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Sinking towards destiny: High throughput measurement of phytoplankton sinking rates through time-resolved fluorescence plate spectroscopy.

ABSTRACT: Diatoms are marine primary producers that sink in part due to the density of their silica frustules. Sinking of these phytoplankters is crucial for both the biological pump that sequesters carbon to the deep ocean and for the life strategy of the organism. Sinking rates have been previously measured through settling columns, or with fluorimeters or video microscopy arranged perpendicularly to the direction of sinking. These side-view techniques require large volumes of culture, specialized equipment and are difficult to scale up to multiple simultaneous measures for screening. We established a method for parallel, large scale analysis of multiple phytoplankton sinking rates through top-view monitoring of chlorophyll a fluorescence in microtitre well plates. We verified the method through experimental analysis of known factors that influence sinking rates, including exponential versus stationary growth phase in species of different cell sizes; Thalassiosira pseudonana CCMP1335, chain-forming Skeletonema marinoi RO5A and Coscinodiscus radiatus CCMP312. We fit decay curves to an algebraic transform of the decrease in fluorescence signal as cells sank away from the fluorometer detector, and then used minimal mechanistic assumptions to extract a sinking rate (m d-1) using an RStudio script, SinkWORX. We thereby detected significant differences in sinking rates as larger diatom cells sank faster than smaller cells, and cultures in stationary phase sank faster than those in exponential phase. Our sinking rate estimates accord well with literature values from previously established methods. This well plate-based method can operate as a high throughput integrative phenotypic screen for factors that influence sinking rates including macromolecular allocations, nutrient availability or uptake rates, chain-length or cell size, degree of silification and progression through growth stages. Alternately the approach can be used to phenomically screen libraries of mutants.

SUBMITTER: Bannon CC

PROVIDER: S-EPMC5626032 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Sinking towards destiny: High throughput measurement of phytoplankton sinking rates through time-resolved fluorescence plate spectroscopy.

Bannon Catherine C CC Campbell Douglas A DA

PloS one 20171003 10

Diatoms are marine primary producers that sink in part due to the density of their silica frustules. Sinking of these phytoplankters is crucial for both the biological pump that sequesters carbon to the deep ocean and for the life strategy of the organism. Sinking rates have been previously measured through settling columns, or with fluorimeters or video microscopy arranged perpendicularly to the direction of sinking. These side-view techniques require large volumes of culture, specialized equip ...[more]

PMID: 28972987

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Project description:Combining multiple stimuli-responsive functionalities into the polymer design is an attractive approach to improve nucleic acid delivery. However, more in-depth fundamental understanding how the multiple functionalities in the polymer structures are influencing polyplex formation and stability is essential for the rational development of such delivery systems. Therefore, in this study the structure and dynamics of thermosensitive polyplexes were investigated by tracking the behavior of labeled plasmid DNA (pDNA) and polymer with time-resolved fluorescence spectroscopy using fluorescence resonance energy transfer (FRET). The successful synthesis of a heterofunctional poly(ethylene glycol) (PEG) macroinitiator containing both an atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) initiator is reported. The use of this novel PEG macroinitiator allows for the controlled polymerization of cationic and thermosensitive linear triblock copolymers and labeling of the chain-end with a fluorescent dye by maleimide-thiol chemistry. The polymers consisted of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM, N), hydrophilic PEG (P), and cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, D) block, further referred to as NPD. Polymer block D chain-ends were labeled with Cy3, while pDNA was labeled with FITC. The thermosensitive NPD polymers were used to prepare pDNA polyplexes, and the effect of the N/P charge ratio, temperature, and composition of the triblock copolymer on the polyplex properties were investigated, taking nonthermosensitive PD polymers as the control. FRET was observed both at 4 and 37 °C, indicating that the introduction of the thermosensitive PNIPAM block did not compromise the polyplex structure even above the polymer's cloud point. Furthermore, FRET results showed that the NPD- and PD-based polyplexes have a less dense core compared to polyplexes based on cationic homopolymers (such as PEI) as reported before. The polyplexes showed to have a dynamic character meaning that the polymer chains can exchange between the polyplex core and shell. Mobility of the polymers allow their uniform redistribution within the polyplex and this feature has been reported to be favorable in the context of pDNA release and subsequent improved transfection efficiency, compared to nondynamic formulations.

Dataset Information

Sinking towards destiny: High throughput measurement of phytoplankton sinking rates through time-resolved fluorescence plate spectroscopy.

Publications

Sinking towards destiny: High throughput measurement of phytoplankton sinking rates through time-resolved fluorescence plate spectroscopy.

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