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Updated: Aug 15, 2025
Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment
Published on: January 7, 2019
Label-Free Characterization of Macrophage Polarization Using Raman Spectroscopy
Max Naumann1,2, Natalie Arend1, Rustam R Guliev1
1Leibniz Institute of Photonic Technology Jena, Member of Leibniz Health Technologies, Member of the Leibniz Centre for Photonics in Infection Research, LPI, Albert-Einstein-Str. 9, 07745 Jena, Germany.
Abstract:
Macrophages are important cells of the innate immune system that play many different roles in host defense, a fact that is reflected by their polarization into many distinct subtypes. Depending on their function and phenotype, macrophages can be grossly classified into classically activated macrophages (pro-inflammatory M1 cells), alternatively activated macrophages (anti-inflammatory M2 cells), and non-activated cells (resting M0 cells). A fast, label-free and non-destructive characterization of macrophage phenotypes could be of importance for studying the contribution of the various subtypes to numerous pathologies. In this work, single cell Raman spectroscopic imaging was applied to visualize the characteristic phenotype as well as to discriminate between different human macrophage phenotypes without any label and in a non-destructive manner. Macrophages were derived by differentiation of peripheral blood monocytes of human healthy donors and differently treated to yield M0, M1 and M2 phenotypes, as confirmed by marker analysis using flow cytometry and fluorescence imaging. Raman images of chemically fixed cells of those three macrophage phenotypes were processed using chemometric methods of unmixing (N-FINDR) and discrimination (PCA-LDA). The discrimination models were validated using leave-one donor-out cross-validation. The results show that Raman imaging is able to discriminate between pro- and anti-inflammatory macrophage phenotypes with high accuracy in a non-invasive, non-destructive and label-free manner. The spectral differences observed can be explained by the biochemical characteristics of the different phenotypes.
Insights
Single cell Raman spectroscopic imaging rapidly distinguishes macrophage phenotypes (M1, M2, M0) without labels. This non-destructive method accurately identifies pro-inflammatory and anti-inflammatory cells for pathology research.
Area of Science:
- Immunology
- Cell Biology
- Spectroscopy
Background:
- Macrophages are crucial innate immune cells with diverse roles, classified into M1 (pro-inflammatory), M2 (anti-inflammatory), and M0 (resting) phenotypes.
- Accurate, rapid, label-free characterization of macrophage phenotypes is vital for understanding their role in various diseases.
Purpose of the Study:
- To apply single cell Raman spectroscopic imaging for label-free, non-destructive visualization and discrimination of human macrophage phenotypes (M0, M1, M2).
Main Methods:
- Human peripheral blood monocytes were differentiated into M0, M1, and M2 macrophages, confirmed by flow cytometry and fluorescence imaging.
- Single cell Raman spectroscopic imaging was performed on chemically fixed cells.
- Chemometric methods, including N-FINDR unmixing and PCA-LDA discrimination, were used to analyze Raman spectra.
Main Results:
- Raman imaging successfully visualized characteristic phenotypes of M0, M1, and M2 macrophages.
- High accuracy was achieved in discriminating between pro-inflammatory (M1) and anti-inflammatory (M2) macrophage phenotypes.
- Spectral differences correlated with the distinct biochemical characteristics of each macrophage phenotype.
Conclusions:
- Single cell Raman spectroscopic imaging offers a powerful, non-invasive, and label-free approach for classifying macrophage phenotypes.
- This technique has significant potential for advancing research into the contribution of macrophage subtypes to various pathologies.
