Cytochrome c, an essential protein integral to the electron transport chain within cellular mitochondria, plays a crucial role in the intricate process of apoptosis, or programmed cell death. An early event in apoptosis involves the release of cytochrome c from the mitochondria's intermembrane space into the cytoplasm. This paper explores the detection of cytochrome c during apoptosis using Raman spectroscopy, with a specific focus on its release from the mitochondria of human microglial cells (HTHμ). Raman spectroscopy, a non-invasive and label-free analytical technique, allows the examination of biomolecular changes based on their chemical properties. Our experimental approach induced apoptosis in HTHμ cells using methamphetamine (METH) and utilized Raman spectroscopy on both control and apoptotic samples. Through the analysis of spectra by singular value decomposition (SVD), which reveals subtle trends and facilitates biological interpretation, distinct spectral features corresponding to cytochrome c were identified. This evidence supports the concept of cytochrome c release from the mitochondria during apoptosis. The label-free nature and high sensitivity of Raman spectroscopy position it as a promising technique for studying apoptosis in biomedical research and contributing to the development of innovative diagnostic approaches for apoptotic-related disorders.
Programmed cell death, or apoptosis, can be triggered in C6 glial cells through exposure to the drug methamphetamine. Non-invasive, quantitative tracking of apoptotic glial cell morphology can be difficult, as many cellular samples are weakly scattering, and therefore traditional bright field images may be of low contrast. Higher contrast images may be found through incorporation of the quantitative phase delay a beam can undergo due to transmission through a sample. In addition, quantitative phase information can be used, non-invasively, to track meaningful morphological quantities over time. Digital holographic microscopy (DHM) and utilization of the transport of intensity equation (TIE) are two label-free, high-resolution phase imaging techniques. DHM quantitatively retrieves phase through measurement of a hologram, or the interference pattern created when combining object and reference beams. The TIE quantifies the relationship between a field’s phase and intensity upon propagation. Solving the TIE requires measurement of an in-focus intensity, and images in symmetric planes about focus. On a setup capable of simultaneous data collection for both techniques, phase reconstructions were retrieved of C6 rat glial cells undergoing methamphetamine induced apoptosis. The two techniques’ measurements of total optical volume of cell clusters were compared over time. Additionally, the behavior of cells’ index of refraction during apoptosis was explored through optical diffraction tomography (ODT) retrieved reconstructions. Through these reconstructions, both cell volume and cell optical volume were tracked. The average relative refractive index behavior measured by ODT was extended to extrapolate volume from the TIE/DHM optical volume measurements.
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.
Transport of intensity (TIE) and digital holographic microscopy (DHM) are imaging techniques capable of real-time high resolution phase reconstructions. DHM is a widely used technique that provides phase maps through numerical reconstruction of light propagation of captured hologram intensities generated by interference between an object and a reference beam. TIE is a bright-field compatible technique that yields phase reconstructions through intensity measurements of a single object beam at symmetric planes about the focal plane. A TIE setup is simpler than DHM due to its non-interferometric nature and may yield a higher resolution reconstruction than DHM. Since TIE is a somewhat less-mature technique, we have developed a setup capable of both TIE and DHM measurements and simultaneously measured the volume changes of biological cells using both techniques. The setup is based on a modified bright-field microscope, with the addition of laser illumination for the DHM measurements. Live C6 glial cells were monitored as a hydrogen peroxide solution was introduced to the sample media to produce a visible and measurable decrease in cell volume through apoptosis. This decrease in volume was simultaneously measured by TIE and DHM, and the results were directly compared. Additionally, volume changes in C6 glial cells undergoing methamphetamine-induced apoptosis were tracked and compared.
We used phase microscopy and Raman spectroscopic measurements to assess the response of in vitro rat C6 glial cells following methamphetamine treatment in real time. Digital holographic microscopy (DHM) and three-dimensional (3-D) tomographic nanoscopy allow measurements of live cell cultures, which yield information about cell volume changes. Tomographic phase imaging provides 3-D information about the refractive index distribution associated with the morphology of biological samples. DHM provides similar information, but for a larger population of cells. Morphological changes in cells are associated with alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopy measurements provide information about chemical changes within the cells. Our Raman data indicate that the chemical changes in proteins preceded morphological changes, which were seen with DHM. Our study also emphasizes that tomographic phase imaging, DHM, and Raman spectroscopy are imaging tools that can be utilized for noninvasive simultaneous monitoring of morphological and chemical changes in cells during apoptosis and can also be used to monitor other dynamic cell processes.
Non-invasive live cell measurements are an important tool in biomedical research. We present a combined digital holography/Raman spectroscopy technique to study live cell cultures during apoptosis. Digital holographic microscopy records an interference pattern between object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information about the sample. When the phase is mapped across the sample and converted into height information for each pixel, a three dimensional image is obtained. The measurement of live cell cultures by digital holographic microscopy yields information about cell shape and volume, changes to which are reflective of alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopy, on the other hand, is sensitive to rotational and vibrational molecular transitions, as well as intermolecular vibrations. Therefore, Raman spectroscopy provides complementary information about cells, such as protein, lipid and nucleic acid content, and, particularly, the spectral signatures associated with structural changes in molecules. The cell cultures are kept in the temperature-controlled environmental chamber during the experiment, which allows monitoring over multiple cell cycles. The DHM system combines a visible (red) laser source with conventional microscope base, and LabVIEW-run data processing. We analyzed and compared cell culture information obtained by these two methods.
This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug “Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.
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