Microscopic-based analysis of nuclei in spheroids via SUNSHINE: An on-chip workflow integrating optical clearing, fluorescence calibration and supervoxel segmentation

Multicellular spheroids (MCSs) are increasingly employed as 3D cell culture models in biomedical research due to their ability to effectively replicate in vivo cell interactions, making them suitable for high-throughput drug screening. Accurate cell counting is critical for data normalization, therapeutic evaluation, and exploration of culture conditions; however, affordable software solutions for 3D cell counting using microscopic images are limited. To fill this gap, we created SUNSHINE, an innovative on-chip analytical workflow that uniquely merges optical clearing, histogram matching (HM)-assisted fluorescence calibration, and simple linear iterative clustering (SLIC) supervoxel segmentation. This tool offers an efficient method for analyzing the characteristics and counts of fluorescence-labeled nuclei within MCSs. While optical clearing improves the penetration depth of microscopic imaging, deeper regions of thicker samples often yield faint fluorescence signals. SUNSHINE resolves this issue through the HM image post-processing algorithm. Moreover, SLIC is an effective alternative to traditional contour-wise segmentation, enabling the identification of irregularly shaped fluorescent nuclei. We found that SUNSHINE generated results comparable to commercial software like Imaris and machine learning (ML)-based tools, such as StarDist and Cellpose, in our analysis of the effects of seeding density and cell type on spheroid growth. We also used it to measure the volume and spatial distribution of nuclei, focusing on the hypoxic and peripheral regions of spheroids. Overall, this study finds that SUNSHINE serves as a valuable and economical approach for characterizing cellular activity and interactions in 3D, diminishing the reliance on costly proprietary software.