Analysis of autofluorescence using full-spectrum cytometry to discriminate and monitor microalgae and bacteria

The autofluorescence of algal pigments enables non-invasive, high-throughput characterization of microalgae at single-cell resolution. We applied full-spectrum cytometry, imaging flow cytometry, and cell sorting to analyze the spectral and morphological diversity among major microalgal groups and 102 Chlorophyta strains. The distinct spectral signatures from chlorophylls, carotenoids, and phycobiliproteins enabled clear separation of major pigment-defined algal groups, particularly those containing phycobiliproteins. Furthermore, principal component analysis of Volvocales (Chlorophyta) revealed three spectral clusters supported by corresponding differences in cell size and shape. Additionally, in Gonium cultures, we observed that spectral signatures in the yellow-green region were altered in the presence of bacteria, suggesting that interactions between the algal host and bacteria affect pigment-related fluorescence. Spectral heterogeneity observed within monocultures was linked to pigment accumulation, cell size, and morphological variability. These findings establish full-spectrum cytometry as a powerful method for profiling pigment composition, physiology, and structural diversity in microalgae, with broad applications in microbial ecology, environmental monitoring, and biotechnology. • Full-spectrum cytometry resolved algal taxa by distinct pigment autofluorescence. • Volvocales strains formed three clusters by geographical area and emission spectra. • Autofluorescent subpopulations in Chlamydomonas reflected cell state variations. • Carotenoids and prodigiosin accumulation were detected in algae and bacteria. • Axenic strains differ in their spectral signatures.