Quantum dot conjugates for SEM of bacterial communities

Abstract

Biologically compatible quantum dot (QD) nanoparticles are hybrid inorganic-organic materials with increasing popularity as fluorescent probes for studying biological specimens. QDs have several advantageous optical features compared to fluorescent dyes and they are electron-dense, allowing for correlated fluorescence and electron microscopic imaging. Despite these features, widespread use of QDs as biological probes has generally been limited by the complex chemistry required for their synthesis and the conjugation. In this work, we show that easily prepared quantum dot (QD) probes provide excellent contrast for fluorescent confocal and environmental scanning electron microscopy (ESEM) analysis of pure microbial cultures and microbial communities. Two conjugation strategies were employed in order to specifically target the QDs to bacterial cell surfaces. The first was biotinylation of the bacteria followed by labeling with commercially available QDs incorporating the high-affinity partner for biotin (QD-streptavidin). Second, we designed a novel QD probe for Gram negative bacteria: QD-polymyxin B (PMB), which binds to lipopolysaccharide (LPS) in the Gram negative cell wall. Pure cultures of Gram positive and Gram negative strains were used to illustrate that QDs impart electron density and irradiation stability to the cells, and so no other preparation apart from QD labeling is required. The techniques were then extended to a set of recently characterized microbial communities of perennial cold springs in the Canadian High Arctic, which live in close association with unusual sulfur crystals. Using correlated confocal and and ESEM, we were able to image these organisms in living samples and illustrate their relationship to the minerals.