Label-free multimodal nonlinear optical microscopy identifies markers of macrophage polarization

Tumor-associated macrophages are key immune cells in the tumor microenvironment. They promote or inhibit tumor progression based on their polarization state: M0 (resting/nonpolarized), M1 (proinflammatory/anti-tumorigenic), and M2 (anti-inflammatory/pro-tumorigenic). Traditional classification methods are invasive and marker dependent. This study employs multimodal nonlinear optical imaging, combining label-free SRS and TPEF microscopy, to noninvasively identify macrophage activation states in in vitro murine models at the single-cell level. Synchronized laser pulses for SRS and TPEF microscopy were generated using a dual-output fiber optical parametric oscillator. SRS imaging at the 2930 cm^-1 and 2855 cm^-1 Raman shifts visualized the lipid and protein distribution at the sub-cellular level. Simultaneously, TPEF imaging visualized NAD(P)H and FAD autofluorescence signals, enabling optical metabolic measurements of the cellular redox state. SRS microscopy identified lipidome alterations as key markers and provided insights in the different lipid/protein ratios that characterize the macrophage phenotypes. ORR analysis revealed distinct metabolic signatures and heterogeneous metabolic populations. Our results highlight the potential of non-invasive multimodal label-free imaging for advancing the understanding of macrophage polarization mechanisms, potentially enabling the development of innovative macrophage-targeted cancer therapies.