Applications of Imaging Flow Cytometry for Microalgae.

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.

Development of flow cytometric procedures for the efficient isolation of improved lipid accumulation mutants in a Chlorella sp. microalga.

The successful development of microalgae-based biofuel production will rely on improvements in the amount and rate of fuel molecule precursor accumulation. Mutagenesis and selection is an attractive approach to improve fuel molecule productivity. Previous screening methods have been laborious, the numbers of mutants isolated have been small, and overall performance of mutants may have been compromised by the presence of deleterious secondary mutations generated by random mutagenesis that affect other cellular processes and growth. We report an improved method of isolating high triacylglycerol (TAG) accumulating mutants of a Chlorella sp., KAS603, using flow cytometric-based selection. In addition to selection for high TAG accumulating strains, the method requires that growth of mutants be competitive with other cells in the population. Not only is growth competitive, but there is improved performance in TAG accumulation with repeated selection, suggesting purification from deleterious secondary mutations. The procedure resulted in the isolation of mutants with far higher efficiency (thousands of fold) that outperformed wild type substantially better (1.8-2.5-fold) than with previous methods. This opens the door to new approaches to the characterization of genes involved in TAG accumulation and other cellular processes.

In-depth proteome analysis of Arabidopsis leaf peroxisomes combined with in vivo subcellular targeting verification indicates novel metabolic and regulatory functions of peroxisomes.

Peroxisomes are metabolically diverse organelles with essential roles in plant development. The major protein constituents of plant peroxisomes are well characterized, whereas only a few low-abundance and regulatory proteins have been reported to date. We performed an in-depth proteome analysis of Arabidopsis (Arabidopsis thaliana) leaf peroxisomes using one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry. We detected 65 established plant peroxisomal proteins, 30 proteins whose association with Arabidopsis peroxisomes had been previously demonstrated only by proteomic data, and 55 putative novel proteins of peroxisomes. We subsequently tested the subcellular targeting of yellow fluorescent protein fusions for selected proteins and confirmed the peroxisomal localization for 12 proteins containing predicted peroxisome targeting signals type 1 or 2 (PTS1/2), three proteins carrying PTS-related peptides, and four proteins that lack conventional targeting signals. We thereby established the tripeptides SLM> and SKV> (where > indicates the stop codon) as new PTS1s and the nonapeptide RVx(5)HF as a putative new PTS2. The 19 peroxisomal proteins conclusively identified from this study potentially carry out novel metabolic and regulatory functions of peroxisomes. Thus, this study represents an important step toward defining the complete plant peroxisomal proteome.