Final Report Summary - OPTICMYELIMET (Non-linear Optical Imaging of Myelin and Metabolism in living tissues) Summary:The overall goal of OpticMyeliMet project is to develop non-invasive multi-modal non-linear optical (NLO) methods to simultaneously probe myelin and cellular metabolism during the evolution of Multiple sclerosis (MS) pathology. The myelin sheath plays a crucial role in the vertebrate nervous system, by providing energetic support for neurons. MS is characterized by the occurrence of disruption of myelin (demyelination), as well as cellular energetic failure and neurodegeneration. Therefore the development of remyelination strategies remains a crucial therapeutic objective. MS diagnosis and evolution are usually followed by Magnetic Resonance Imaging (MRI), which allows rapid identification of demyelinating MS lesions but with a poor spatial resolution and specificity for single myelin fibers. Potent label-free and non-invasive optical methods to investigate myelin and metabolism pathology and repair at the sub-cellular scale are the key tools for the analysis of demyelinating lesions in MS. OpticMyeliMet project proposes to develop and optimize an ensemble of advanced optical methods to study normal and pathological myelin and cellular redox states in diverse experimental conditions, and to visualize and longitudinally quantify myelin and metabolic states in the brain cortex. Third Harmonic Generation (THG) and Coherent anti-Stokes Raman Scattering (CARS) allow the visualization of myelin sheets without labeling, while two-photon microscopy and Fluorescence Lifetime Microscopy (FLIM) allows the quantification of the intrinsic metabolic coenzyme NADH and the metabolic state of single cells. The project aims to achieve a multi-parametric imaging of myelinated tissues on a "multiscale" level, providing an experimental and theoretical framework to relate the imaging data to the myelin organization at the macromolecular (sub-µm) to tissue (tens-to-hundreds of µm) scales. Results:The first objective of OpticMyeliMet is the implementation of advanced nonlinear optical (NLO) imaging combining several contrast modalities through spatial and temporal control of the pulsed excitation beams. We developed an efficient multimodal 2PEF-CARS-THG-FLIM microscopy by performing spatial and temporal shaping of the excitation beams.Specifically we have developed THG and CARS microscopy within the same microscopy platform. We have implemented CARS microscopy to probe different vibrational (chemical) frequencies: lipids and water and off resonance. We have characterized THG and CARS in a multilamellar vesicle (MLV), which is constituted by multiple layers of lipids, as a model for myelin in the nervous system. Within the same experimental setup we have implemented multicolor imaging by wavelength mixing for efficiently excite endogenous fluorophores. We implemented polarization-sensitive p-THG and p-CARS to probe the structural conformation and molecular organization of lipids. P-THG and p-CARS have been implemented and optimized for different excitation wavelengths. We improved the efficiency of p-THG data Analysis by developing a new fast way of analyzing the data, based on Fast Fourier Transform (FFT). We developed Time Correlated Single Photon Counting (TCSPC) for Fluorescence Lifetime Microscopy (FLIM) imaging by taking advantage of the existing non-linear microscopes equipped with programmable time-gated photon counting detection. A programmable time correlated single photon counting (TCSPC) system has been implemented and programmed to carry out real-time Fluorescence Lifetime Microscopy (FLIM). TCSPC system for FLIM imaging has been integrated in the same microscopy platform used to acquire THG and CARS. We have written a new real time and fast software for the analysis of FLIM based on Fast Fourier Transform (FFT). We have established the optimal experimental condition for Fluorescence Lifetime imaging performance of the NADH fluorophore. In particular we assessed the sensitivity of the FLIM system, lifetime resolution, acquisition speed and the shortest lifetime that can be measured with different numbers of channels of the time gated detection system. We tested TCSPC FLIM on a living system (developing Zebrafish embryo) and we implemented simultaneously THG and FLIM in the same microscopy platform. Finally we have also implemented a TCSPC FLIM system with two Photomultipliers, to perform simultaneously FLIM at different spectral emission bands. We implemented a fast switching scheme between two beams of different polarization states. The second objective of OpticMyeliMet consists in using the above described NLO microscopies to characterize myelination and metabolic processes in normal and pathological tissues. We characterized of THG signal as a function of myelin structure. We recorded and compared the THG signal as a function of myelin content and structure in vitro systems. The experiments in PLP-GFP tissues has been performed in conjunction with 2PEF imaging, to enable comparison of the coherent THG signals with the myelin density measured from the GFP image. We showed the sensitivity of THG to myelin structure and density both at the large scale of brain slice and the level of single axons within cortex. We compared the optical THG signal from myelin with GFP-tagged myelin in brain slices and in an organotypic culture sample. We showed the sensitivity of THG toward myelin density and distribution in in healthy and pathological brain slices both in the Cortex and in the Corpum Callosum.In the future se aim to perform the same characterization in CARS to obtain complementary information. We also continue to explore in depth the myelin structure at a submicrometer scale.We have shown experimentally that p-THG is sensitive to lipid organization and order in a model system (MLV); We have characterized the cellular redox state in biological models by a Two Photon Fluorescence Lifetime Microscopy. Cellular redox-ratio has been quantified through Fluorescence Lifetime Microscopy (FLIM), via the NAD(P)H/NAD(P)+ ratio measurement. We have determined the minimal illumination conditions that are permitting 2p-FLIM of NADH with minimal photo-perturbation of the living system. We performed control experiments on cells in vitro to demonstrate the effect of metabolic drugs on the free/bound NADH relative concentration and lifetime. We equipped the 2PEF-CARS-THG-FLIM microscope with a temperature and gas control chamber to perform measurements in living tissues, so we will soon to perform longitudinal measurements in live myelinated tissues.The third objective of OpticMyeliMet is to correlate the “large-scale” myelin MRI images with the “micron and sub-micron-scale” optical microscopy images, to achieve a better understanding of myelin changes observed with MRI. We recorded large scale THG images of a fixed brain slices in one plane. We equipped the 2PEF-CARS-THG-FLIM microscope with a translation stage to acquire automated images. We will soon acquire CARS and THG images of an entire brain slide to compare the NLO contrast with the MRI contrast.The fourth objective of OpticMyeliMet id to develop optical approaches to perform long term chronic optical imaging in the cortex of in vivo model of de- and re-myelination. We started to characterize the experimental conditions for epidetected THG and CARS imaging of myelin in fixed brain slices. Conclusions and impact:In Conclusion OpticMyeliMet has developed innovative non-invasive NLO techniques using endogenous contrasts to address a major issue in public health, both in terms of fundamental research and medical diagnosis. Disruption of the myelin sheath is indeed involved in several neuropathies of the central (CNS) and peripheral (PNS) nervous system, while oxidative stress and metabolic alterations are an important hallmark of several neurodegenerative diseases such Alzheimer and Parkinson. These innovative NLO techniques might be used in the future in several fields of fundamental and translational neuroscience. They are unique powerful tools to monitor in vitro and in vivo de- and re-myelination and single cells metabolic changes / oxidative injury at multiple scales. The perspective of comparing NLO and MRI contrast will provide a better understanding of MRI contrasts in terms of myelin characterization will have a great impact on the medical community as well as on the vast population of patients, by introducing a break-through in the availability of quantitative diagnostic tools.
