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The fluorescence signal from the mounted samples was visualised under a fluorescence microscope with a × 20 objective.
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The mounted samples were immersed in juice medium to obtain a constant potential, which is referred as the Open Circuit Potential.
The mounted samples were immediately transferred into the AFM liquid cell, while avoiding dewetting.
The mounted samples were imaged using anLSM 510 confocal microscope (Zeiss;Hamburg, Germany), with a 100× or a 40× objective lens.
The mounted samples were stained with 1% toluidine blue for light microscopy.
The mounted samples were scanned with an LSM 510 microscope (Carl Zeiss, Jena, Germany).
The test began when the mounted samples were added to the saliva medium.
The mounted samples were then cleared through a series of 25%.
Prior to imaging, mounted samples were kept at room temperature for 1 hr or stored at −20°.
The mounted samples were then cleaned to remove debris and dust by a cycle of acetone, isopropanol, and water rinsing prior to blow drying with dry nitrogen.
The mounted samples were then outlined with silver solution and subjected to carbon coating using Speedivac carbon coating unit (Model 12E6/1598) to make them electrically conductive.
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