DIY enzyme labelled fluorescence alcohol (ELFA) standard production protocol to quantify single-cell phosphatase activity (SCPA) of microplankton.

Extracellular enzyme activities (EEA) are crucial components of microbial food web interactions and biogeochemical cycles in aquatic ecosystems. They also represent relevant biological traits in the ecophysiology of phytoplankton and other components of microbial plankton. To assess species-specific and (sub-)population-level characteristics of phytoplankton EEA at the single-cell level and close-to-in-situ conditions solely the enzyme labelled fluorescence (ELF)-based substrates have been used, because they become fluorescent and precipitate around the enzyme activity location upon enzymatic cleavage. However, the enzyme-labelled fluorescence alcohol (ELFA) standard is no longer commercially available, hence standard curves cannot be run anymore and single-cell phosphatase activity (SCPA) is no longer quantifiable. Therefore, we introduce a simple protocol for an ELFA standard do it yourself (DIY) production to enable quantifying microplankton SCPA again. This protocol is based on fluorescence measurements easily available to environmental enzyme activity laboratories, and it circumvents any need for chemical synthesis equipment and knowledge. The method is based on a controlled reaction of the ELF-phosphate (ELFP) substrate with commercially available alkaline phosphatase, which efficiently turns all the substrate into ELFA product. The ELFA product was dried out and dissolved again in dimethyl sulfoxide (DMSO) for storage. The ELFA concentration of that standard-to-be ELFA solution in DMSO was determined by linear regression between a low concentration dilution series of ELFA solution measured fluorimetrically and parallel measurements of a series of phosphatase-catalysed reactions at an overlapping ELFP concentration range. Finally, the fluorescence- and concentration-stable ELFA solution in DMSO with a known concentration constitutes the ELFA standard that is necessary to quantify bulk (fluorimeter) and single-cell (microscope and flow cytometer) phosphatase activity in microplankton.

3D restoration microscopy improves quantification of enzyme-labeled fluorescence-based single-cell phosphatase activity in plankton.

The ELF or fluorescence-labeled enzyme activity (FLEA) technique is a culture-independent single-cell tool for assessing plankton enzyme activity in close-to-in situ conditions. We demonstrate that single-cell FLEA quantifications based on two-dimensional (2D) image analysis were biased by up to one order of magnitude relative to deconvolved 3D. This was basically attributed to out-of-focus light, and partially to object size. Nevertheless, if sufficient cells were measured (25-40 cells), biases in individual 2D cell measurements were partially compensated, providing useful and comparable results to deconvolved 3D. We also discuss how much caution should be used when comparing the single-cell enzyme activities of different sized bacterio- and/or phytoplankton populations measured on 2D images. Finally, a novel method based on deconvolved 3D images (wide field restoration microscopy; WFR) was devised to improve the discrimination of similar single-cell enzyme activities, the comparison of enzyme activities between different size cells, the measurement of low fluorescence intensities, the quantification of less numerous species, and the combination of the FLEA technique with other single-cell methods. These improvements in cell enzyme activity measurements will provide a more precise picture of individual species' behavior in nature, which is essential to understand their functional role and evolutionary history.

A comparative study of fluorescence-labelled enzyme activity methods for assaying phosphatase activity in phytoplankton. A possible bias in the enzymatic pathway estimations.

We compared different fluorescence-labelled enzyme activity (FLEA) methods for assaying phosphatase activity in phytoplankton. Unfixed and liquid incubations are devised. We demonstrated that the presence of intracellular labelling was persistent, which could point out a source of bias in ectoenzymatic activities measurements based either on the FLEA or classical methods.