an assay for real-time monitoring of extracellular ph in dental biofilms

An Assay for Real-Time Monitoring of Extracellular pH in Dental Biofilms

Within a few hours after biofilm collection, after preparing a salivary solution according to the text protocol, titrate the solution to pH 7.0, and add glucose to a concentration of 0.4%. Pipette 100 microliters of solution per biofilm to be analyzed into a glass bottom 96-well plate for microscopy.
Add 5 microliters of the ratiometric dye per well. Place the 96-well plate on the microscope stage. Then, turn on the microscope, and the 543 laser line. Warm up the incubator to 37 degrees Celsius. Use the same microscope settings as for the calibration of the dye.
Next, with a slim pair of tweezers, pick up one or more glass slabs and place them in the saliva-filled wells -- one slab per well with the biofilms facing downward. Under &#34;Scan Control&#34; &#8594; &#34;Single&#34; acquire single images, or to acquire Z-stacks under &#34;Scan Control&#34;, &#34;Z Settings&#34;, &#34;Num Slices&#34;, choose the number of slices to be imaged and use &#34;Mark First,&#34; &#34;Mark Last&#34; to mark the first and last slice.
To follow pH changes in a microscopic field of view over time, under &#34;Stage and Focus Control,&#34; &#34;Mark Pos&#34; to mark the XY position, and take repeated images at consecutive time points.
To export the images as .tiff files, use &#34;Macro&#34; &#8594; &#34;File Batch Export&#34; from the microscope software. Mark the files to be exported and save red and green channel images in separate folders as .tiff files. Then, rename the files in both folders to give them sequential numbers.
After importing the red and green image series into software such as daime, under &#34;Segment&#34; &#8594; &#34;Automatic segmentation&#34; &#8594; &#34;Custom Threshold,&#34; segment the green channel images with individually chosen brightness thresholds. Choose the brightness threshold with care so that all bacteria, 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.
It is crucial to select brightness thresholds that differentiate accurately between cells and extracellular matrix, and to verify visually that all bacterial cells have been removed from the images during image processing.
Under &#34;Segment&#34; &#8594; &#34;Transfer Object Layer,&#34; transfer the object layer of the segmented green channel images to the corresponding red channel images. Use the &#34;Object Editor&#34; function to reject and delete all objects in the red and green channel images, so that only the extracellular matrix is left in the biofilm images.
Next, import the background images into ImageJ and use &#34;Analyze&#34; &#8594; &#34;Histogram&#34; to determine the average fluorescence intensity in the background images taken with the laser turned off. Now, import the biofilm images into ImageJ. Under &#34;Process&#34; &#8594; &#34;Math&#34; &#8594; &#34;Subtract&#34; subtract the appropriate background from the red and green images. Then, use &#34;Process&#34; &#8594; &#34;Image Calculator&#34; to divide the green image series G1 by itself.
Now, 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.
Under &#34;Process&#34; &#8594; &#34;Filters&#34; &#8594; &#34;Mean&#34;; radius: one pixel, apply the &#39;Mean&#39; filter to compensate for detector noise. Then, divide the green image series by the red image series. This results in a green-to-red ratio for every remaining pixel in the extracellular space of the images.
From &#34;Image&#34; &#8594; &#34;Lookup tables&#34; use false coloring for graphic representation of the ratios in the images. Then use &#34;Analyze&#34; &#8594; &#34;Histogram&#34; to calculate the mean ratio for each image.
Finally, convert the green-to-red ratios to pH values according to the text protocol.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. 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 occlusion.</li>
	<li>Drill recessions in the buccal flanges of the acrylic splint 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 enamel.</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 cheeks. 
	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>
2. Biofilm pH Imaging
<ol>
	<li>Prepare salivary solution by adding dithiothreitol to collected saliva according to the method of de Jong et al. 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. 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; &#8594; &#34;Single&#34;) or z-stacks (&#34;Scan Control&#34; &#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; &#8594; &#34;Z Settings&#34; &#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; &#8594; &#34;Z Settings&#34; &#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; &#8594; &#34;Mark Pos&#34;) and take repeated images at consecutive time points (&#34;Scan Control&#34; &#8594; &#34;Single&#34;). Regularly take images with the laser power set to zero for background subtraction.</li>
</ol>
3. 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; &#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). Segment the green channel images with individually chosen brightness thresholds (Segment &#8594; Automatic segmentation &#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 &#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;Histogram). Subtract the appropriate background from the red and green images (Process &#8594;Math &#8594;Subtract).</li>
	<li>Still in ImageJ, divide the green image series (G1) by itself (Process &#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 3.3, the fluorescent intensity is 0 in these areas. Step 3.5 is necessary to convert the value 0 to NaN, which allows for ratio calculation in step 3.6.</li>
	<li>Apply the &#39;Mean&#39; filter (Process &#8594;Filters &#8594;Mean; radius: 1 pixel) to compensate for detector noise. Divide the green image series by the red image series (Process &#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 &#8594;Lookup Tables). Calculate the mean ratio for each image (Analyze &#8594;Histogram).</li>
	<li>Convert the green/red ratios to pH values according to the function fitted.</li>
</ol>

Source: Schlafer, S., et al. <a href="https://app.jove.com/v/53622">Ratiometric Imaging of Extracellular pH in Dental Biofilms.</a> J. Vis. Exp. (2016)
This video demonstrates a real-time assay for monitoring extracellular pH in dental biofilms using a pH-sensitive ratiometric probe. When an in situ-grown bacterial biofilm is exposed to glucose, a reduction in extracellular pH near the bacterial biomass protonates the probe, leading to its increased concentration inside the bacteria. The disparity in emission wavelength between the protonated and unprotonated probe is utilized to calculate the pH across the extracellular matrix of the biofilm and track pH changes over time.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Collection of In Situ Grown ...

Take a multi-well plate containing a neutral pH salivary solution supplemented with glucose.
Add a dual-emission ratiometric, pH-sensitive dye.
Place the plate on a confocal microscope stage.
Introduce a glass slab with an adhered dental biofilm into the well facing down.
Bacteria in the biofilm ferment glucose, generating organic acids and lowering pH in the vicinity.
The acidic pH protonates the dye, causing its internalization and up-concentration within bacteria, staining them, while the deprotonated dye remains extracellular.
Over time, the pH becomes acidic in other areas of the biofilm.
Upon laser excitation, the protonated dye exhibits a shift in fluorescence emission compared to its deprotonated form.
Capture fluorescent images at specified intervals.
Using appropriate software, eliminate bacterial-derived fluorescence signals.
Calculate the ratio of fluorescent emission intensities from the extracellular matrix at two specific wavelengths, correlating to pH values.
Determine the decrease in extracellular biofilm pH following glucose metabolism by bacteria.

11 Views. An Assay for Real-Time Monitoring of Extracellular pH in Dental Biofilms

An Assay for Real-Time Monitoring of Extracellular pH in Dental Biofilms

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