Selective study of the electron transport chain components in living mitochondria is essential for fundamental biophysical research and for the development of new medical diagnostic methods. However, many important details of inter-and intramembrane mitochondrial processes have remained in shadow due to the lack of non-invasive techniques. Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria. We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase. Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric nearfield and large contact area between mitochondria and nanostructured surfaces.Mitochondria are organelles of fundamental importance for cellular energy production, metabolic regulation, aging and cell survival under stress [1][2][3] . Normal function of mitochondria and their pathological changes, including production of reactive oxygen species (ROS), are heavily dependent on the redox state of the electron transport chain (ETC) cytochromes and cytochrome c in particular 4,5 . At present, most of the studies of isolated mitochondria and mitochondria in cells are performed by fluorescent microscopy, absorption spectroscopy and measurements of O 2 consumption 3,[6][7][8] . The fluorescent microscopy with small fluorescent dyes (rhodamin and MitoTracker-family, etc.) or fluorescent proteins (GFP, YFP, RFP) can provide general information about changes in the potential of the inner mitochondrial membrane (Δ Φ ), the mitochondrial volume, and the co-localization of certain mitochondrial components with a molecule of interest
SERS studies of intact erythrocytes and functional mitochondria are demonstrated for the first time using silver–silica beads prepared by aerosol pyrolysis with aqueous diamminesilver(i) hydroxide as a source of silver nanoparticles for SiO2 microspheres.
This is a review of relevant Raman spectroscopy (RS) techniques and their use in structural biology, biophysics, cells, and tissues imaging towards development of various medical diagnostic tools, drug design, and other medical applications. Classical and contemporary structural studies of different water-soluble and membrane proteins, DNA, RNA, and their interactions and behavior in different systems were analyzed in terms of applicability of RS techniques and their complementarity to other corresponding methods. We show that RS is a powerful method that links the fundamental structural biology and its medical applications in cancer, cardiovascular, neurodegenerative, atherosclerotic, and other diseases. In particular, the key roles of RS in modern technologies of structure-based drug design are the detection and imaging of membrane protein microcrystals with the help of coherent anti-Stokes Raman scattering (CARS), which would help to further the development of protein structural crystallography and would result in a number of novel high-resolution structures of membrane proteins—drug targets; and, structural studies of photoactive membrane proteins (rhodopsins, photoreceptors, etc.) for the development of new optogenetic tools. Physical background and biomedical applications of spontaneous, stimulated, resonant, and surface- and tip-enhanced RS are also discussed. All of these techniques have been extensively developed during recent several decades. A number of interesting applications of CARS, resonant, and surface-enhanced Raman spectroscopy methods are also discussed.
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