The study of cell death mechanisms via simultaneous Raman and transport of intensity phase-imaging techniques

Non-invasive methods of tracking morphological cell changes are based on measurements of phase, which is proportional to the cell thickness and allows calculation of cell volume. Additionally, Raman micro-spectroscopy is widely used for the mapping of chemical composition within live biological samples, such as cells, organoids, and tissues. We have previously reported the use of Raman spectroscopy and Digital Holographic microscopy (DHM) to study cell death induced by methamphetamine treatment. Here, we have replaced DHM with another method that is capable of real-time high resolution phase reconstruction. Assembling or altering a system to make the measurements required to solve the Transport-of-Intensity Equation (TIE) is easier than implementing a DHM setup. For the full phase retrieval, TIE requires only the data collected in the focal plane and in two planes symmetrically positioned about the focus. Furthermore, TIE is robust to reduced spatial and temporal coherence. Since TIE can utilize incoherent sources of illumination, we implemented a TIE setup within an existing Raman microscope, which provided near simultaneous chemical composition and morphological cell data. This setup is well-suited to study another form of programmed cell death, ferroptosis, which is the main cause of tissue damage driven by iron overload and lipid peroxidation. Previously, only invasive cell biological assays were used to monitor the expression level and subcellular location of proteins known to bind iron or be involved in ferroptosis. In this work, our group applied Raman spectroscopic techniques to study MDA-MB-231 breast cancer cells treated with an activator and/or inhibitor of ferroptosis.