Label-free analysis of living cell cultures at the single cell level utilizing artificial neural network supported simultaneous bright field and off-axis digital holographic microscopy

Label-free multimodal microscopy techniques provide complementary information about cellular processes and the morphology of living single cells. The combination of laser-based off-axis digital holographic microscopy (DHM)-based quantitative phase imaging (QPI) with broadband bright-field (BF) microscopy facilitates labelfree minimally invasive determination of biophysical cell properties such as dry mass, refractive index and morphology related parameters. In addition, the detection of small absorbing intracellular organelles as well as colocalization of absorbance and phase changes within a sample by noise-reduced BF intensity images is provided. Here, we report on a high-speed capable multimodal microscopy approach for robust label-free analysis of living cell cultures at single cell level. Based on a previously reported concepts, BF images and laser-based digital offaxis holograms were captured simultaneously by grayscale dynamic range multiplexing utilizing a commercial optical microscope with an attached DHM module and a single monochrome camera. BF and QPI images were recovered from multiplexed images by spatial filtering-based demultiplexing and weighted image subtraction. To achieve rapid and automated single cell segmentation without manual labelling of training data, BF images were evaluated with a pretrained artificial neural network utilizing the freely available Cellpose algorithm and employing a graphics processing unit (GPU). The segmentation masks obtained were then transferred to the QPI images and used in the procedure to calculate biophysical and morphology-related single cell parameters sets such as projected area, circularity, and dry mass. Data from living adherent tumor cells and mouse fibroblast as well as results from experiments with tumor cells in flow demonstrate our approach to be applicable in high-throughput cell culture analysis and high-rate multimodal imaging flow cytometry.