Fig. 1.

Representation of the 3D pipeline summarizing the concept of our experiments. Spheroids from T-47D, 5-8F, and Huh-7D12 cell lines with a similar size range (approx. 250 µm) were cleared with Clear^T, Clear^T2, CUBIC, ScaleA2, and Sucrose protocols, and the nuclei of the cells were labeled with DRAQ5 staining. For imaging, a Leica SP8 digital light-sheet microscope was used, yielding z-stack images. Ten experts evaluated the sharpness of the fluorescent images, and we compared their scoring with the tested quality metrics. Correlations between the quality metrics and human evaluation were calculated using Pearson’s correlation, and the metric with the best correlation was used to compare the efficacy of the optical clearing protocols applied on three types of spheroids originating from different cell lines.