Light-quality-dependent pigment remodeling and ¹³C incorporation dynamics in Haematococcus pluvialis revealed by...

Light quality critically regulates pigment remodeling and carbon allocation in Haematococcus pluvialis, yet stage-resolved, single-cell quantification of these processes remains limited. Bulk pigment assays, confocal Raman spectroscopy, Raman chemical imaging, and ¹³C-based Raman stable isotope probing (Raman-SIP) were integrated to characterize pigment dynamics and de novo carbon incorporation across cultivation time and division stages under white, red, and blue light. A spectral correction was established to reduce carotenoid interference in the chlorophyll-related region, enabling clearer separation of pigment pools and improved agreement with bulk measurements. Blue light selectively promoted carotenoid accumulation, increased single-cell heterogeneity, and induced stage-dependent spatial reorganization, whereas red light preferentially enhanced chlorophyll-related signals. Raman-SIP revealed progressive ¹³C incorporation into pigment-associated molecular structures under all light treatments. Blue light produced the highest labeling fraction for the carotenoid-associated 1156/1137 cm^-1 pair on Day 7 (52 %), whereas red light showed the highest corrected labeling fraction for the chlorophyll-related 1521/1490 cm^-1 pair (51 %). Raman imaging further revealed pronounced intracellular heterogeneity and hotspot-like carotenoid distributions under blue light, with transmission electron microscopy providing ultrastructural context. This integrated, stage-resolved single-cell framework directly links light-quality regulation to pigment remodeling and carbon allocation dynamics in H. pluvialis.