Name: ratiometric imaging of extracellular ph in dental biofilms
Uploaded: 2016-03-09T14:00:00
Description: Watch this Scientific Journal Video about Ratiometric Imaging of Extracellular pH in Dental Biofilms at JoVE.com

The authors would like to thank Javier E. Garcia and Lene Gr&#248;nkj&#230;r for technical assistance and Merete K. Raarup for fruitful discussions. This work was funded by Aarhus University Research Foundation and the Simon Spies Foundation.

The experimental protocol was reviewed and approved by the Ethics Committee of Aarhus County (M-20100032).
1. Confocal Microscopic Calibration of the Ratiometric Dye
<ol>
	<li>For image acquisition, use an inverted confocal microscope equipped with an incubator, a 63X/1.2-numeric aperture water immersion objective, a 543 nm laser line and a META detector.</li>
	<li>Prepare HEPES buffer stock solutions (50 mM, adjusted to pH 4.5-8.5 in steps of 0.1 pH units). Pipette 100 &#181;l&#160;of each solution into the wel...

<ol>
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Microscopic monitoring of biofilm pH provides several advantages, as compared to electrode or microelectrode measurements4-6. Microscopic techniques permit to determine pH with a high spatial resolution and allow capturing both horizontal and vertical pH gradients in biofilms without disturbing the biofilm mechanically. Previous attempts of microscopic pH monitoring, however, failed to differentiate between extracellular and intracellular pH in the biofilms1,7,9. Due to bacterial homeostasis, intrac...

The method described here allows monitoring extracellular pH in dental biofilms in the range between 4.5 and 7, using the ratiometric dye seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) in combination with confocal laser scanning microscopy and digital image analysis. The employed fluorescent dye is pH-sensitive and displays a shift in its fluorescent emission depending on the state of protonation. The fluorescent emission of the protonated molecule peaks at 580 nm, and the emission of the deprotonated molecule at 640 nm1. The ratio of the fluorescent emission intensities in two detection windows comprising the two emission peaks (576 - 608 nm and 629 - 661 nm) thus reflects pH in the liquid phase, irrespective of dye concentration. With a pKa of ~6.4 the dye is suitable to visualize pH in moderately acidic environments.
PH in bacterial biofilms is of central importance for all metabolic processes. In the case of dental biofilms, pH in the extracellular matrix is the key virulence factor for the development of dental caries. Extended periods with low pH at the biofilm-tooth interface lead to slow demineralization of the underlying enamel2. Due to the complex three-dimensional architecture of biofilms, metabolites, including organic acids, are not uniformly distributed across the biofilm. Highly and less acidogenic microenvironments may be found in close spatial proximity3.
For decades, vertical pH gradients in biofilms were recorded with the help of microelectrodes4-6. While they offer a good spatial resolution due to their small tip size, they are not well-suited to monitor horizontal gradients. Moreover, insertion of the electrode disturbs the biofilm mechanically. Quantitative fluorescent microscopic techniques offer the advantage of visualizing pH changes in different areas of a biofilm without mechanical interference. Different microscopic fields of view can be selected freely and imaged repeatedly over prolonged periods1,7-9. However, when interpreting microscopic biofilm images, it is important to distinguish between fluorescence deriving from the microbial biomass and fluorescence deriving from the extracellular space. In acidic conditions, pH inside bacterial cells is different from pH in the extracellular matrix, as the bacteria actively transport protons across their cell membrane at the expense of adenosine triphosphate10. In the context of dental caries, intracellular bacterial pH does not have a direct impact on the underlying enamel whereas low extracellular pH leads to demineralization. Averaging pH in microscopic images that contain both bacteria-free areas and bacteria leads to erroneous results. The use of other stains along with the pH-sensitive dye in order to visualize the bacterial biomass and differentiate between extra- and intracellular areas brings about the risk of fluorescent contamination of the extracellular space and false measurements11.
The present manuscript therefore describes the use of the ratiometric dye in a double function; both as a pH marker and as a universal bacterial stain. As the dye is up-concentrated in bacterial cells, the combination of confocal microscopic imaging and an accurate digital image analysis procedure allows determining extracellular pH in the range between 4.5 and 7.0 in thin dental biofilms.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Zeiss LSM 510 META</td>
			<td style="width:305px;">Zeiss</td>
			<td style="width:243px;">N/A</td>
			<td style="width:301px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">C-Apochromat 63X water immersion objective</td>
			<td style="width:305px;">Zeiss</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">XL Incubator</td>
			<td style="width:305px;">PeCON</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">SNARF-4F 5-(and-6)-Carboxylic Acid</td>
			<td style="width:305px;">Life Technologies</td>
			<td>S23920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Dimethyl sulfoxide</td>
			<td style="width:305px;">Life Technologies</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">HEPES</td>
			<td style="width:305px;">Life Technologies</td>
			<td>11344-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Costar 96-well black clear-bottom plate</td>
			<td style="width:305px;">Fisher Scientific</td>
			<td>07-200-567</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:409px;">Custom-made glass slabs (4x4x1 mm; 1,200 grit)</td>
			<td style="width:305px;">Menzel</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Alginate impression material</td>
			<td style="width:305px;">GC Corporation</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Acrylic Adjusting Logic Sets/set of acrylic dental burs</td>
			<td style="width:305px;">Axis Dental</td>
			<td style="width:243px;">LS-906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Orthodontic retainer containers</td>
			<td style="width:305px;">Spark Medical Equipment Co., Ltd</td>
			<td style="width:243px;">SK-WDTC01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Sticky wax</td>
			<td style="width:305px;">Dentsply</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chewing paraffin wax&#160;</td>
			<td>Ivoclar Vivadent AG</td>
			<td style="width:243px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">Dithiothreitol</td>
			<td style="width:305px;">Sigma Aldrich</td>
			<td>D0632</td>
			<td>Used during preparation of salivary solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">0.45 &#181;m and 0.2 &#181;m syringe filters</td>
			<td style="width:305px;">Sigma Aldrich</td>
			<td>CLS431220; CLS431219&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:409px;">daime</td>
			<td style="width:305px;">University of Vienna, Austria</td>
			<td style="width:243px;">http://dome.csb.univie.ac.at/daime</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:409px;">ImageJ</td>
			<td>NIH, Bethesda, Maryland, USA</td>
			<td style="width:243px;">http://imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
	</tbody>
</table>

ratiometric imaging of extracellular ph in dental biofilms

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms. pH ratiometry exploits a pH-dependent shift in the fluorescent emission of pH-sensitive dyes. Calculation of the emission ratio at two different wavelengths allows determining local pH in microscopic images, irrespective of the concentration of the dye. Contrary to microelectrodes the technique allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) is employed to monitor extracellular pH in in vivo grown dental biofilms of unknown species composition. Upon exposure to glucose the dye is up-concentrated inside all bacterial cells in the biofilms; it is thus used both as a universal bacterial stain and as a marker of extracellular pH. After confocal microscopic image acquisition, the bacterial biomass is removed from all pictures using digital image analysis software, which permits to exclusively calculate extracellular pH. pH ratiometry with the ratiometric dye is well-suited to study extracellular pH in thin biofilms of up to 75 &#181;m thickness, but is limited to the pH range between 4.5 and 7.0.

The presented method allows monitoring extracellular pH drops in different microenvironments of dental biofilms in the pH range from 4.5 to 7 in real-time. If the experimental conditions are chosen as described above, pH starts to drop in all areas of the biofilms shortly after exposure to glucose.
When pH in a biofilm drops, bacterial cells become visible within short time (&#60;1 min), as the ratiometric dye is upconcentrated ...

Watch this Scientific Journal Video about Ratiometric Imaging of Extracellular pH in Dental Biofilms at JoVE.com

Ratiometric Imaging of Extracellular pH in Dental Biofilms

A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time.

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites ...

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