Christopher J. Hintz

USE OF THE FLUORESCENT CALCITE MARKER CALCEIN TO LABEL FORAMINIFERAL TESTS

We describe a novel application of the fluorescent compound calcein (Bis[N,N-bis(carboxymethyl)aminomethyl]-fluorescein), which was used to fluorescently label foraminiferal calcite. Foraminifers that were incubated in a 10 mg L−1 solution of calcein and seawater precipitated normal-looking chambers during and after calcein incubation, which lasted up to three weeks. The survival rate of specimens incubated in calcein was similar to that of control specimens; some specimens reproduced during or after calcein exposure. Thus, this calcein-tagging method is non-lethal. Chambers precipitated during calcein incubation fluoresced a yellow-green when viewed with epifluorescence or laser scanning confocal microscopy (470 nm excitation, 500 nm emission). When viewed alternatively with reflected light, chambers formed after calcein incubation were easily distinguished from calcein-marked chambers, because calcite precipitated after calcein exposure does not fluoresce. Fluorescence is retained through fixation and air drying, thus the signal can be viewed in archived specimens. The method was executed on specimens from 15 species collected from three habitats with diferent environmental conditions. Results indicate that calcein is incorporated by all 15 species. The method has a number of potential applications, including experiments aimed at identifying benthic foraminifers that are faithful recorders of paleoceanographic proxies, as well as field studies to assess locations and chronology of foraminiferal calcification.

The Carbon and Oxygen Stable Isotopic Composition of Cultured Benthic Foraminifera

Abstract Laboratory cultures of several species of benthic foraminifera were grown under controlled physical and chemical conditions during months-long experiments carried out at the University of South Carolina in 2001 and 2002. A dozen experimental culture chambers contained a c. 1–3 mm layer of trace-metal free silica substrate, and were continuously flushed with water from a large (1600 L) seawater reservoir with known, constant temperature and composition (δ18O(water), carbonate system chemistry, and trace element concentrations). Each year, in most of the culture chambers, one or more species reproduced, producing hundreds of juveniles which grew into size classes ranging from 100 to 500 microns. Bulimina aculeata was the most successful species in the 2001 cultures, and both B. aculeata and Rosalina vilardeboana were abundant in 2002. We determined the shell C and O isotopic composition of the cultured foraminifera, and compared these isotopic values with the water chemistry of the culture chambers, and also with the shell chemistry of field specimens collected from sites on the North Carolina and South Carolina (USA) continental margin. The cultured foraminifera showed substantial offsets from the δ13C of system water dissolved inorganic carbon (−0.5 to −2.5‰, depending on species) and smaller offsets (0 to −0.5‰) from the predicted δ18O of calcite in equilibrium with the culture system water at the growth temperature. These offsets reflect at least three factors: species-dependent vital effects; ontogenetic variations in shell chemistry; and the aqueous carbonate chemistry ([CO3−] or pH) of the experimental system.

Construction, figures of merit, and testing of a single-cell fluorescence excitation spectroscopy system

Characterization of phytoplankton community composition is critical to understanding the ecology and biogeochemistry of the oceans. One approach to taxonomic characterization takes advantage of differing pigmentation between algal taxa and thus differences in fluorescence excitation spectra. Analyses of bulk water samples, however, may be confounded by interference from chromophoric dissolved organic matter or suspended particulate matter. Here, we describe an instrument that uses a laser trap based on a Nikon TE2000-U microscope to position individual phytoplankton cells for confocal fluorescence excitation spectroscopy, thus avoiding interference from the surrounding medium. Quantitative measurements of optical power give data in the form of photons emitted per photon of exposure for an individual phytoplankton cell. Residence times for individual phytoplankton in the instrument can be as long as several minutes with no substantial change in their fluorescence excitation spectra. The laser trap was found to generate two-photon fluorescence from the organisms so a modification was made to release the trap momentarily during data acquisition. Typical signal levels for an individual cell are in the range of 10(6) photons/s of fluorescence using a monochromated 75 W Xe arc lamp excitation source with a 2% transmission neutral density filter.