Development of a scanning confocal laser microscopic technique to examine the structure and composition of marme snow

A technique was developed to image marine snow particles by scanning confocal laser microscopy (SCLM). This method allows structural and compositional characterization of fully hydrated marine snow particles with minimal disruption of the particle structure. High specificity fluorescent stains permitted sequential imaging of selected polysaccharides (concanavalin A: specific for mannose and glucose polymer:;), proteins [5-(4,6-dichlorotriazin-2-yl) aminofluorescein], and DNA (propidium iodide). Chl a autofluorescence can rtlso be imaged. SCLM produced optical slices of marine snow particles that were suited for a number of image-processing applications. These applications include high-resolution fluorescently derived optical slices and composite images produced from combined optical slices. Initial observations suggest that marine snow particles found in the oligotrophic Pacific Ocean vary in composition and structure. Variations in polysaccharide and protein composition of marine snow particles may be related to particle morphology. When combined with other conventional analysis, such as epifluorescent and electron microscopy, LSCM can provide important contributions to characterizing composition and structure of marine snow. Organic aggregates, or marine snow, are typically composed of an assemblage of organic polymers, phytoplankton, bacteria, fecal pellets, mineral materials, and other suspended matter characteristic of a given water mass (Silver and Alldredge 1981). Marine aggregates can form de novo from living organisms such as abandoned mucus feeding structures that continue to scavenge fine suspended particles or from the physical aggregation of suspended particles due to collision and flocculation (AIldredge and Silver 1988; Shanks and Edmondson 1989). Thorium isotopic disequilibrium studies suggest that a repeating aggregation-disaggregation mechanism is responsible for the fate of fine particles and dissolved constituents in the ocean (Bacon and Anderson 1982). Degradation of organic coatings or matrices associated with marine aggregates could facilitate the disaggregation phase. Recent studies have examined the relative importance of extracellular polysaccharides in the marine environment. Alldredge et al. (1993) identified a class of seemingly invisible transparent exopolymer particles (TEP), composed primarily of polysaccharides, that facilitate the formation of marine aggregates. In the oligotrophic environment, microbially produced exopolymers may be important in concentrating nutrients, thus promoting the attraction and attachment of periphytic marine microorganisms (Costerton et al. 1978; Corpe 1980). These polymers are able to absorb and Acknowledgments This project was supported by National Science Foundation research grant OCN 92-17514. We thank M. Sykes and D. McGee for technical assistance, and the captain and crew of the RV Moana Wave for ship support. We gratefully acknowledge SOEST (Univ. Hawaii) Confocal Microscopy and the Analytical Electron Microscopy Facility. We also thank L. Campbell, E Mackenzie, and two anonymous reviewers for their helpful comments. This manuscript is based in part on a thesis submitted by C.EH. in partial fullfillment of the M.S. degree atthe University of Hawaii. SOEST contribution number 455 1. concentrate a number of dissolved and colloidal compounds in seawater such as amino acids and proteins (Dugan et al. 1970; Joyce and Dugan 1970), as well as a number of metals (Corpe 1975; Brown and Lester 1982; Cowen and Silver 1984). Metals have been found closely associated with bacterial exopolymers in the sediments (Nealson 1983) and in the water column (Cowen and Silver 1984; Cowen and Bruland 1985; Cowen and Li 1991). Relative protein abundance has been used as a proxy for the degree of decomposition of organic material collected by sediment traps (Hecky et al. 1973; Haake et al. 1993). While some compositional information on marine snow is available, there is a dearth of information on the structural characteristics of these aggregates. As a first step, Alldredge et al. (1990) examined marine snow strength as a function of its size. Th#:ir study concluded that the strength or resistance of marine snow to physical breakup increased exponentially with decreasing size. Throughout the size spectra of marine snow there are varied morphologies that can enhance or inhibit the disaggregating processes. As a result laf the keen interest in marine snow dynamics, several methods have been developed to characterize and image marine aggregates. Initially, marine snow structure was characterized via visual description and in situ macrophotographs (Alldredge and Silver 1988; Riebeseli 199 1). Macrophotography was helpful in characterizing the gross morphology of marine snow, yet these images provide only coarse structm-al information and no indication of composition. Conventional light microscopy can provide limited information on marine snow structure. Epifluorescence microscopy with the help of compositionally specific stains has provided compositional information (Silver and Alldredge 198 l), yet fluorescent “haze” can obliterate much of the three-dimensional structural characteristics. Scanning electron microscopy (SEM) and transmission electron micros-