Epi-Fluorescence Microscopy

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Analytical Method

Water samples of 50 ml (nanoplankton) and 300-500 ml (microplankton) are taken at each of 8 depths in the euphotic zone (surface to 175 m) from the same CTD profiles (and bottles) that sample the microbial community by HPLC pigments and flow cytometry. The 50-ml samples are preserved with 2-ml of 10% paraformaldehyde and stained with proflavin (0.33% w/v). The larger samples are preserved with 300 µl of alkaline Lugols solution followed by 10 ml of buffered formalin and 500 µl of sodium thiosulfate (modified protocol from Sherr and Sherr, 1993); and then stained with proflavin (0.33% w/v). Preserved samples are slowly (~5 psi) filtered onto either black 0.8-µm (50 ml) or 8.0-µm (300-500 ml) Nuclepore filters overlaying 20-µm Millipore backing filters to facilitate even cell distributions. During filtration, the samples are drawn down until approximately 1-2 ml remain in the filtration tower. Concentrated DAPI (50 mg ml^-1) is added and allowed to sit briefly before filtering the remainder of the sample until dry. Filters are mounted onto glass slides with immersion oil and cover slips. Prepared slides are placed in a dark box and kept frozen at -80^oC until analysis.

Microscopical fields on the thawed slides are viewed and digitized at 630X (nanoplankton) or 200X (microplankton) with an automated Zeiss Axiovert 200M compound microscope equipped for epifluorescence microscopy and a 14 mega-pixel color CCD digital camera. For each slide, at least 25 random positions are imaged separately on four color channels with narrow band filters sets (green, blue, red and orange, respectively, for FITC, DAPI, chlorophyll a and phycoerythrin). The digital channel images are merged and analyzed using Image-Pro software. All object cells of >1.2 mm length are segmented from the background based upon green fluorescence (proflavin) threshold values, and cell length, width, feret min and max, roundness, radius, perimeter, and fluorescence measurements are quantified in list mode files for each color channel. Measurement calibration settings are applied to each image.

Autotrophic and heterotrophic cells are distinguished by the presence/absence of Chl a. Groups (including diatoms, dinoflagellages, prymnesiophytes and cyanobacteria) are enumerated manually and/or with neural network software based on characteristic sizes, shapes and fluorescence values. The biovolume of each cell is calculated using the formula of a prolate sphere: π*length*width²*6^-1. Carbon per cell is calculated from biovolumes using carbon conversion factors from Eppley et al. (1970) for eukaryotic protists, and 240 fg C µm^-3 for cyanobacteria (Worden et al., 2004).

References

Eppley, R.W., F.M.H. Reid and J.D.H. Strickland. 1970. Estimates of phytoplankton crop size, growth rate, and primary production, in The Ecology of the Plankton off La Jolla California in the Period April Through September, 1967, edited by H. J. D. Strickland, Bull. Scripps Inst. Oceanogr. 17, 33-42.

Sherr, E. B., Sherr, B. F., 1993. Preservation and storage of samples for enumeration of heterotrophic protists. In: P. Kemp, E. Sherr, B. Sherr and J. Cole (Editors), Handbook of Methods in Aquatic Microbial Ecology. Lewis Publishers, Boca Raton, FL, pp. 207-212.

Worden, A.Z., J. K.. Nolan, J.K. and B. Palenik. 2004. Assessing the dynamics and ecology of marine picophytoplankton: The importance of the eukaryotic component. Limnol. Oceanogr. 49: 168-179.