Significance: A major obstacle to studying resident microglia has been their similarity to infiltrating immune cell types and the lack of unique protein markers for identifying the functional state. Given the role of microglia in all neural diseases and insults, accurate tools for detecting their function beyond morphologic alterations are necessary.
Aims: We hypothesized that microglia would have unique metabolic fluxes in reduced nicotinamide adenine dinucleotide (NADH) that would be detectable by relative changes in fluorescence lifetime imaging microscopy (FLIM) parameters, allowing for identification of their activation status. Fluorescence lifetime of NADH has been previously demonstrated to show differences in metabolic fluxes.
Approach: Here, we investigate the use of the label-free method of FLIM-based detection of the endogenous metabolic cofactor NADH to identify microglia and characterize their activation status. To test whether microglial activation would also confer a unique NADH lifetime signature, murine primary microglial cultures and adult mice were treated with lipopolysaccharide (LPS).
Results: We found that LPS-induced microglia activation correlates with detected changes in NADH lifetime and its free-bound ratio. This indicates that NADH lifetime can be used to monitor microglia activation in a label-free fashion. Moreover, we found that there is an LPS dose-dependent change associated with reactive microglia lifetime fluxes, which is also replicated over time after LPS treatment.
Conclusion: We have demonstrated a label-free way of monitoring microglia activation via quantifying lifetime of endogenous metabolic coenzyme NADH. Upon LPS-induced activation, there is a significant change in the fluorescence lifetime following activation. Together, these results indicate that NADH FLIM approaches can be used as a method to characterize microglia activation state, both in vitro and ex vivo.
The excited state lifetime of a fluorophore together with its fluorescence emission spectrum provide information that can yield valuable insights into the nature of a fluorophore and its microenvironment. However, it is difficult to obtain both channels of information in a conventional scheme as detectors are typically configured either for spectral or lifetime detection. We present a fiber-based method to obtain spectral information from a multiphoton fluorescence lifetime imaging (FLIM) system. This is made possible using the time delay introduced in the fluorescence emission path by a dispersive optical fiber coupled to a detector operating in time-correlated single-photon counting mode. This add-on spectral implementation requires only a few simple modifications to any existing FLIM system and is considerably more cost-efficient compared to currently available spectral detectors.
Microglia are central nervous system (CNS) resident macrophages. They play a prominent role in virtually all neurodegenerative and traumatic brain injuries. Visualizing microglia using label-free methodologies will allow a better understanding of how microglia participate in CNS disorders in the absence of perturbations from external fluorescent dyes. Fluorescence Lifetime Imaging (FLIM) of NADH is an effective tool for monitoring cell intrinsic metabolic changes, and can imply metabolic state based on free/bound NADH lifetime quantification. Recently, FLIM based NADH lifetime quantification was used to characterize macrophages and other immune cell types. Here, we use a lifetime-based, label-free method to characterize microglia in vitro. We have found that there is a unique and statistically significant difference (p<0.05, n=5) in the NADH lifetime and free/bound NADH levels in microglia compared to other CNS cell types. Activated (i.e. inflammatory) microglia play a particularly important role in CNS diseases compared to quiescent microglia, and to our knowledge, there are no markers that can uniquely identify activated microglia, and distinguish them from related immune cell types. Thus, we have extended our NADH FLIM-based approach to differentiate quiescent microglia from activated microglia and found that there is a statistically significant difference (p<0.05,n=5) in NADH mean lifetime in the activated cells. Identifying microglia in a mixed population of CNS cells, and distinguishing activated from quiescent microglia will pave the way to better understanding their roles in the healthy and diseased brain.
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