Multimodal segmentation of dynamic subcellular features using quantitative phase imaging and FRET-based sensors [Invited]

Understanding cellular responses to mechanical environmental stimuli is important for cellular mechanotransduction studies. While fluorescence microscopy has been used for aiding mechanotransduction research due to its molecular sensitivity, the ability of quantitative phase imaging (QPI) to visualize morphology has yet to be widely applied, perhaps due to its limited specificity. Here, we seek to expand on previous work which combined quantitative phase imaging with a molecularly sensitive Förster resonance energy transfer (FRET) construct by developing additional analysis techniques. This work seeks to characterize the response of individual cells to mechanical stimulus through a novel, to the best of our knowledge, QPI-guided cellular segmentation algorithm. The multimodal imaging instrument and analysis techniques are employed to examine cellular responses to hypo-osmotic stimulus by observing the calcium ion flux using a FRET-based sensor coupled with a mapping of intracellular mass reorganization using QPI. The combined imaging modality enables a discrimination of cell response by localized region, revealing distinct behavior between regions and relative to a control group. Our novel analysis techniques can be used to identify cell expansion and cell region specific responses in both modalities due to the stimulus. With the broad array of FRET sensors under development, the complementary addition of QPI offers new avenues for studying cell responses to a range of environmental stimuli.