The overall goal of this Confocal Microscopy based Ratiometric Imaging Technique is to determine and visualize Extracellular pH in Bacterial Biofilms in real time. This method helps to answer some key questions in the field of Biofilm research such as where exactly acid production in Biofilms are cures and how pH micro environments are conserved over time in distinct areas of the Biofilm. The main advantage of this technique is that Extracellular pH can be recorded in real time with high spatial resolution in all three dimensions.

To carry out Confocal Microscopic Calibration of the Ratiometric Dye use an inverted Confocal Microscope equipped with an incubator, a 63x 1.2 Numerical Aperture Water Emergent Objective, a 543 Nanometer Laser Line, and a Meta Detector. Prepare HEPES Buffer Stock Solutions Adjusted to pH 4.5 to 8.5 in Steps of 0.1 pH Units. Then pipette 100 microliters of each solution into the wells of a clear bottom, 96-Well plate or Fluorescent Microscopy.

While wearing nitro gloves, add 5 microliters of a one millimolar Stock Solution of C-SNARF-4 in Dimethyl Sulfoxide to each well of HEPES Buffer. Then place the 96-Well plate on the microscope. Next, turn on the microscope and open the microscope software.

Click on the following panels, Acquire Laser, Acquire Micro, Acquire Config, Acquire Scan, and Acquire Stage. Then warm up the incubator to 37 degrees celsius. In the Laser Control window turn on the 543 Nanometer Laser Line by clicking the on button.

Then in the Microscope Control window, chose the 63x 1.2 Numerical Aperture Water Emergent Objective. Under Configuration Control, ChS, set the Meta Detector to simultaneously monitor fluorescents within the 576 to 608 Nanometer or Green, and 629 to 661 Nanometer, or Red intervals. Under Configuration Control Excitation, adjust the laser power.

Then under Scan Control, Pinhole, set the Pinhole to yield an Optical Slice Thickness of 1.6 micrometers. Acquire an image of each HEPES Buffer Solution, 5 micrometers above the glass bottom of the 96-Well Plate. After every third image, set the laser power to zero and take an image for background subtraction.

After performing the Calibration Experiment in triplicate, determine average Fluorescent Intensities and standard deviations and plot calculated ratios for each pH value from the three replicate experiments according to the text protocol. To prepare the Acrylic Splints for collecting In Situ-grown Dental Biofilm Samples, use Dental Acrylic Burs to drill recessions at least 1.5 millimeters in depth in the Buccal Flanges of the Acrylic Splint to allow insertion of glass slabs. For a Biofilms collection, use custom-made non-fluorescent glass slabs with s surface roughness of 1, 200 grit in order to mimic the colonization pattern on natural enamel.

After sterilizing the glass slabs by autoclaving, Use Sticky Wax to mount them in the depressions in the Buccal Flanges of each side. Slightly recess to the Acrylic Surface to protect the Biofilms from sheer forces exerted by movement of the cheeks.Next. insert the appliance in the mouth of a previously selected volunteer.

Then instruct the volunteer to retain the appliance intraorally throughout the experimental period. During toothbrushing, an intake of food and beverages other than water, instruct the volunteer to store the appliance in an orthodontic retainer container with a piece of wet paper tissue at room temperature. Also instruct the volunteer to not touch the Buccal Acrylic Flanges with the glass slabs while place and removing the appliance.

At the end of the experimental period, carefully remove the glass slabs from the appliance. Use a knife to remove the Sticky Wax around the slabs and use a pair of tweezers to transfer them to a sealable container containing wet paper tissue with the Biofilm facing upward into microscopic analysis. 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 percent. Pipette 100 microliters of solution per Biofilm to be analyzed into a glass bottom, 96-Well Plate for Microscopy. Add 5 microliters of the Ratio metric 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 Scan Control, Single, Acquire Single Images. Or to Acquire Z Stacks, under Scan Control, Z Settings, Num Slices, choose the number of slices to be imaged and use Mark First, Mark Last to mark the first and last slice. To follow pH changes in a Microscopic Field of View over time, under Stage and Focus Control, Mark Pos, to mark the X, Y position.

And take repeated images at consecutive time points. To Export the images as TIF files, use Macro, File Batch Export, from the Microscope software. Mark the files to be exported and save Red and Green channel images in separate folders as TIF 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 Dyme. Under segment, automatic segmentation, custom threshold, 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 bacteria cells have been removed from the images during image processing. Under Segment, Transfer Object Layer, Transfer the Object Layer of the segmented Green Channel Images to the corresponding Red Channel Images. Use the Object editor 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 Analyze, Histogram, to determine the Average Fluorescence Intensity in the background images taken with the laser turned off. Now Import the Biofilm images into ImageJ. Under Process, Math, Subtract, Subtract the appropriate background from the Red and Green Images.

Then us Process, Image Calculator, to divide the Green Image Series, G1, by itself. Now multiply the resulting Image Series, J2, with a 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 Dyme. Proceed in the same away with a Red Image Series.

Under Proces, Filters, Mean, Radius 1 Pixel apply the Mean filter to compenstate 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 Image, look up Tables, use false coloring for Graphic Representation of the ratios in the images. Then use Analyze, Histogram, to calculate the Mean ratio for each image. Finally, convert the Green to Red ratios to pH values according to the text protocol.

When pH in Biofilms drops, for example, after the addition of glucose, bacterial cells become visible within a short time as the concentration of the Ratiometric Dye increases in the cells. Before acid production in the Biofilms starts, no difference can be seen between Extra-and Intracellular Fluorescence. As shown here, this image Exported into the software Dyme, has been segmented with a carefully chosen Brightness Threshold in order to obtain a good concurrence between the Bacterial Biomass and the objects recognized by the program.

As demonstrated in this figure, if segmented appropriately, deletion of objects in Dyme removes all fluorescent signals that derive from Bacterial Cells. Image Processing and ImageJ results in Green, Red ratios for pixels in the Extracellular space of the images. Using the Calibration Curve, these ratios were converted to pH values.

Envisionalized with false coloring for graphic representation. This graph illustrates that for each processed image, the average pH was calculated and the pH development was followed over time. When segmenting the images in Dyme, it is crucial to choose Brightness Thresholds that recognized all bacterial cells and, if present, human epithelial cells as objects.

Once mastered, this technique allows the reliable recording of extra set of that buy for pH. You want three dimensions, in real time. By modifying the procedure, pH recordings can be performed under float conditions.

Which provides additional insight into the dynamic developing of pH micro environments and Biofilms. The techniques has been developed recently. And it can pave the way for Biofilm Researchers to monitor Extracellular pH accurately in a variety of different Biofilms.