New approaches in imaging of reactive oxygen species in animal models of neurodegenerative diseases
ROSim-consortium

Giniatullin Rashid, UEF, Courtney Michael, UEF, Khirug Leonard, UH, Koistinaho Jari, UEF

Free radicals, also known as reactive oxygen species (ROS), exert a variety of pivotal actions in health and disease. Despite the growing number of researchers employing photonics techniques to visualize endogenous ROS in living cells and tissues in real time, these studies are hampered by the lack of reliable methods for accurate detection of ROS. The current protocols do not allow reproducible, calibrated ROS measurement due to poor photostability of existing dyes. To overcome these limitations, we propose new ROS imaging protocols based on two cutting-edge developments in biophotonics: 1) total internal reflection fluorescence (TIRF) microscopy, a powerful near-field optical sectioning technique with nano-scale spatial resolution in cultured cells, and 2) multiphoton microscopy, an advanced non-linear optical method that offers unsurpassed light penetration in brain slices and in vivo imaging of intact brain through the cranial window. The key advantage of both techniques applied to ROS imaging is that they ensure a dramatic reduction of the volume in which dye excitation occurs, in comparison to conventional wide-field or confocal microscopy. Thus, all irreversibly photooxidised and/or photobleached dye molecules are quickly replaced by fresh ones diffusing into focus, and the baseline fluorescence remains stable. This reversibility makes it possible, for the first time, to compare the effects of several repetitive applications of ROS-inducing drugs to the same cell or tissue area. We will further complement conventional ROS dyes with ROS-sensitive fluorescent proteins that can be genetically targeted to selected cells or even to specific intracellular organelles. Our multidisciplinary approach will not only allow us to develop new, strictly-controlled imaging technologies but also to immediately test them in our ongoing research projects in animal models of neurodegenerative diseases where the role of ROS has been implicated. The proposed project will produce new knowledge on the ROS-mediated pathogenesis of neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, migraine and stroke.