Spectral and Imaging Flow Cytometry in Phytoplankton Research

Abstract

Spectral and imaging flow cytometry are emerging technologies that allow quantifying spectral, fluorescent, and/or morphological parameters of heterogeneous cellular populations. The protocol describes a detailed step-by-step analysis of microalgae using these techniques and examples from our laboratory (Aphanizomenon sp., Cryptomonas pyrenoidifera, and Chlorella sp.). Moreover, the chapter will be helpful to scientists who want to perform spectral flow cytometry and apply principal component analysis.

Unlocking autofluorescence in the era of full spectrum analysis: Implications for immunophenotype discovery projects

“…Strategies for AF correction or removal out of total fluorescence measurements have been explored in the past, including the development of special instruments or its compensation as a new parameter in multicolor staining using AF‐correlating detectors ( 21 , 27 , 28 , 29 ). In recent years, with improvements in the quality of optical and electronics design and the availability of spectral cytometers (Cytek Aurora and Sony SP6800) ( 13 , 30 , 31 ), the development of strategies to capture and extract AF signatures from polychromatic panel measurements without compromising data or panel size has become a widely shared goal. Although the addition of AF as a new reference during unmixing calculations adds complexity to pre‐defined panels due to unanticipated spectral overlap with fluorochromes tagging critical rare markers, novel post‐acquisition algorithms to mathematically resolve fluorochromes in polychromatic assays ( 31 , 32 , 33 , 34 , 35 ) when applied to fine measurements of spectral signatures, enhance the quality of the processed data.…”

Probing complexity of microalgae mixtures with novel spectral flow cytometry approach and “virtual filtering”

“…However, a spreading spillover from prominent Chl a led to insufficient resolution of different microalgae taxa. The SFC-VF approach [20, 24] allows the creation of “virtual bandpass filters” with no hardware modification and without spectral unmixing. As a result, it was possible to narrow or to widen spectral signal that is taken into consideration from ∼10 nm to ∼300 nm bandwidth (for SP6800 instrument) and to achieve significant discrimination of algal populations.…”

“…Freshwater cultures D. divergens, Aphanizomenon sp . and C. pyrenoidifera were maintained in DY-V medium (modified from [20] at 14°C, 14°C and 20°C, respectively, under 150 µmoles/ m2/sec light and 12/12 L/D cycle. Chlorella sp .…”

“…The critical advantage of spectral flow cytometry (SFC) is that a measurement of complete spectrum happens from single cells with rates of hundreds and thousands of events per sec [17-19]. Moreover, SFC analysis makes possible additional differentiation of heterogeneous algal mixtures by size and granularity in the manner similar to conventional flow cytometry (FCM) [17, 20]. The emission spectrum information for every single cell can be combined with light scattering data through sequential gating on combinations of standard dot plots and histograms.…”