a1 Department of Biomedical Engineering, McGill University, Montréal, QC H3A 2B4, Canada
a2 Jet Propulsion Laboratory, California Institute of Technology, 4800 Oak Grove Dr., Pasadena, CA 91109, USA
a3 Donald Bren School of Environmental Science and Management, University of California, Santa Barbara, CA 93106-5131, USA
Few simple labeling methods exist for simultaneous fluorescence and electron microscopy of bacteria and biofilms. Here we describe the synthesis, characterization, and application of fluorescent nanoparticle quantum dot (QD) conjugates to target microbial species, including difficult to label Gram-negative strains. These QD conjugates impart contrast for both environmental scanning electron microscopy (ESEM) and fluorescence microscopy, permitting observation of living and fixed bacteria and biofilms. We apply these probes for studying biofilms extracted from perennial cold springs in the Canadian High Arctic, which is a particularly challenging system. In these biofilms, sulfur-metabolizing bacteria live in close association with unusual sulfur mineral formations. Following simple labeling protocols with the QD conjugates, we are able to image these organisms in fully-hydrated samples and visualize their relationship to the sulfur minerals using both ESEM and fluorescence microscopy. We then use scanning transmission electron microscopy to observe precipitated sulfur around individual cells and within the biofilm lattice. All combined, this information sheds light on the possible mechanisms of biofilm and mineral structure formation. These new QD conjugates and techniques are highly transferable to many other microbiological applications, especially those involving Gram-negative bacteria, and can be used for correlated fluorescence and electron microscopy.
(Received August 31 2009)
(Accepted December 01 2009)