Dynamic metabolic reprogramming among neurons and glial cells can be characterized by label-free two photon excited fluorescence intensity and lifetime (FLIM) imaging of engineered brain tissue models consisting of human neurons, astrocytes, and microglia tri-cultures. Lipofuscin is a significant contributor to the overall fluorescence detected. Its removal is important for accurate recovery of metabolic function metrics. Results reveal the important function of glial cells to reduce oxidative stress in neurons over fourteen weeks of co-culture. Distinct metabolic profiles and dynamic changes are also evident for neurons and glial cells. Our study highlights that engineered brain tissue models in combination with label-free two photon imaging offer an excellent platform for understanding cell-cell interactions and are well-suited to improve understanding of the role of metabolic and mitochondrial dysfunction in neurodegenerative diseases.
Multi-photon excited intensity and lifetime fluorescence images relying on endogenous contrast can be analyzed to quantify contributions from key metabolic co-enzymes and associated metabolic function and mitochondrial organization metrics. The high spatio-temporal resolution and context of these non-destructive measurements can be used to provide important insights related to a wide range of samples, conditions and disease models. Corresponding images are acquired from mitochondria, engineered tissues, excised and in vivo human tissues. Recent studies highlight the value of multi-parametric, label-free, metabolic assessments to improve our understanding of traumatic brain injury, (pre)cancer development, and vitiligo lesions.
In this project, we aim to build an unlabeled, two-photon imaging-based approach to identify cellular biomarkers in the context of traumatic brain injuries with subcellular imaging resolutions. So far, we have identified NAD(P)H, FAD, LipDH, and Lipofuscin, four main contributors from the brain cells. And we built a math model to quantify the fluorophore contributions directly from the two photon images. Therefore, we can calculate cellular redox state, the lipofuscin level, which is associated with oxidative stress, as well as the mitochondrial organization, cell-matrix interactions using established optical biomarkers in the lab in a more robust way.
Engineered brain tissue models with human derived cells are a promising platform for improving our understanding of brain function. Our study aims to develop a label-free, two-photon imaging focused approach that enables us to assess important morphological and functional changes that occur in such brain tissue models over time. We acquired spectral, intensity, and lifetime images of the same tissues over two months. Our results indicate that such dynamic monitoring of the cellular and matrix/scaffold components of such tissues is feasible, but complex because multiple fluorophores are present. Thus, a multi-modal, multi-wavelength approach is necessary to quantify meaningful functional changes.
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