Correlative multiscale 3D imaging of mouse primary and metastatic tumors by sequential light sheet and confocal fluorescence microscopy

Three-dimensional (3D) optical microscopy permits <i>in situ</i> interrogation of the tumor microenvironment (TME) in volumetric tumors for research while light sheet and confocal fluorescence microscopy are often used to achieve macroscopic and microscopic 3D images of tissues, respectively. Although each technique offers distinct fields of view (FOVs) and spatial resolution, the combination of the two to obtain correlative multiscale 3D images from the same tumor tissues has not yet been explored. We established a workflow that enables the tracking and 3D imaging of region of interests (ROIs) within tumor tissues through sequential light sheet and confocal fluorescence microscopy. This approach allowed for quantitative 3D spatial analysis of the immune response in the TME at multiple spatial scales and facilitated the direct localization of a metastatic lesion within a mouse brain. Our method offers an approach for correlative multiscale 3D optical microscopy with the potential to provide new insights into comprehensive research in disease mechanism or drug response.