Phasor analysis of NADH FLIM identifies pharmacological disruptions to mitochondrial metabolic processes in the rodent cerebral cortex

March Phasor analysis of NADH FLIM identifies pharmacological disruptions to mitochondrial metabolic processes in the rodent cerebral cortex Carlos A. Go mez 0 1 2 3 Jason Sutin 0 1 2 3 Weicheng Wu 0 1 2 3 Buyin Fu 0 1 2 3 Hana Uhlirova 0 2 3 Anna Devor 0 1 2 3 David A. Boas 0 1 2 3 Sava SakadzÏić 0 1 2 3 Mohammad A. Yaseen 0 1 2 3 0 Funding: This study was funded by National Institute on Aging, R00AG042026, Dr Mohammad Abbas Yaseen; National Institute of Neurological Disorders and Stroke , R01-NS057476 , David Boas; National Institute of Neurological Disorders and Stroke (US) , R01NS091230 , Sava Sakadzic; National Institute of Neurological Disorders and Stroke (US) , P01-NS055104, David Boas; National 1 Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School , Charlestown, MA , United States of America, 2 Department of Neurosciences and Radiology, UC San Diego, La Jolla, CA, United States of America, 3 Central European Institute of Technology, Brno University of Technology , Brno , Czech Republic 2 Editor: Vadim E. Degtyar, University of California Berkeley , UNITED STATES 3 Institute of Biomedical Imaging and Bioengineering , R01-EB000790 , David Boas; National Institute of Biomedical Imaging and Bioengineering , R01-EB021018, Anna Devor; and CEITEC, 2020 LQ1601, Hana Uhlirova Investigating cerebral metabolism in vivo at a microscopic level is essential for understanding brain function and its pathological alterations. The intricate signaling and metabolic dynamics between neurons, glia, and microvasculature requires much more detailed understanding to better comprehend the mechanisms governing brain function and its diseaserelated changes. We recently demonstrated that pharmacologically-induced alterations to different steps of cerebral metabolism can be distinguished utilizing 2-photon fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotide (NADH) fluorescence in vivo. Here, we evaluate the ability of the phasor analysis method to identify these pharmacological metabolic alterations and compare the method's performance with more conventional nonlinear curve-fitting analysis. Visualization of phasor data, both at the fundamental laser repetition frequency and its second harmonic, enables resolution of pharmacologically-induced alterations to mitochondrial metabolic processes from baseline cerebral metabolism. Compared to our previous classification models based on nonlinear curve-fitting, phasor±based models required fewer parameters and yielded comparable or improved classification accuracy. Fluorescence lifetime imaging of NADH and phasor analysis shows utility for detecting metabolic alterations and will lead to a deeper understanding of cerebral energetics and its pathological changes. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Introduction The electrochemical signaling that takes place between neurons, glia, and cerebral microvasculature during healthy brain function is extraordinarily complex and energetically demanding. To maintain this intricate, relentless activity, the brain is vitally dependent on a well-regulated, uninterrupted supply of metabolites from the bloodstream and continuous oxidative metabolism within neurons and glia [ 1 ]. For many pathological conditions, including neurodegenerative diseases, cancer, and stroke, alterations in cerebral energy metabolism constitute a hallmark of disease onset or advancement [ 2,3 ]. Such alterations, including relative shifts from oxidative to glycolytic metabolism, reduced mitochondrial membrane potential, and oxidative stress, manifest with the onset and progression of these disorders and are often associated with impairments of blood flow and metabolite supply [4±7]. Understanding these disease-related variations is essential for development of novel biomarkers and therapeutic targets. Among existing technologies for investigating cerebral metabolism, 2-photon microscopy (2PM) uniquely enables minimally invasive observation of multiple facets of blood flow and energy metabolism at cellular and subcellular resolutions in vivo, where the structural and functional connections remain undisturbed. For in vivo imaging, 2PM offers distinct advantages over other methods such as ultrasound, X-ray computed tomography, magnetic resonance imaging, and positron emission tomography. Benefits include high axial and lateral resolutions, deeper optical penetration relative to single-photon based confocal microscopy, and broader 2-photon absorption spectra of many common fluorophores, which enables simultaneous excitation of multiple fluorophores with distinct emission spectra using a single excitation wavelength. [8±10]. Applying 2PM for measuring endogenous fluorescence of reduced nicotinamide adenine dinucleotide (NADH) has demonstrated utility as a minimally invasive techni (...truncated)