Label-free acoustic and optical microscopy of live tumor spheroids in hydrogel for high-throughput 3D In-vitro drug screening

3D cell cultures, including spheroids, have become essential tools in cancer research and drug discovery due to their ability to more accurately mimic in-vivo tissue environments compared to traditional 2D cultures. However, imaging these thick, complex structures remains a challenge, as conventional optical microscopy techniques are limited by shallow depth penetration. This study explores the complementary use of gradient light interference microscopy (GLIM) and scanning acoustic microscopy (SAM) for label-free imaging of 3D spheroid clusters embedded in hydrogels. GLIM offers high-resolution optical imaging but struggles with depth in dense samples, while SAM provides greater depth penetration and a larger field of view, albeit with lower resolution. By correlating SAM and GLIM imaging, this study demonstrates how the two techniques can be synergistically used to enhance the visualization of spheroids, capturing both large-scale structural features and fine cellular details. The benefits make such a platform suitable for screening high-number multi-well plates and evaluating necrotic and angiogenic features from the core of the thick sample. Such platforms have the potential of combining acoustic and optical imaging modalities for high-throughput screening and physical characterization in 3D cell culture research, advancing our understanding of drug efficacy in complex biological systems.