Spectral cytometry approach for characterization of algal assemblages using autofluorescent signatures

Traditional methods for studying algal assemblages, such as microscopy and pigment analysis, are limited in throughput and resolution. In contrast, advancements in full-spectrum cytometry enabled high-throughput, single-cell algae analysis based on their size and spectral fingerprints. Algal assemblages can be characterized by strong and highly heterogenous autofluorescence signals, resulting from main photosynthetic and accessory pigments. In this work, spectral and imaging cytometry were used to characterize the autofluorescence-based spectral signatures of major phytoplankton groups and to assess intra- and interspecies spectral variability. Comparative analysis of nine major phytoplankton groups revealed differences in fluorescence intensity ratios, particularly between phycobiliprotein(PBP)-containing and non-phycobiliprotein-containing taxa Next, principal component analysis (PCA) of spectral data from more than 50 Chlamydomonas spp. isolates from different geographic regions revealed three distinct spectral clusters, with subsequent imaging cytometry analysis demonstrating variations in cell morphology, particularly differences in cell size and structure. Besides, distinct autofluorescent populations were observed within individual Chlamydomonas spp.
Using scattering parameters and fluorescent intensities to spectral and imaging cytometry we were able to distinguish several populations. attribute the differences we observed to the differences in viability cell death, characterized with increased intensity in 400 – 640nm region. In addition, Haematococcus sp. cultures were analyzed under normal and stress conditions, revealing increased fluorescence in carotenoid-associated spectral regions. These observations suggest that autofluorescence not only possibly reflects physiological and metabolic states but may also be associated with structural diversity within phytoplankton populations. With the development of standardized spectral libraries, this work demonstrates improved characterization of algal communities. Together, these findings highlight the potential of spectral and imaging cytometry as powerful tools for studying pigment composition, cellular states, and stress responses in algal cultures.