Fig. 5: Qualitative comparison of the HCS plate and the traditional imaging method where the samples were placed into Petri dishes for LSFM.

a Fluorescent images of T-47D spheroids stained with DRAQ5 were used as a reference to compare image quality. Randomly selected spheroids screened in the HCS plate (left image) and in Petri dish (right image) were visualised where images were selected from the top, middle, and bottom regions. Scale bars represent 100 µm. b To characterise the general blurriness of the whole z-stack, the intensity variance metric was used both for the inside circle and for the whole spheroid. Results are shown in a boxplot, showing that no significant differences were measured between the HCS plate and the Petri. n = 5 for each group. The Kolmogorov-Smirnov test was used. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. c Preparation and screening times were measured for both techniques and displayed on a stacked bar plot. Starting from the darkest colour the 4 categories are Sample preparation, Change sample (this category does not apply to the HCS plate), Calibration, and Imaging. The total time was calculated for 10 samples for each method. d Example image of a cleared T-47D spheroid labelled with DRAQ5 for nuclei (purple) and Phalloidin 488 for actin (green) and optically cleared with Sucrose protocol. A total of 5184 nuclei were segmented using BIAS. Scale bar represents 100 µm. e By co-culturing T-47D breast cancer cells with MRC-5 fibroblasts and EA.hy926 endothelial cells, a hydrogel-based tumour model was created to track the tumour microenvironment in real time. CellTracker dyes (orange, deep red, and green) are used to detect cells. The tumour, endothelial, and fibroblast cells are marked by red, green, and white labels, respectively. Scale bar represents 50 µm. f A ground truth dataset of HeLa Kyoto - MRC-5 co-cultures was created using the HCS plate. Scale bar represents 50 µm. Box plots were visualised using Tukey’s method, with medians shown as central lines, boxes representing interquartile ranges, and whiskers extending to 1.5× interquartile range.