Abstract
To date, in situ visualization of microbial density has remained an open problem. Here, functionalized buckyballs (e.g., C60-pyrrolidine tris acid) are shown to be a versatile platform that allows internalization within a microorganism without either adhering to the cell wall and cell membrane or binding to a matrix substrate such as soil. These molecular probes are validated via multi-scale imaging, to show association with microorganisms via fluorescence microscopy, positive cellular uptake via electron microscopy, and non-specific binding to the substrates through a combination of fluorescence and autoradiography imaging. We also demonstrate that cysteine-functionalized C60-pyrrolidine tris acid can differentiate live and dead microorganisms.
Highlights
Soil hosts most of the biodiversity in the environment, where each cubic centimeter of soil matrix can contain hundreds of thousands of microorganisms that cohabitate in a complex assemblage of mineral and organic matter[1]
In a previous study[18], our lab demonstrated that guanidinium-rich molecular transporters (GR-MoTrs) can internalize in different strains of algae by crossing both the cell wall and the lipid membrane; we later learned, that these molecular transporters are sticky to the matrix substrate
C60-pyrrolidine tris acid is a derivative of fullerene C60 (Fig. 1) and possesses three key properties: (i) containing three carboxyl groups that allow for further decoration; (ii) being extremely small (1–2 nm for a single molecule, 10–20 nm for a cluster of molecules), which facilitates intercellular movement and actions; and (iii) maintaining an intact carbon cage that retains enough hydrophobicity to inhibit adherence of C60-pyrrolidine tris acid to the soil matrix and organic matter
Summary
Soil hosts most of the biodiversity in the environment, where each cubic centimeter of soil matrix can contain hundreds of thousands of microorganisms that cohabitate in a complex assemblage of mineral and organic matter[1]. To meet the requirements of in situ imaging, synthesized probes must (i) penetrate the cell wall and lipid membrane, (ii) be non-sticky to the soil matrix, and (iii) differentiate between living and dead microorganisms. Our approach for visualizing microorganisms with the potential ability to monitor microbial activities in soil matrix in situ is based on a fullerene C60 derivative: C60-pyrrolidine tris acid, a spherical multi-carbon molecule that is known better as a buckyball. There are numerous applications of its variants, such as carbon nanotubes (CNTs)[28], megatubes[29], nano onions[30], and fullerene rings[31,32] These probes can be functionalized with a fluorescent tag for imaging microbial species, using fluorescence microcopy as one of the needed steps for validation and characterization (e.g., localization, uptake). The net result is that (a) experiments can be designed to assess viability and density of microorganisms dynamically, and (b) initiating the design of new imaging instruments can be better justified for visualizing microbial species in an opaque soil environment
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