Microalgal cytometric analysis in the presence of endogenous autofluorescent pigments

Herein, we tested two different approaches to measure algal fluorescence in the presence of a strong autofluorescent signal: 1) by separating dyes between different excitation lasers in order to reach minimal spectral overlap with the autofluorescent signal using flow and imaging cytometry and 2) through full spectrum analysis, virtual filtering and spectral unmixing of dye combinations and algal pigments' autofluorescence via spectral flow cytometry. For this purpose, we used viability dyes from the SYTOX family and lipophilic dyes. Among the dyes tested, the SYTOX Blue (SB) dye had minimal overlap with chlorophyll fluorescence and can be combined with autofluorescence assessment and lipophilic dyes (validated with Emiliania huxleyi algal monocultures). Imaging cytometry provided a detailed characterization of algal subpopulations stained with a combination of fluorescent dyes. A spectral flow cytometer allowed us to analyze environmental phytoplankton samples stained with fluorescent dyes in the presence of strong and heterogeneous autofluorescence from intrinsic algal pigments. We concluded that the multi-color staining of algal samples can be achieved in the presence of strong and diverse algal autofluorescence using dyes with minimal spectral overlap, a multi-laser approach (flow and imaging cytometry) and/or virtual filter and spectral flow cytometry instrumentation. This can open a new page for analytical and cell sorting algal applications.