Advanced Mounting Technique for Improved Imaging and Analysis of Embedded Spheroids and Migrating Cells in Light Sheet Fluorescence Microscopy

ABSTRACT For the investigation of three‐dimensional (3D) tumor spheroids and migrating cells embedded in a hydrogel using light sheet fluorescence microscopy (LSFM), a novel approach has been developed. LSFM has become a powerful tool for visualizing 3D structures of fluorescently labeled biological samples, providing high‐resolution imaging with minimal phototoxicity. Unlike conventional widefield or confocal microscopy methods, LSFM selectively illuminates a sample using a thin light sheet, thereby minimizing out‐of‐focus signals and facilitating quick volumetric data collection. However, sample mounting for LSFM still requires improvement, as conventional strategies, such as capillary‐based methods, present challenges especially for larger or delicate specimens, and may include mechanical stress. In this work, a novel sample holder compatible with the Zeiss Lightsheet 7 microscope is presented, specifically designed for the analysis of spheroids embedded in collagen gels. The novel holder enables dual‐sided illumination, improving effective image quality and facilitating detailed analysis of cell migration within the collagen matrix. Images acquired with this setup are analyzed using Arivis Vision4D to assess parameters such as cell number, cell density, and cell distance. The results demonstrated significant improvements not only in studying cancer cell migration and early metastatic behavior but also in evaluating chemotherapy within 3D extracellular matrix models.