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

Three-dimensional (3D) optical microscopy, combined with advanced tissue clearing, permits in situ interrogation of the tumor microenvironment (TME) in large volumetric tumors for preclinical cancer research. Light sheet (also known as ultramicroscopy) and confocal fluorescence microscopy are often used to achieve macroscopic and microscopic 3D images of optically cleared tumor tissues, respectively. Although each technique offers distinct fields of view (FOVs) and spatial resolution, the combination of these two optical microscopy techniques to obtain correlative multiscale 3D images from the same tumor tissues has not yet been explored. To establish correlative multiscale 3D optical microscopy, we developed a method for optically marking defined regions of interest (ROIs) within a cleared mouse tumor by employing a UV light-activated visible dye and Z-axis position-selective UV irradiation in a light sheet microscope system. By integrating this method with subsequent tissue processing, including physical ROI marking, reversal of tissue clearing, tissue macrosectioning, and multiplex immunofluorescence, we established a workflow that enables the tracking and 3D imaging of 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 of a mouse mammary tumor following cancer immunotherapy at multiple spatial scales. The workflow also facilitated the direct localization of a metastatic lesion within a whole mouse brain. These results demonstrate that our ROI tracking method and its associated workflow offer a novel approach for correlative multiscale 3D optical microscopy, with the potential to provide new insights into tumor heterogeneity, metastasis, and response to therapy at various spatial levels.