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Updated: Sep 28, 2025
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Autofluorescence Imaging to Evaluate Cellular Metabolism
Published on: November 15, 2021
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Impacts of autofluorescence on fluorescence based techniques to study microglia
Haozhe Zhang1,2, Chen Tan1, Xiaoyue Shi1
1Department of Pharmacology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, Henan, China.
BMC Neuroscience
|April 1, 2022
Summary
Microglia autofluorescence interferes with fluorescence techniques. Phycoerythrin (PE) antibodies are best for flow cytometry, and autofluorescence should be removed for intracellular staining and excluded during live imaging analysis.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia, the immune cells of the central nervous system, accumulate autofluorescent granules over time.
- This autofluorescence can significantly impact fluorescence-based experimental techniques used to study microglia.
Purpose of the Study:
- To investigate the effects of microglia autofluorescence on common fluorescence-based methods.
- To compare the efficacy of different fluorophore-conjugated antibodies in flow cytometry.
- To characterize the source and spectral properties of microglia autofluorescence.
Main Methods:
- Flow cytometry with FITC, PE, and APC conjugated antibodies.
- Confocal imaging and spectral analysis of fixed brain tissue.
- Live imaging of GCaMP6s expressing microglia in brain slices.
Main Results:
- Phycoerythrin (PE) conjugated antibodies showed superior performance over FITC and APC in flow cytometry due to reduced interference from autofluorescence.
- Microglia autofluorescence originates from cytoplasmic granules and exhibits a multi-peak emission spectrum.
- Autofluorescence reduced calcium signal amplitudes in live imaging and required exclusion from regions of interest.
Conclusions:
- Microglia autofluorescence is a critical consideration for experimental design and data interpretation in fluorescence-based studies.
- Lipofuscin removal agents are recommended for intracellular staining.
- Careful ROI selection is necessary during live imaging to account for autofluorescence interference.
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