Technical Reference #1776

1776.

Functional Tomographic Fluorescence Imaging of pH Microenvironments in Microbial Biofilms by Use of Silica Nanoparticle Sensors Gabriela Hidalgo; Andrew Burns; Erik Herz; Anthony G. Hay; Paul L. Houston; Ulrich Wiesner; Leonard W. Lion, Cornell University, Applied and Environmental Microbiology, 75(1776), (2009)
Link To Paper

Materials & Methods:
C dot sensors. C dot sensors with 70- 30- and 10-nm diameters were grown via modified Sto¨ber silica nanoparticle syntheses (31 50). Amination was per-FIG. 1. Sensor schematic and calibration. (a) Schematic diagram showing the core-shell architecture of a C dot sensor that highlights the reference dye (Cy5) sequestered in the core and coated by a sensor dye (fluorescein) shell. (b) Peak fluorescein emission collected for solutions of known pH values ranging from 4.5 to 9.0. Also shown is the molecular structure of the dye in its dianionic charge state where it exhibits a quantum yield of approximately 93% and in its monoanionic state where its quantum yield is approximately 37%. VOL. 75 2009 IMAGING OF pH MICROENVIRONMENTS IN MICROBIAL BIOFILMS 7427 formed by using 3-aminopropyl triethoxysilane as a surface-silanizing agent. The 10-nm-diameter bare silica particles which were ultimately used for all of the functional 3D reconstructions shown here consist of a particle core covalently incorporating Cy5 as an environmentally insensitive reference dye via coupling to a reactive silane surrounded by a shell covalently incorporating fluorescein dye (fluorescein isothiocyanate [FITC]) through coupling to a reactive silane. FITC was chosen for its pKa of 6.4 which is within the range of interest for this biological system. At pH 9 FITC exists in the dianionic charge state and exhibits a quantum yield of approximately 93% while at pH 4.5 the dye is protonated to the monoanionic state with a quantum yield of approximately 37% (Fig. 1b) (18). Cy5 has a quantum yield that is constant within this pH range making it an ideal internal standard. Additional information on the synthesis and characteristics of these particles may be found elsewhere (4 5 31).

Microscopic Technique
confocal fluorescence microscopy

Cell Type(s)
Escherichia coli PHL628