method that can clear, stain, and image centimetre-size samples in just two days, a significant improvement over previously reported studies.

Cancer diagnosis remains a formidable challenge, largely due to the high rate of false negatives associated with traditional 2D histology. This method, which involves observing only a tiny fraction of the sample, often leads to the underestimation of metastasis size and, consequently, incorrect diagnoses. However, advancements in imaging technology have ushered in new methodologies, including 3D pathology, which offers a more comprehensive understanding of biological sample structures by integrating 3D clearing, staining, and imaging techniques. This approach represents a significant advancement in pathology, allowing for the analysis and accurate diagnosis of entire samples. Despite these advancements, existing 3D pathology methods are time-consuming, requiring weeks to months for completion, which is impractical for clinical applications. This study introduces a novel 3D staining and clearing protocol combined with automated robotic systems and 3D imaging techniques to elevate cancer detection within a feasible timeframe. A total of 14 samples were processed. Our method facilitates the 3D reconstruction of entire biological samples in an efficient, reliable, and accurate manner, encompassing steps for clearing, staining, and imaging. The protocol is adaptable for reverse clearing, enabling the sample to be rehydrated and used for secondary processes with standard 2D clearing and staining. The iDISCO protocol, employed for the clearing step, is effectively paired with IMAI’s mechanical antibody injection and 3D imaging using a light-sheet microscope (MesoSPIM). Analysis of the images is conducted using ImageJ (FIJI software), with a wide variety of lens zoom settings in the MesoSPIM enabling an in-depth understanding of the sample's morphology and detailed exploration of microscopic structures. This Master’s thesis presents a ground-breaking method that can clear, stain, and image centimetre-size samples in just two days, a significant improvement over previously reported studies.