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 wells of a clear-bottom 96-well plate for fluorescent microscopy.</li>
	<li>Wear nitrile gloves when handling the ratiometric dye C-SNARF-4. Prepare a 1 mM stock solution of the dye in dimethyl sulfoxide. Add 5 &#181;l of the stock solution to each well with HEPES buffer. Place the 96-well plate on the microscope.</li>
	<li>Turn on the microscope. Open the microscope software. Click the following panels: Acquire &#8594; Laser; Acquire&#160;&#8594; Micro; Acquire&#160;&#8594; Config; Acquire&#160;&#8594; Scan; Acquire&#160;&#8594; Stage. Warm up the incubator to 37 &#176;C.</li>
	<li>Turn on the 543 nm laser line by clicking on the 543 nm laser and the &#34;On&#34; button in the &#34;Laser Control&#34; window. Choose the 63X/1.2-numeric aperture water immersion objective in the &#34;Microscope Control&#34; window.</li>
	<li>Set the META detector to simultaneously monitor fluorescence within 576- to 608-nm (green) and 629- to 661 nm (red) intervals (&#34;Configuration Control&#34;&#160;&#8594; &#34;ChS&#34;). Adjust the laser power (&#34;Configuration Control&#34;&#160;&#8594; &#34;Excitation&#34;). Set the pinhole to yield an optical slice thickness of 1.6 &#181;m (&#34;Scan Control&#34;&#160;&#8594; &#34;Pinhole&#34;).</li>
	<li>Acquire an image of each HEPES buffer solution, 5 &#181;m above the glass bottom of the 96-well plate. Note: As soon as the focus plane is situated below the glass bottom, no fluorescent light can be seen on the screen. After every third image, set the laser power to zero and take an image for background subtraction.</li>
	<li>Perform the calibration experiment in triplicate (1.2-1.7).</li>
	<li>Determine the average fluorescent intensity and standard deviation in all red and green images.
	<ol>
		<li>Calculate the ratio R and standard error of mean, SR, for each image according to equations (1) and (2) 
		(1) <img alt="Equation1" src="/files/ftp_upload/53622/53622equation1.jpg" /> 
		(2) <img alt="Equation2" src="/files/ftp_upload/53622/53622equation2.jpg" /> 
		g, r, sg and sr are the averages and standard deviations in the respective green and red images. bg, br, sbg and sbr are the corresponding values for the background images. n2 is number of pixels imaged.</li>
		<li>Plot the calculated ratios for each pH value from the three replicate calibration experiments in a diagram and construct a fitted curve from this series of data points (i.e. using the software SigmaPlot 13). Make a mathematical function from the fitted curve that can convert ratios into pH values10.</li>
	</ol>
	</li>
</ol>
2. Collection of In Situ Grown Dental Biofilm Samples
<ol>
	<li>Select volunteers that fulfill inclusion and exclusion criteria relevant for the study. Make alginate impressions of their upper and lower dental arch. Make cast models from these impressions and manufacture an acrylic splint in the lower jaw. Design the splint with buccal acrylic flanges connected by a lingual orthodontic wire that allows the volunteer to bite into normal occlusion12.</li>
	<li>Drill recessions in the buccal flanges of the acrylic splint (Figure 1) with the help of dental acrylic burs to allow insertion of glass slabs for biofilm collection. The depth of the recessions should be at least 1.5 mm, while the width and length of the recessions may vary depending on the number of glass slabs to be inserted.</li>
	<li>For biofilm collection, use custom-made non-fluorescent glass slabs (4 x 4 x 1 mm3) with a surface roughness of grit 1,200 in order to mimic the colonization pattern on natural enamel11.</li>
	<li>Sterilize the glass slabs by autoclaving prior to mounting. Mount the glass slabs with sticky wax in the depressions in the buccal flanges of each side slightly recessed to the surface of the acrylic surface in order to protect the biofilm from shear forces exerted by movement of the cheeks11. 
	Note: The number of glass slabs placed in a recession may vary between 3 and 14, depending on the aim of the study.</li>
	<li>Insert the appliance in the mouth of the volunteer. Instruct the volunteer to retain the appliance intra-orally throughout the experimental period. Instruct the volunteer to store the appliance in an orthodontic retainer container with a piece of wet paper tissue (to keep it humid) at room temperature during tooth brushing and intake of food and beverages other than water. Instruct the volunteer to not touch the buccal acrylic flanges with the glass slabs while placing and removing the appliance. 
	Note: The experimental period may vary depending on the aim of the study (one day to several weeks).</li>
	<li>Carefully remove the glass slabs from the appliance at the end of the experimental period. Remove the sticky wax around the slabs with a knife and transfer them with a pair of tweezers to a closed container, the biofilm facing upward, until microscopic analysis. Keep the container humid with wet paper tissue. Perform pH imaging within few hours after biofilm collection.</li>
</ol>
3. Biofilm pH Imaging
<ol>
	<li>Prepare salivary solution by adding dithiothreitol to collected saliva according to the method of de Jong et al.13. Titrate the salivary solution to pH 7.0 and add glucose to a concentration of 0.4 % (wt/vol). Pipette 100 &#181;l per biofilm to be analyzed into a glass-bottom 96-well plate for microscopy. Add 5 &#181;l of the ratiometric dye per well.</li>
	<li>Place the 96-well plate on the microscope stage. Turn on the microscope and the 543 nm laser line. Warm up the incubator to 37 &#176;C. Use the same microscope settings as for the calibration of the dye (see steps 1.5-1.6). Wait for 30 min, until the 96-well plate has reached working temperature.</li>
	<li>Pick up one or more glass slabs with a slim set of tweezers and place them in the saliva-filled wells, one slab per well, with the biofilms facing downward.</li>
	<li>Acquire single images (&#34;Scan Control&#34;&#160;&#8594; &#34;Single&#34;) or z-stacks (&#34;Scan Control&#34;&#160;&#8594; &#34;Start&#34;) spanning the depth of the biofilms in different areas. To acquire z-stacks choose the number of slices to be imaged (&#34;Scan Control&#34;&#160;&#8594; &#34;Z Settings&#34;&#160;&#8594; &#34;Num Slices&#34;) and mark the z-position for the first and the last slice in the microscope software (&#34;Scan Control&#34;&#160;&#8594; &#34;Z Settings&#34;&#160;&#8594; &#34;Mark First&#34;; &#34;Mark Last&#34;). 
	Note: Z-stacks with a depth of up to 75 &#181;m can be acquired with good contrast between extracellular and intracellular areas.</li>
	<li>To follow pH changes in a microscopic field of view over time, mark the x-y-position in the microscope software (&#34;Stage and Focus Control&#34;&#160;&#8594; &#34;Mark Pos&#34;) and take repeated images at consecutive time points (&#34;Scan Control&#34;&#160;&#8594; &#34;Single&#34;). Regularly take images with the laser power set to zero for background subtraction.</li>
</ol>
4. Digital Image Analysis
<ol>
	<li>To export the microscopic images as TIF files, use the file batch export of the microscope software (&#34;Macro&#34;&#160;&#8594; &#34;File Batch Export&#34;). Mark the files to be exported and save red and green channel images in separate folders as TIF-files (&#34;Start Batch Export&#34;). Rename the files in both folders giving them sequential numbers.</li>
	<li>Import the red and green image series into software such as daime (digital image analysis in microbial ecology)14. Segment the green channel images with individually chosen brightness thresholds (Segment&#160;&#8594; Automatic segmentation&#160;&#8594; Custom threshold). Choose the brightness thresholds with care (typically between 20 and 80), so that all bacteria (brighter than the extracellular matrix), but not the matrix will be recognized as objects during segmentation. Verify visually that the areas recognized as objects correspond well to the bacterial biomass.</li>
	<li>Transfer the object layer of the segmented green channel images to the corresponding red channel images (Segment&#160;&#8594; Transfer object layer). Use the object editor function to reject and delete all objects in the red and green channel images. Now only the extracellular matrix is left in the biofilm images. Export the processed image series as TIF files.</li>
	<li>Import the image series into ImageJ (http://rsb.info.nih.gov/ij; v.1.47). Determine the average fluorescence intensity in the background images taken with the laser turned off (Analyze &#8594;&#160; Histogram). Subtract the appropriate background from the red and green images (Process &#8594;&#160; Math &#8594;&#160; Subtract).</li>
	<li>Still in ImageJ, divide the green image series (G1) by itself (Process&#160;&#8594; Image calculator). Then multiply the resulting image series (G2) with the green image series (G1). This will yield an image series (G3), where NaN is assigned to all pixels belonging to areas that were recognized as objects in daime. Proceed in the same way with the red image series (R1/R1 = R2; R2 x R1 = R3). 
	Note: As the bacterial biomass was removed from the images in step 4.3, the fluorescent intensity is 0 in these areas. Step 4.5 is necessary to convert the value 0 to NaN, which allows for ratio calculation in step 4.6.</li>
	<li>Apply the &#39;Mean&#39; filter (Process&#160;&#8594; Filters&#160;&#8594; Mean; radius: 1 pixel) to compensate for detector noise. Divide the green image series by the red image series (Process&#160;&#8594; Image calculator). This results in a green/red ratio for every remaining pixel in the extracellular space of the images. Use false coloring for graphic representation of the ratios in the images (Image&#160;&#8594; Lookup Tables). Calculate the mean ratio for each image (Analyze&#160;&#8594; Histogram).</li>
	<li>Convert the green/red ratios to pH values according to the function fitted under 1.9.2). Note: An example for calibration data and fitted curve can be seen in Schlafer et al, 201511.</li>
</ol>

<ol>
	<li>Hunter, R. C., Beveridge, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+pH-sensitive+fluoroprobe+(C-SNARF-4)+for+pH+microenvironment+analysis+in+Pseudomonas+aeruginosa+biofilms.">Application of a pH-sensitive fluoroprobe (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms.</a> Appl. Environ. Microbiol. 71 (5), 2501-2510 (2005).</li><li>Takahashi, N., Nyvad, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caries+ecology+revisited:+microbial+dynamics+and+the+caries+process.">Caries ecology revisited: microbial dynamics and the caries process.</a> Caries Res. 42 (6), 409-418 (2008).</li><li>Schlafer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH+landscapes+in+a+novel+five-species+model+of+early+dental+biofilm.">pH landscapes in a novel five-species model of early dental biofilm.</a> PLoS. One. 6 (9), e25299 (2011).</li><li>von Ohle, O. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+microsensor+measurement+of+local+metabolic+activities+in+ex+vivo+dental+biofilms+exposed+to+sucrose+and+treated+with+chlorhexidine.">Real-time microsensor measurement of local metabolic activities in ex vivo dental biofilms exposed to sucrose and treated with chlorhexidine.</a> Appl. Environ. Microbiol. 76 (7), 2326-2334 (2010).</li><li>Revsbech, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+microbial+communities+with+electrochemical+microsensors+and+microscale+biosensors.">Analysis of microbial communities with electrochemical microsensors and microscale biosensors.</a> Methods Enzymol. 397, 147-166 (2005).</li><li>Vanhoudt, P., Lewandowski, Z., Little, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iridium+oxide+pH+microelectrode.">Iridium oxide pH microelectrode.</a> Biotechnol. Bioeng. 40 (5), 601-608 (1992).</li><li>Franks, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+strategy+for+three-dimensional+real-time+imaging+of+microbial+fuel+cell+communities:+monitoring+the+inhibitory+effects+of+proton+accumulation+within+the+anode+biofilm.">Novel strategy for three-dimensional real-time imaging of microbial fuel cell communities: monitoring the inhibitory effects of proton accumulation within the anode biofilm.</a> Energy &amp; Environmental Science. 2 (1), 113-119 (2009).</li><li>Hidalgo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tomographic+fluorescence+imaging+of+pH+microenvironments+in+microbial+biofilms+by+use+of+silica+nanoparticle+sensors.">Functional tomographic fluorescence imaging of pH microenvironments in microbial biofilms by use of silica nanoparticle sensors.</a> Appl. Environ. Microbiol. 75 (23), 7426-7435 (2009).</li><li>Vroom, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth+penetration+and+detection+of+pH+gradients+in+biofilms+by+two-photon+excitation+microscopy.">Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy.</a> Appl. Environ. Microbiol. 65 (8), 3502-3511 (1999).</li><li>Bender, G. R., Sutton, S. V., Marquis, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid+tolerance,+proton+permeabilities,+and+membrane+ATPases+of+oral+streptococci.">Acid tolerance, proton permeabilities, and membrane ATPases of oral streptococci.</a> Infect. Immun. 53 (2), 331-338 (1986).</li><li>Schlafer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+imaging+of+extracellular+pH+in+bacterial+biofilms+using+C-SNARF-4.">Ratiometric imaging of extracellular pH in bacterial biofilms using C-SNARF-4.</a> Appl. Environ. Microbiol. 81 (4), 1267-1273 (2015).</li><li>Dige, I., Nilsson, H., Kilian, M., Nyvad, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+identification+of+streptococci+and+other+bacteria+in+initial+dental+biofilm+by+confocal+laser+scanning+microscopy+and+fluorescence+in+situ+hybridization.">In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization.</a> Eur. J Oral Sci. 115 (6), 459-467 (2007).</li><li>de Jong, M. H., van der Hoeven, J. S., van OS, J. H., Olijve, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+oral+Streptococcus+species+and+Actinomyces+viscosus+in+human+saliva.">Growth of oral Streptococcus species and Actinomyces viscosus in human saliva.</a> Appl. Environ. Microbiol. 47 (5), 901-904 (1984).</li><li>Daims, H., Lucker, S., Wagner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daime,+a+novel+image+analysis+program+for+microbial+ecology+and+biofilm+research.">daime, a novel image analysis program for microbial ecology and biofilm research.</a> Environ. Microbiol. 8 (2), 200-213 (2006).</li><li>Liu, Y. L., Nascimento, M., Burne, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+toward+understanding+the+contribution+of+alkali+generation+in+dental+biofilms+to+inhibition+of+dental+caries.">Progress toward understanding the contribution of alkali generation in dental biofilms to inhibition of dental caries.</a> Int. J Oral Sci. 4 (3), 135-140 (2012).</li></ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>Zeiss LSM 510 META</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>C-Apochromat 63X water immersion objective</td><td>Zeiss</td><td>N/A</td><td></td></tr><tr><td>XL Incubator</td><td>PeCON</td><td>N/A</td><td></td></tr><tr><td>SNARF-4F 5-(and-6)-Carboxylic Acid</td><td>Life Technologies</td><td>S23920</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Life Technologies</td><td>D12345</td><td></td></tr><tr><td>HEPES</td><td>Life Technologies</td><td>11344-041</td><td></td></tr><tr><td>Costar 96-well black clear-bottom plate</td><td>Fisher Scientific</td><td>07-200-567</td><td></td></tr><tr><td>Custom-made glass slabs (4 x 4 x 1 mm&lt;sup&gt;3&lt;/sup&gt;; 1,200 grit)</td><td>Menzel</td><td>N/A</td><td></td></tr><tr><td>Alginate impression material</td><td>GC Corporation</td><td>N/A</td><td></td></tr><tr><td>Acrylic Adjusting Logic Sets/set of acrylic dental burs</td><td>Axis Dental</td><td>LS-906</td><td></td></tr><tr><td>Orthodontic retainer containers</td><td>Spark Medical Equipment Co., Ltd</td><td>SK-WDTC01</td><td></td></tr><tr><td>Sticky wax</td><td>Dentsply</td><td>N/A</td><td></td></tr><tr><td>Chewing paraffin wax&amp;nbsp;</td><td>Ivoclar Vivadent AG</td><td>N/A</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma Aldrich</td><td>D0632</td><td>Used during preparation of salivary solution</td></tr><tr><td>0.45 &amp;micro;m and 0.2 &amp;micro;m syringe filters</td><td>Sigma Aldrich</td><td>CLS431220; CLS431219&amp;nbsp;</td><td></td></tr><tr><td>daime</td><td>University of Vienna, Austria</td><td>http://dome.csb.univie.ac.at/daime</td><td></td></tr><tr><td>ImageJ</td><td>NIH, Bethesda, Maryland, USA</td><td>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 ...

ratiometric-imaging-of-extracellular-ph-in-dental-biofilms

Research

JoVE Journal

Immunology and Infection

9.9K Views.  Aarhus University.  Een pH-gevoelige ratiometrische kleurstof wordt gebruikt in combinatie met confocale laser scanning microscopie en digitale beeldanalyse extracellulaire pH in tandheelkundige biofilms in real-time te volgen. 

Video: Ratiometric Imaging of Extracellular pH in Dental Biofilms

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

Chemistry

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1)&#160;selection of fluorescent dyes having minimal spectral overlap is complicated, and 2)&#160;quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH3 resin composites. Specimens were subsequently immersed for 60 sec in either Biot&#232;ne PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; &#945;=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p&#60;0.05) on both resin composites, whereas LTO did not produce differences (p&#62;0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.

A protocol for the concurrent quantification and comparison of three cellular and extracellular components within biofilms is presented. The methodology involves the use of confocal laser scanning microscopy, biofilm structural analysis and visualization software, and statistical analysis software.

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified ...

In vitro Monitoring of Extracellular pH in Real-Time

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including the intestinal diseases ulcerative colitis (UC) and Crohn's Disease (CD). The pH in the distal colon in individuals with active UC can range between a pH of 5 and 6, whereas healthy individuals maintain colonic pH in the range of 6.8-7.4. Extracellular pH has been shown to influence both intestinal epithelial cells and the infiltrating immune cells. More specifically, extracellular acidosis significantly impacts PMN. At pH below 6.5, there are increases in the production of H2O2, inhibition of apoptosis, and increases in the functional lifespan of PMN. Given the significant presence of PMN and extracellular acidification at sites of inflammation, we developed a novel model that allows for the monitoring of extracellular pH during PMN transepithelial migration in real time. Here, we describe this model and how it can be utilized to measure both the apical and basal pH during PMN trafficking. This model can be utilized to monitor extracellular pH under a wide range of conditions; including, hypoxia, PMN transepithelial migration, and for extended periods of time.

In vitro Monitoring of Extracellular pH in Real-Time

This article represents a useful in vitro assay to measure changes in extracellular pH during neutrophil (PMN) transepithelial migration (TEM)

Early accumulation of neutrophils (PMN) is a hallmark of acute intestinal inflammation. This acute inflammation is either resolved or progresses to ...

Biochemistry

High Spatial Resolution Chemical Imaging of Implant-Associated Infections with X-ray Excited Luminescence Chemical Imaging Through Tissue

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will allow successful eradication with antibiotics without an extra cost for a second surgery. Herein, we describe XELCI as a technique with high X-ray resolution, implant specificity, and chemical sensitivity to noninvasively image chemical concentrations near the surface of implanted medical devices. The devices are coated with chemically reporting surfaces. This chemically responsive surface consists of two layers coated on an implantable medical device; a pH-sensitive layer (bromothymol blue or bromocresol green incorporated hydrogel) which is coated over a red-light emitting scintillator (Gd2O2S: Eu) layer for monitoring. A focused X-ray beam irradiates a spot on the implant, and the red light generated by the scintillator (with 620 nm and 700 nm peaks) is transmitted through the sensing layer which alters the spectral ratio depending on the pH. An image is generated by scanning the X-ray beam across the implant and measuring the spectral ratio of light passing through the tissue point-by-point. We used this imaging technique for monitoring implant-associated infections previously on the bone surface of the femur with a modified implantable plate sensor. Now we are studying pH changes that occur from tibial intramedullary rod infections. Two different types of intramedullary rod designs are used in pre-pilot rabbit studies, and we learned that the XELCI technique could be used to monitor any chemical changes that occur not only on the bone surface but also inside the bone. Thus, this enables noninvasive, high spatial resolution, low background local pH imaging to study implant-associated infection biochemistry.

Here, we present a protocol for high-resolution optical detection of chemical information around implanted medical devices with X-ray excited luminescence chemical imaging (XELCI). This novel imaging technique is developed in our lab which enables studying implant-associated infection biochemistry.

Microbial infections associated with implantable medical devices are a major concern in fracture fixation failure. Early diagnosis of such infection will ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.

This manuscript describes a simple method to measure the phagosomal pH and area as well as the cytoplasmic pH of human and mouse neutrophils using the ratiometric indicator seminaphthorhodafluor (SNARF)-1, or S-1. This is achieved using live-cell confocal fluorescence microscopy and image analysis.

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 &#181;m or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.

The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms.

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Biology

Semi-Automated Planimetric Quantification of Dental Plaque Using an Intraoral Fluorescence Camera

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area of a tooth that is covered by plaque deposits. Compared to clinical indices, the PPI has a higher discriminatory power, but traditional planimetry is a time-consuming analysis, as the plaque-covered and clean tooth areas have to be determined manually for each image using image-processing software. Here, we present a method for the semi-automated planimetric quantification of dental plaque, which allows for the rapid processing of up to 1,000 images simultaneously. The method exploits the enhanced contrast between disclosed plaque, sound tooth surfaces, and soft tissues in fluorescence images acquired with an intraoral camera. Careful execution of the clinical procedures and accurate image acquisition are crucial steps for the successful semi-automated identification of the plaque-covered areas. The method is suitable for planimetry on sound facial and oral tooth surfaces, on most composite resin restorations, and on teeth with orthodontic brackets, but not on metallic restorations. Compared to traditional PPI recordings, semi-automated planimetry considerably reduces the amount of time spent on the analysis, as well as the subjective human input, thus increasing the reproducibility of planimetric measurements.

This study presents a semi-automated digital image analysis procedure for the planimetric quantification of disclosed dental plaque based on images acquired with an intraoral fluorescence camera. The method allows for the rapid and reliable quantification of dental plaque in the research environment.

Dental plaque accumulation is quantified using clinical indices or, otherwise, the planimetric plaque index (PPI),&#160;which measures the relative area ...

Ratiometric Imaging of Extracellular pH in Dental Biofilms

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