AI-Driven Analysis for Real-Time Detection of Unstained Microscopic Cell Culture Images

Staining-based assays are widely used for cell analysis but are invasive, alter physiology, and prevent longitudinal monitoring. Label-free, morphology-based approaches could enable real-time, non-invasive drug testing, yet detection of subtle and dynamic changes has remained difficult. We developed a deep learning framework for stain-free monitoring of leukemia cell cultures using automated bright-field microscopy in a semi-automated culture system (AICE3, LABMaiTE, Augsburg, Germany). YOLOv8 models were trained on images from K562, HL-60, and Kasumi-1 cells, using an NVIDIA DGX A100 GPU for training and tested on GPU and CPU environments for real-time performance. Comparative benchmarking with RT-DETR and interpretability analyses using Eigen-CAM and radiomics (RedTell) was performed. YOLOv8 achieved high accuracy (mAP@0.5 > 98%, precision/sensitivity > 97%), with reproducibility confirmed on an independent dataset from a second laboratory and an AICE3 setup. The model distinguished between morphologically similar leukemia lines and reliably classified untreated versus differentiated K562 cells (hemin-induced erythroid and PMA-induced megakaryocytic; >95% accuracy). Incorporation of decitabine-treated cells demonstrated applicability to drug testing, revealing treatment-specific and intermediate phenotypes. Longitudinal monitoring captured culture- and time-dependent drift, enabling separation of temporal from drug-induced changes. Radiomics highlighted interpretable features such as size, elongation, and texture, but with lower accuracy than the deep learning approach. To our knowledge, this is the first demonstration that deep learning resolves subtle, drug-induced, and time-dependent morphological changes in unstained leukemia cells in real time. This approach provides a robust, accessible framework for label-free longitudinal drug testing and establishes a foundation for future autonomous, feedback-driven platforms in precision oncology. Ultimately, this approach may also contribute to more precise and adaptive clinical decision-making, advancing the field of personalized medicine.