Use of quantum dot luminescent probes to achieve single-cell resolution of human oral bacteria in biofilms

Abstract

Oral biofilms are multispecies communities, and in their nascent stages of development, numerous bacterial species engage in interspecies interactions. Better insight into the spatial relationship between different species and how species diversity increases over time can guide our understanding of the role of interspecies interactions in the development of the biofilms. Quantum dots (QD) are semiconductor nanocrystals and have emerged as a promising tool for labeling and detection of bacteria. We sought to apply QD-based primary immunofluorescence for labeling of bacterial cells with in vitro and in vivo biofilms and to compare this approach with the fluorophore-based primary immunofluorescence approach we have used previously. To investigate QD-based primary immunofluorescence as the means to detect distinct targets with single-cell resolution, we conjugated polyclonal and monoclonal antibodies to the QD surface. We also conducted simultaneous QD conjugate-based and fluorophore conjugate-based immunofluorescence and showed that these conjugates were complementary tools in immunofluorescence applications. Planktonic and biofilm cells were labeled effectively by considering two factors: the final nanomolar concentration of QD conjugate and the amount of antibody conjugated to the QD, which we define as the degree of labeling. These advances in the application of QD-based immunofluorescence for the study of biofilms in vitro and in vivo will help to define bacterial community architecture and to facilitate investigations of interactions between bacterial species in these communities.

FIG. 1.

Planktonically grown S. gordonii DL1 cells labeled with the antibody conjugate anti-DL1-Alexa Fluor 546 (A) or anti-DL1-QD655 (B), showing comparable labeling with the two antibody conjugates as revealed by epifluorescence microscopy. Inserts are transmitted light images; bar, 5 μm.

FIG. 2.

S. gordonii DL1 cells labeled with anti-DL1-QD655 conjugates are brighter and more photostable than those labeled with anti-DL1-Alexa Fluor 546. x axis, number of consecutive images under constant fluorescence excitation; y axis, autoexposure time in milliseconds.

FIG. 3.

Confocal scanning laser microscopic analysis showing effect of DOL and nanomolar concentration of QD conjugates in obtaining single-cell resolution of S. gordonii DL1 biofilms. Biofilms labeled with the general nucleic acid stain SYTO9 (green) or antibody conjugates (red): A, anti-DL1-Alexa Fluor 546 at 5 μg protein/ml; B, anti-DL1-QD655 at 5 μg protein/ml and 10 nM QD concentration; C, anti-DL1-QD655 at 10 μg protein/ml and 20 nM QD concentration; D, anti-DL1-QD655 at 15 μg protein/ml and 30 nM QD concentration with DOL of 3. Images are single optical sections.

FIG. 4.

Confocal scanning laser microscopic images of 4-h S. gordonii DL1 biofilm showing simultaneous and equivalent efficiency of labeling of cells with both anti-DL1-QD655 conjugate (15 μg protein/ml with DOL of 3) and anti-DL1-Alexa Fluor 546 conjugate (5 μg protein/ml). Maximum projection image of the QD655 channel (A, blue), Alexa Fluor 546 channel (B, red), and overlay (C, purple [red plus blue]) showing absence of interference of labeling by conjugates. The biofilm was first labeled with anti-DL1-QD655 conjugate and then with anti-DL1-Alexa Fluor 546 conjugate; bar, 4 μm.

FIG. 5.

Penetration of EPS by MAb SWLA1.2-QD605 conjugate in biofilms grown for 18 h with 28-fold-diluted BHI containing 3 mM sucrose in a flow of 0.2 ml/min. Confocal scanning laser microscopic analysis of biofilms stained with calcofluor (A), general nucleic acid stain SYTO9 (B), or MAb SWLA1.2-QD605 conjugate (C) and overlay image of all channels (D). All images are maximum projection; bar, 4 μm.

FIG. 6.

Mixed-species communities revealed in undisturbed plaque. (A) Primary antibody anti-RPS-QD655 conjugate (red) and general nucleic acid stain 4′,6′-diamidino-2-phenylindole (blue). Spatial relationship of two species revealed in undisturbed plaque. (B) Primary antibody anti-RPS-QD655 conjugate (red) and primary antibody antiveillonella-QD605 conjugate (green). Confocal scanning laser micrographs of 6-h oral biofilms developed on an enamel surface in vivo. Maximum projection images.