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Updated: Dec 11, 2025
Autofluorescence Imaging to Evaluate Cellular Metabolism
Published on: November 15, 2021
Microglia activation visualization via fluorescence lifetime imaging microscopy of intrinsically fluorescent
Md Abdul K Sagar1, Jonathan N Ouellette1, Kevin P Cheng1
1University of Wisconsin-Madison, Department of Biomedical Engineering, Madison, Wisconsin, United States.
Abstract:
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.
Insights
Researchers developed a label-free method using NADH fluorescence lifetime imaging microscopy (FLIM) to monitor microglia activation. This technique detects metabolic changes in microglia, aiding in the study of neurological diseases.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Microglia identification is challenging due to similarities with other immune cells, hindering functional state assessment.
- Accurate tools are needed to detect microglial function beyond morphology, especially given their role in neural diseases.
Purpose of the Study:
- To investigate if unique metabolic fluxes in reduced nicotinamide adenine dinucleotide (NADH) in microglia can be detected by fluorescence lifetime imaging microscopy (FLIM).
- To establish a label-free method for identifying microglia activation status using NADH FLIM parameters.
Main Methods:
- Utilized label-free FLIM to detect endogenous NADH in murine primary microglial cultures and adult mice.
- Induced microglia activation using lipopolysaccharide (LPS) to observe changes in NADH lifetime and free-bound ratio.
Main Results:
- LPS-induced microglia activation correlated with detectable changes in NADH lifetime and its free-bound ratio.
- Demonstrated an LPS dose-dependent and time-dependent change in reactive microglia lifetime fluxes.
- Confirmed that NADH lifetime can serve as a marker for microglia activation.
Conclusions:
- NADH FLIM provides a label-free method for monitoring microglia activation status.
- Significant changes in NADH fluorescence lifetime occur upon LPS-induced activation.
- NADH FLIM is a viable approach for characterizing microglia activation both in vitro and ex vivo.
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