Abstract
SIGNIFICANCE: Three-dimensional (3D) visualization of multicellular tumor spheroids (MCTS) in fluorescence microscopy can rapidly provide qualitative morphological information about the architecture of these cellular aggregates, which can recapitulate key aspects of their in vivo counterpart. AIM: The present work is aimed at overcoming the shallow depth-of-field (DoF) limitation in fluorescence microscopy while achieving 3D visualization of thick biological samples under study. APPROACH: A custom-built fluorescence microscope with an electrically focus-tunable lens was developed to optically sweep in-depth the structure of MCTS. Acquired multifocus stacks were combined by means of postprocessing algorithms performed in the Fourier domain. RESULTS: Images with relevant characteristics as extended DoF, stereoscopic pairs as well as reconstructed viewpoints of MCTS were obtained without segmentation of the focused regions or estimation of the depth map. The reconstructed images allowed us to observe the 3D morphology of cell aggregates. CONCLUSIONS: Computational multifocus fluorescence microscopy can provide 3D visualization in MCTS. This tool is a promising development in assessing the morphological structure of different cellular aggregates while preserving a robust yet simple optical setup.