A Suite of Stains: Characterization of four fluorophores as complementary tools for visualizing neutral lipids in an extremophilic green alga

Abstract

Understanding lipid metabolism in algae is critical to advancing our knowledge on fundamental algal physiology and for harnessing these organisms as platforms for the sustainable production of high-energy lipids. BODIPY is the most prevalently used fluorescent dye for the visualization of lipid droplets (LDs) in algae; however, its limitations warrant exploration of alternatives. Here we evaluate and compare four lipophilic fluorophores (BODIPY, DAF, Cou, DPAS) for their effectiveness in staining LDs in the extremophilic green alga Chlamydomonas priscui. We assess each dyes photophysical properties, synthetic accessibility, LD specificity, cellular toxicity, and suitability for microscopy and flow cytometry. All four dyes successfully stain LDs, but their performance diverges under different experimental conditions. BODIPY permits long-term incubation allowing quantification in time-course studies but exhibits poor LD specificity and high susceptibility to photobleaching. DAF enables polarity-sensitive staining but is highly toxic on prolonged exposure or during cellular stress. Cou and DPAS yield strong LD-specific signals with low cytotoxicity, making them ideal for studies involving environmental stress. However, DPAS requires room-temperature incubation, pointing toward greater potential utility for non-extremophilic algae. These results expand the toolbox for lipid biotechnology research in extremophiles and underscore the importance of tailoring dye selection and experimental conditions to algal physiology.

Plant phenotyping relevance

藻類細胞の脂質滴を可視化・定量する蛍光染色法を比較評価し、顕微鏡およびフローサイトメトリーへの適用性、特異性、毒性、光退色を検証しているため、表現型取得法が中心である。

Code and data availability

The paper reports fluorescence microscopy and flow cytometry phenotyping of lipid droplets in the alga C. priscui using four fluorophores. No public dataset, image collection, code repository, or trained model is deposited; the only availability statement directs readers to contact the corresponding author. No public,纸

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