Profiling macrophage responses to biochemical stimuli via stimulated Raman scattering microscopy

Macrophages play a critical role in immune responses and tumor progression, with their polarization states influencing their functional behavior in the tumor microenvironment. To investigate macrophage polarization in a label-free and nondestructive manner, we employed multispectral sparse Stimulated Raman Scattering (SRS) microscopy, informed by spontaneous Raman spectroscopy. This technique enabled the rapid and quantitative imaging of the chemical composition of murine macrophages with high spatial resolution. We identified distinct biochemical signatures associated with the M0, M1, and M2 polarization states, characterized by variations in protein, lipid, and nucleic acid content. Notably, M1 macrophages exhibited the highest lipid content and the lowest protein and nucleic acids concentrations. These findings highlight the potential of label-free SRS microscopy for studying macrophage polarization and plasticity with enhanced molecular contrast and speed, making it suitable for dynamic studies.