Applications of Imaging Flow Cytometry for Microalgae

Abstract

The ability to image large numbers of cells at high resolution enhances flow cytometric analysis of cells and cell populations. In particular, the ability to image intracellular features adds a unique aspect to analyses, and can enable correlation between molecular phenomena resulting in alterations in cellular phenotype. Unicellular microalgae are amenable to high-throughput analysis to capture the diversity of cell types in natural samples, or diverse cellular responses in clonal populations, especially using imaging cytometry. Using examples from our laboratory, we review applications of imaging cytometry, specifically using an Amnis(®) ImageStream(®)X instrument, to characterize photosynthetic microalgae. Some of these examples highlight advantages of imaging flow cytometry for certain research objectives, but we also include examples that would not necessarily require imaging and could be performed on a conventional cytometer to demonstrate other concepts in cytometric evaluation of microalgae. We demonstrate the value of these approaches for (1) analysis of populations, (2) documentation of cellular features, and (3) analysis of gene expression.

Hidden Secrets Behind Dots: Improved Phytoplankton Taxonomic Resolution Using High‐Throughput Imaging Flow Cytometry

“…Thompson & van den Engh (64) could show that with a multiple laser excitation and subsequent multi-dimensional scaling approach using five different laser/filter combinations, it was possible to distinguish several Synechococcus isolates based on their different pigmentation. Hildebrand et al (46) and Dashkova et al (45) already presented some applications for phytoplankton analysis with this instrument, like live-dead discrimination, apoptosis detection, ROS-detection, lipid accumulation, GFP localization, and esterase activity. Even if phytoplankton fluorescence patterns can be derived from natural samples, high diversity of species in combination with pigment variability makes it much more difficult to unambiguously identify species.…”

“…Therefore, main limitations of standard FCM are the lack of precise size and morphological information including determination of spatial arrangement of intracellular components (38), allowing the identification of individual species in a complex community with similar functional properties (traits). Therefore, main limitations of standard FCM are the lack of precise size and morphological information including determination of spatial arrangement of intracellular components (38), allowing the identification of individual species in a complex community with similar functional properties (traits).…”

“…To date, several studies have examined natural phytoplankton communities using IFC, such as FlowCam (Fluid Imaging Technologies, Inc., Scarborough) (39-41), CytoSense (CytoBuoy b.v., Woerden, the Netherlands) (42,43) or FlowCytobot (McLANE Research Laboratories, Inc., East Falmouth) (44). Since 2009, the ImageStream X Mk II (Luminex, Austin, Texas) IFC has been available and used mainly for biomedical research, only occasionally has it been used for phytoplankton analysis (45,46). Nevertheless, certain technical limitations still occur.…”

Microalgal cytometric analysis in the presence of endogenous autofluorescent pigments

“…This was sufficient to prevent the saturation of images. Alternatively, [25] suggests adding an attenuation filter to the Imagestream optical configuration in order to decrease chlorophyll signal spillover. However, chlorophyll autofluorescence was still detectable in the appropriate fluorescent channels (imaging cytometer: channel 5 and 11; bandpass 702/85) and adjusting channels (data not shown) after the laser power decrease and had to be spectrally compensated.…”

Role of flow cytometry for the improvement of bioprocessing of oleaginous microorganisms

“…detailed FACS principles and applications with special emphasis on microalgal strain development. A few more reviews, by Hyka et al, Hildebrand et al . and Daskova et al, have discussed various flow cytometry methods including imaging flow cytometry, in line with applied microalgal biotechnology.…”