Deep learning-driven automatic nuclei segmentation of live-cell chromatin-sensitive partial wave spectroscopic microscopy imaging

Chromatin-sensitive partial wave spectroscopic (csPWS) microscopy offers a noninvasive glimpse into the mass density distribution of cellular structures at the nanoscale, leveraging the spectroscopic information. Such capability allows us to analyze the chromatin structure and organization and the global transcriptional state of the cell nuclei for the study of its role in carcinogenesis. Accurate segmentation of the nuclei in csPWS microscopy images is an essential step in isolating them for further analysis. However, manual segmentation is error-prone, biased, time-consuming, and laborious, resulting in disrupted nuclear boundaries with partial or over-segmentation. Here, we present an innovative deep-learning-driven approach to automate the accurate nuclei segmentation of label-free (without any exogenous fluorescent staining) live cell csPWS microscopy imaging data. Our approach, csPWS-seg, harnesses the convolutional neural networks-based U-Net model with an attention mechanism to automate the accurate cell nuclei segmentation of csPWS microscopy images. We leveraged the structural, physical, and biological differences between the cytoplasm, nucleus, and nuclear periphery to construct three distinct csPWS feature images for nucleus segmentation. Using these images of HCT116 cells, csPWS-seg achieved superior performance with a median intersection over union (IoU) of 0.80 and a Dice similarity coefficient (DSC) score of 0.89. The csPWS-seg outperformed the segmentation performance over several other commonly used deep learning-based segmentation models for biomedical imaging, such as U-Net, SE-U-Net, Mask R-CNN, and DeepLabV3+, marking a significant improvement in segmentation accuracy. Further, we analyzed the performance of our proposed model with four loss functions: binary cross-entropy loss, focal loss, Dice loss, and Jaccard loss separately, as well as a combination of all of these loss functions. The csPWS-seg with focal loss or a combination of these loss functions provided the same best results compared to other loss functions. The automatic and accurate nuclei segmentation offered by the csPWS-seg not only automates, accelerates, and streamlines csPWS data analysis but also enhances the reliability of subsequent chromatin analysis research, paving the way for more accurate diagnostics, treatment, and understanding of cellular mechanisms for carcinogenesis.