Anette Aakj&#230;r Thomsen and Javier E. Garcia are acknowledged for excellent technical support. The authors thank Rubens Spin-Neto for fruitful discussions on image analysis.

The protocol for saliva collection was reviewed and approved by the Ethics Committee of Aarhus County (M-20100032).
1. Cultivation of Cross-kingdom Biofilms
<ol>
	<li>Grow S. mutans DSM 20523 and C. albicans NCPF 3179 on blood agar plates at 37 &#176;C under aerobic conditions.</li>
	<li>Transfer single colonies of each organism to test tubes filled with 5 mL of brain heart infusion (BHI). Grow for 18 h under aerobic conditions at 37 &#176;C.</li>
	<li>Centrifuge the overnight cultures at 1,200 x g for 5 min. Discard the supernatant, resuspend the cells in physiological saline and adjust the OD550 nm to 0.5 for C. albicans (~107 cells/mL) and S. mutans (~108 cells/mL). Dilute the S. mutans suspension 1:10 with sterile physiological saline (~107 cells/mL).</li>
	<li>Pipette 50 &#181;L of sterile salivary solution, prepared according to the method of de Jong et al.19, into the wells of an optical bottom 96-well plate for microscopy. Incubate for 30 min at 37 &#176;C. Wash the wells 3x with 100 &#181;L of sterile physiological saline. Empty the wells.</li>
	<li>Add 100 &#181;L of C. albicans suspension to each well. Incubate at 37 &#176;C for 90 min. Wash 3x with sterile physiological saline. 
	NOTE:Do not empty the wells completely during washing. Leave a reservoir of 20 &#181;L to avoid excessive shear forces.</li>
	<li>Add 100 &#181;L of heat-inactivated fetal bovine serum (inactivated at 56 &#176;C for 30 min) to each well. Incubate at 37 &#176;C for 2 h. Wash 3x with sterile physiological saline. Empty the wells, leaving a reservoir of 20 &#181;L.</li>
	<li>Add 100 &#181;L of S. mutans suspension (prepared in step 1.3) to each well. Add 150 &#181;L of BHI containing 5% sucrose. Incubate at 37 &#176;C for 24 h or longer. When cultivating older biofilms, change the medium daily to fresh BHI. At the end of the cross-kingdom biofilm growth phase, wash 5x with sterile physiological saline.</li>
</ol>
2. Ratiometric pH Imaging
NOTE:Ratiometric pH imaging needs to be performed immediately after biofilm growth is complete.
<ol>
	<li>For ratiometric pH imaging, use an inverted confocal laser scanning microscope with a 63x oil or water immersion lens, a 543 nm laser line, and a spectral imaging system (i.e., META detector) to allow for the imaging of overlapping fluorescent signals. Use an incubator to warm the microscope stage to 35 &#176;C.</li>
	<li>Set the detector to ensure the detection of green fluorescence from 576&#8722;608 nm and simultaneous detection of red fluorescence from 629&#8722;661 nm. Choose an appropriate laser power and gain to avoid over- and underexposure. 
	NOTE: Exposure of the images is best seen in palette images with false coloring.</li>
	<li>Set the pinhole size to 1 Airy Unit or an optical slice of ~0.8 &#181;m. Set the image size to 512 x 512 pixels and the scan speed to 2. Choose a line average of 2, using the mean option. 
	NOTE:At the beginning of a series of experiments, check that the chosen microscope settings provides a clear contrast between bacterial cells, fungal cell walls, biofilm matrix, and fungal cytoplasm.</li>
	<li>Prepare 100 &#181;L of sterile physiological saline containing 0.4% (w/v) of glucose, titrated to pH 7. Prepare a stock solution of C-SNARF-4 (1 mM in dimethyl sulfoxide). Add the dye to a final concentration of 30 &#181;M. 
	CAUTION: Wear nitrile gloves when handling the ratiometric dye.</li>
	<li>Empty one of the wells with a cross-kingdom biofilm, leaving a reservoir of 20 &#181;L. Add the sterile saline containing glucose and the ratiometric dye. Place the 96-well plate on the microscope stage and start imaging the biofilm.</li>
	<li>Acquire single images or Z-stacks in different locations of the biofilm. Mark the X-Y position in the microscope software to follow pH changes in particular fields of view over time. At regular intervals, take images with the laser turned off to correct for detector offset.</li>
	<li>Repeat steps 2.4&#8211;2.6 for the analysis of each biofilm grown in a different well.</li>
</ol>
3. Calibration of the Ratiometric Dye
NOTE: Calibration of the dye and the fitting of a calibration curve can be performed on a different day than ratiometric pH imaging.
<ol>
	<li>Prepare a series of 50 mM 2-morpholinoethanesulfonic acid (MES) buffer titrated to pH 4.0&#8722;7.8 in steps of 0.2 pH units at 35 &#176;C. Pipette 150 &#181;L of each buffer solution into the wells of an optical-bottom 96-well plate for microscopy.</li>
	<li>Add the dye to the buffer-filled wells of the 96-well plate to a final concentration of 30 &#181;M. Let equilibrate for 5 min.</li>
	<li>Warm the microscope stage to 35 &#176;C. Choose the same microscope settings as for ratiometric pH imaging. Place the 96-well plate on the microscope stage. Focus on the bottom of the wells. Acquire two images (green and red channel) for all buffer solutions, 5 &#181;m above the bottom of the well. At regular intervals, take images with the laser turned off to correct for detector offset.</li>
	<li>Perform the calibration experiment in triplicate.</li>
	<li>Export all images as TIF files. Import them into dedicated image analysis software (i.e., ImageJ20). Subtract the images taken with the laser turned off from the respective images of the buffer solutions by clicking Process | Image Calculator | Subtract). 
	NOTE: If necessary, crop the images to eliminate artifacts at the image borders by performing rectangular selection using Image | Crop.</li>
	<li>Divide the green channel images through the red channel images and calculate the average fluorescence intensities in the resulting images by clicking Analyze | Histogram.</li>
	<li>From the triplicate experiments, plot the average green/red ratios against the pH. Use dedicated software to fit a function to the calibration data (i.e., MyCurveFit).</li>
</ol>
4. Digital Image Analysis
NOTE:Digital image analysis can be performed at any time point after calibration of the dye and ratiometric pH imaging.
<ol>
	<li>Store the green and red channel biofilm images in separate folders and rename both series of files with sequential numbers (e.g., GREEN_0001). Import the images into dedicated image analysis software (i.e., ImageJ). Click Analyze | Histogram to determine the average fluorescence intensity in the images taken with the laser off and subtract the value from the biofilm images by clicking Process | Math | Subtract.</li>
	<li>Import the 2 image series into dedicated image analysis software (i.e., daime21). Perform a threshold-based segmentation of the red channel images (Segment | Automatic segmentation | Custom threshold). Set the &#39;low&#39; threshold above the fluorescence intensity of the fungal cytoplasm and the &#39;high&#39; threshold below the intensity of the fungal cell walls and the bacteria. 
	NOTE:When appropriate thresholds have been chosen, only extracellular areas are recognized as objects.</li>
	<li>Transfer the object layer of the segmented image series to the green channel image series. To do so, click Segment | Transfer object layer. 
	NOTE: If the contrast between extracellular areas and fungal cytoplasm is too weak in the individual color channels, add the green channel image series to the red channel series prior to segmentation by clicking Edit | Image calculator | Addition. Perform the segmentation as described under 4.2 and transfer the object layer of the segmented image series to both the green and the red channel image series.</li>
	<li>Employ the object editor to delete non-object pixels in the red and green channel image series (Visualizer | Object editor | In all images | Delete non-object pixels). Now the biofilm images are cleared from both bacterial and fungal cells. Export the processed image series as TIF files.</li>
	<li>Import both image series to ImageJ. ImageJ assigns an intensity of 0 to all non-object pixels. Remove those pixels by dividing the red image series (R1) by itself (Process | Image calculator | Image 1: R1; Operation: Divide; Image 2: R1) and multiplying the resulting image series (R2) with the original red image series (Process | Image calculator | Image 1: R1; Operation: Multiply; Image 2: R2). A third image series (R3) is created, identical to R1, except for the fact that NaN is assigned to all pixels with an intensity of 0 in R1. Proceed in the same way with the green image series.</li>
	<li>Use the &#39;Mean&#39; filter (Process | Filters | Mean | Radius | 1 pixel) on the red and green channel image series to compensate for detector noise. Divide the green channel image series by the red channel image series (Process | Image calculator | Image 1: G3; Operation: Divide; Image 2: R3). The resulting image series (G3/R3) shows the green/red ratio for all object pixels.</li>
	<li>Calculate the average ratio for each image (Analyze | Histogram). Apply false coloring for better visual representation of the ratios in the images (Image | Lookup Tables). Convert the green/red ratios to pH values employing the function fitted under 3.6.</li>
</ol>

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The authors have nothing to disclose.

Different protocols for the cultivation of cross-kingdom biofilms involving C. albicans and Streptococcus spp. have been described previously9,22,23,24,25. However, the present setup focuses on simple growth conditions, a time schedule compatible with regular working days, a balanced species composition, and the development of a voluminous biofilm matrix. Moreover, 96-well plates were coated with salivary solution to mimic oral conditions of adhesion to some extent.
The use of pH ratiometry with C-SNARF-4 is an inexpensive method for rapid measurements of biofilm pH, the advantages and disadvantages of which have been discussed in detail elsewhere12,26. In brief, the technique allows for the assessment of pH in different locations inside biofilms and thus the monitoring of horizontal pH gradients and pH developments over time27. Vertical pH profiles can be recorded, too, but the penetration depth is limited by the use of confocal microscopy. Hence, pH ratiometry represents a faster, more versatile, and less invasive alternative to pH microelectrodes, which are currently the method of choice for Z-profiling of thick biofilms28. Compared to other microscopy-based methods for pH recordings in biofilms, pH ratiometry with C-SNARF-4 has the advantage of only employing one stain. Therefore, several problems that may arise from the differential fluorescent behavior and the interaction between different dyes are circumvented26.
In cross-kingdom biofilms, threshold-based differentiation between microbial biomass and extracellular areas poses some problems when compared to biofilms that only comprise bacterial or fungal cells. Bacterial cells internalize the ratiometric dye and display a bright fluorescent signal compared to the biofilm matrix, but also compared to fungal cell walls. Fungal cell walls appear somewhat brighter than the biofilm matrix, which in turn shows higher fluorescence than the fungal cytoplasm. In biofilms that only consist of fungal cells, images can be acquired with a high laser power/gain, which results in sufficient contrast between the biofilm matrix and the fungal cytoplasm (Figure S7B). When bacteria are present, laser power/gain must be reduced to avoid overexposure of bacterial cells (Figure S7C). Hence, the contrast between fungal cytoplasm and extracellular areas is not as pronounced, and image settings such as pinhole size, pixel size, and pixel-dwell time must be chosen with great care for ratiometric analysis.
As for all microscopy-based techniques, image quality and acquisition time are inversely correlated. In the present cross-kingdom biofilms, an imaging time of ~30 s, ideally 1 min, was necessary to obtain an appropriate quality for subsequent pH analysis. In comparison, biofilms that only harbor bacteria can be imaged with sufficient quality in 10 s or less29. For microscopy analyses of biofilm growth, structure, or composition, acquisition times of 1 min do not pose a problem, and in many instances, this may also apply to pH recordings. However, extreme pH changes, such as the rapid acidification observed immediately after exposure to glucose (&#916;pH &#62; 1 unit/min), are difficult to monitor in cross-kingdom biofilms.
The present work demonstrates that ratiometric pH recordings with C-SNARF-4 are feasible in cross-kingdom biofilms composed of S. mutans and C. albicans. Future studies may employ the method to analyze pH changes in cross-kingdom biofilms with greater species diversity, especially in biofilms grown in situ (i.e. in children with early childhood caries)30. In this study, the pH in the biofilms was monitored under static conditions and for a limited observation time of 15 min, right after a glucose pulse. Further advancements may include the application of a fluid flow to mimic in situ conditions more closely14,31,32. Moreover, pH ratiometry may be used to study the impact of various nutritional conditions on long-term pH changes in cross-kingdom biofilms and contribute to elucidate the effect of biofilm pH on the underlying host tissues. Again, the demineralization of dental enamel in early childhood caries may serve as a prominent example.

Cross-kingdom biofilms comprising both fungal and bacterial species are involved in several pathologic conditions in the oral cavity. Candida spp. have frequently been isolated from endodontic infections1 and from periodontal lesions2,3. In mucosal infections, streptococcal species from the mitis group have been shown to enhance fungal biofilm formation, tissue invasion, and dissemination in both in vitro and murine models4,5,6,7. Most interestingly, oral carriage of Candida spp. has been proven to be associated with the prevalence of caries in children8. As shown in rodent models, a symbiotic relationship between Streptococcus mutans and Candidas albicans increases the production of extracellular polysaccharides and leads to the formation of thicker and more cariogenic biofilms9,10.
In all of the above-mentioned conditions, early childhood caries in particular, the biofilm pH is of importance for disease progression, and the eminent role of the biofilm matrix for the development of acidogenic microenvironments11 calls for methodologies that allow studying pH changes inside cross-kingdom biofilms. Simple and accurate confocal microscopy-based approaches to monitor pH inside bacterial12 and fungal13 biofilms have been developed. With the ratiometric dye C-SNARF-4 and threshold-based image post-processing, extracellular pH can be determined in real-time in all three dimensions of a biofilm14. Compared to other published techniques for microscopy-based pH-monitoring in biofilms, pH ratiometry with C-SNARF-4 is simple and cheap, because it does not require the synthesis of particles or compounds that include a reference dye15 or the use of two-photon excitation16. The use of just one dye prevents problems with probe compartmentalization, fluorescent bleed-through, and selective bleaching16,17,18 while still allowing for a reliable differentiation between intra- and extracellular pH. Finally, incubation with the dye is performed after biofilm growth, which allows studying both laboratory and in situ-grown biofilms.
The aim of the present work is to extend the use of pH ratiometry and provide a method to study pH changes in cross-kingdom biofilms. As proof of concept, the method is used to monitor pH in dual species biofilms consisting of S. mutans and C. albicans exposed to glucose.

<table><tbody><tr><td>Blood agar plates</td><td>Statens Serum Institut</td><td>677</td><td></td></tr><tr><td>Brain heart infusion</td><td>Oxoid</td><td>CM1135</td><td></td></tr><tr><td>Brain heart infusion + 5 % sucrose</td><td>BDH laboratory supplies</td><td>10274</td><td></td></tr><tr><td>Candida albicans</td><td>National Collection of Pathogenic Fungi</td><td>NCPF 3179</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>daime: digital image analysis in microbial ecology</td><td>Universit&amp;auml;t Wien</td><td>N/A</td><td>Freeware; V2.1; &lt;a href=&quot;https://dome.csb.univie.ac.at/daime&quot; target=&quot;_blank&quot;&gt;https://dome.csb.univie.ac.at/daime&lt;/a&gt;</td></tr><tr><td>Dimethyl sulfoxide</td><td>Life Technologies</td><td>D12345</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco Life technologies</td><td>10270</td><td></td></tr><tr><td>GS-6R refrigerated centrifuge</td><td>Beckman</td><td>N/A</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td>N/A</td><td>Freeware; V1.46r; &lt;a href=&quot;https://imagej.nih.gov/ij&quot; target=&quot;_blank&quot;&gt;https://imagej.nih.gov/ij&lt;/a&gt;</td></tr><tr><td>Java</td><td>Oracle</td><td>N/A</td><td>Freeware necessary to run ImageJ; V8.0; &lt;a href=&quot;https://java.com/en/download&quot; target=&quot;_blank&quot;&gt;https://java.com/en/download&lt;/a&gt;</td></tr><tr><td>&amp;micro;-Plate 96 Well Black</td><td>Ibidi</td><td>89626</td><td></td></tr><tr><td>MyCurveFit</td><td>MyAssays Ltd.</td><td>N/A</td><td></td></tr><tr><td>2-(N-Morpholino)ethanesulfonic acid (MES) buffer</td><td>Bioworld</td><td>700728</td><td></td></tr><tr><td>PHM210 pH-meter</td><td>Radiometer Analytical</td><td></td><td></td></tr><tr><td>Plan-Apochromat 63x oil immersion objective</td><td>Zeiss</td><td>N/A</td><td>NA=1.4</td></tr><tr><td>SNARF&amp;reg;-4F 5-(and-6)-Carboxylic Acid</td><td>Life Technologies</td><td>S23920</td><td></td></tr><tr><td>Sterile physiological saline</td><td>VWR</td><td>6404</td><td></td></tr><tr><td>Streptococcus mutans</td><td>Deutsche Sammlung von Mikroorganismen und Zellkulturen</td><td>DSM 20523</td><td></td></tr><tr><td>Vis-spectrophotometer V-3000PC</td><td>VWR</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>Zeiss LSM 510 META</td><td>Zeiss</td><td>N/A</td><td></td></tr></tbody></table>

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.

After 24 h and 48 h, robust cross-kingdom biofilms developed in the well plates. C. albicans showed varying degrees of filamentous growth, and S. mutans formed dense clusters of up to 35 &#181;m in height. Single cells and chains of S. mutans grouped around fungal hyphae, and large intercellular spaces indicated the presence of a voluminous matrix (Figure S1).
Calibration of the ratiometric dye yields an asymmetrical sigmoidal curve13,14. The extracellular pH in the biofilms dropped quickly in the first 5 min after exposure to glucose at different rates in different microscopic fields of view. Thereafter, acidification slowed down, typically reaching values of 5.5&#8211;5.8 after 15 min (Figure 1). Replicate biofilms showed a similar behavior, with only slight variations in average pH (Figure S2).
The accuracy of the pH calculations is strongly dependent on a careful choice of image settings during acquisition. For the biofilms in question, an optical slice of 0.8 &#181;m proved to be ideal to obtain the best possible contrast between extracellular areas and fungal cytoplasm (Figure S3). Moreover, a pixel size of 0.28 &#181;m (Figure S4), a pixel dwell time of 102.4 &#181;s (Figure S5), and a line average of 2 (Figure S6) provided a good contrast along with an acceptable image acquisition time of ~1 min. During image post-processing, all bacterial and fungal cells were removed from the images, while most of the surrounding extracellular areas were included in the subsequent pH analysis. Depending on the brightness of the images, different upper and lower thresholds had to be chosen (Figure 2).
The pH data shown in Figure 1 and Figure S2 were recorded 5 &#181;m from the biofilm-substratum interface. With increasing distance from the substrate, and depending on the cell density in the biofilms, the fluorescence intensity decreases, resulting in a lower contrast between cells and matrix. However, in the thin biofilms (~35 &#181;m) grown in the present study, contrast at the top of the biofilm allowed reliable image analysis (Figure S7A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60270/60270fig1a.jpg" /> 
Figure 1: Use of pH ratiometry in cross-kingdom biofilms exposed to glucose. (A) A field of view in a stained biofilm was imaged with confocal microscopy and the area covered by bacterial and fungal cells was removed prior to ratiometric analysis. (B) The pH in the extracellular space was calculated and visualized using a lookup table (16 colors). Scale bars = 20 &#181;m. (C) The pH in the 24 h cross-kingdom biofilms dropped rapidly upon exposure to glucose at slightly different rates in different fields of view. Error bars = standard deviation (SD). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60270/60270fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60270/60270fig2.jpg" /> 
Figure 2: Threshold-based image segmentation of stained biofilms. Due to the local pH changes, fluorescence intensity in the biofilms changes over time. (A) and (B) show the same microscopic field of view after 1 min and 15 min of exposure to glucose, respectively. During image segmentation, high and low thresholds need to be chosen adequately to eliminate all areas covered by bacterial and fungal cells. (C) Blue areas were eliminated by the low threshold (40), red areas by the high threshold (115). (D) Only extracellular areas were recognized as objects (surrounded by orange lines). (E) After elimination of the area covered by microbial cells, the pH calculation could be performed in the biofilm matrix. Scale bars = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60270/60270fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/60270/60270supfig1a.jpg" /> 
Figure S1: Typical 24 h cross-kingdom biofilm stained with the ratiometric dye. Many C. albicans cells displayed filamentous growth, while S. mutans (yellow) cells formed dense clusters or localized around fungal hyphae as single cells or chains. Large intercellular spaces indicated the presence of a voluminous biofilm matrix. Scale bars = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60270/60270supfig1a.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 2" src="/files/ftp_upload/60270/60270supfig2.jpg" /> 
Figure S2: The pH development in 24 h cross-kingdom biofilms exposed to glucose. (A), (B), and (C) The pH was determined ratiometrically in three replicate biofilms for 15 min after exposure to glucose. After a rapid pH drop in the first 5 min, acidification slowed down, and levels of 5.5&#8722;5.8 were reached after 15 min. Slightly different rates were observed in different microscopic fields of view (each represented by a line). Calibration of the ratiometric probe was only performed once. Error bars = SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60270/60270supfig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 3" src="/files/ftp_upload/60270/60270supfig3a.jpg" /> 
Figure S3: Impact of optical slice thickness on image contrast. Images of stained biofilms were acquired with (A) a pinhole size of 2 Airy Units and an optical slice thickness of 1.6 &#181;m, and (B) 1 Airy Unit and an optical slice of 0.8 &#181;m. At 1 Airy Unit, a higher laser power/gain was needed for image acquisition, but the contrast between fungal cytoplasm and extracellular areas improved. Scale bars = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60270/60270supfig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 4" src="/files/ftp_upload/60270/60270supfig4.jpg" /> 
Figure S4: Impact of resolution on image contrast. Images of stained biofilms were acquired with pixel sizes of (A) 1.12 &#181;m, (B) 0.56 &#181;m, (C) 0.28 &#181;m, (D) 0.14 &#181;m, and (E) 0.11 &#181;m. Reduction of the pixel size below 0.28 &#181;m did not improve the contrast between extracellular areas and fungal cytoplasm. Scale bars = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60270/60270supfig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 5" src="/files/ftp_upload/60270/60270supfig5.jpg" /> 
Figure S5: Impact of scan speed on image contrast. Images of stained biofilms were acquired with pixel dwell times of (A) 12.8 &#181;s, (B) 25.6 &#181;s, (C) 51.2 &#181;s, (D) 102 &#181;s, and (E) 164 &#181;s. Increasing the pixel dwell time beyond 102 &#181;s did not improve the contrast between extracellular and intracellular areas. Scale bars = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60270/60270supfig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 6" src="/files/ftp_upload/60270/60270supfig6.jpg" /> 
Figure S6: Impact of averaging on image contrast. Images of stained biofilms were acquired with line averages (mean) of (A) 1, (B) 2, and (C) 4. A line average of 2 provided the best compromise between contrast and acquisition time. Scale bars = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60270/60270supfig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 7" src="/files/ftp_upload/60270/60270supfig7.jpg" /> 
Figure S7: Image contrast in purely fungal biofilms and cross-kingdom biofilms. (A) The top of a stained cross-kingdom biofilm was imaged 30 &#181;m from the interface. The contrast between extracellular and intracellular areas was lower than at the biofilm base but still sufficient for ratiometric pH analysis. (B) A C. albicans monospecies biofilm was imaged for pH ratiometry. Laser power/gain could be increased to optimize the contrast between fungal cell walls and cytoplasm. (C) In cross-kingdom biofilms, the brightly fluorescent bacteria precluded further increase of laser power/gain. Hence, the contrast between fungal cell walls and cytoplasm is less pronounced than in purely fungal biofilms. Scale bars = 20 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60270/60270supfig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy at JoVE.com

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

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, ...

monitoring-extracellular-ph-cross-kingdom-biofilms-using-confocal

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.4K Views.  Aarhus University. Het protocol presenteert een eenvoudig uit te voeren methode voor de teelt van cross-kingdom biofilms bestaande uit StreptococcuS. mutans en Candida albicans. De daaropvolgende confocale microscopiegebaseerde pH-ratiometrie maakt nauwkeurige monitoring van de pH-ontwikkelingen in de extracellulaire matrix van die biofilms mogelijk.De zorgvuldige keuze van de parameters voor het verkrijgen van afbeeldingen is van het grootste belang om beeldgegevens te verkrijgen met voldoende contrast ...

Video: Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

Neutrophil Extracellular Traps: How to Generate and Visualize Them

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria1, fungi2 and parasites3. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent4. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase5. 
Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility6.
We describe methods to isolate Neutrophil Granulocytes from peripheral human blood7 and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil Extracellular Traps (NETs) are an important innate immune mechanism to fight pathogenic bacteria, fungi and parasites. Here we describe methods to isolate neutrophil granulocytes from human blood and to activate them to form NETs. We present preparation techniques to visualize NETs in light and electron microscopy.

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

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 ...

Methods for Characterizing the Co-development of Biofilm and Habitat Heterogeneity

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Biofilms have complex interactions with their surrounding environment. To comprehensively investigate biofilm-environment interactions, we present here a series of methods to create heterogeneous chemical environment for biofilm development, to quantify local flow velocity, and to analyze mass transport in and around biofilm colonies.

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is ...

Bioengineering

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

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.

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 ...

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 ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Biology

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

Source: Schlafer, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/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 ...

Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy

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