Mean intensity of enamel and dentin for teeth cultured under the host kidney capsule, and compared to mature, <i>in situ</i> grown teeth.

<p><b>NOTES:</b></p><p>Enamel <i>p</i> values: NBCe1^+/+/NBCe1^+/−  = 0.94; NBCe1^+/−/NBCe1^−/−  = 0.14; NBCe1^+/+/NBCe1^−/−  = 0.63; <i>in situ</i> wild type/NBCe1^+/+  = 0.17.</p><p>Dentin <i>p</i> values: NBCe1^+/+/NBCe1^+/−  = 0.89; NBCe1^+/−/NBCe1^−/−  = 0.36; NBCe1^+/+/NBCe1^−/−  = 0.36; <i>in situ</i> wild type/NBCe1^+/+  = 0.02.</p

<i>In situ</i> derived 2-week old mandibular first molar.

<p>Micro-CT 3D image (panel A) and cross-sectional slices (panels B–D) of an <i>in situ</i> collected 2-week old mandibular first molar tooth used for comparison with the kidney capsule grown teeth. Avizo software was used to analyze µCT data. Scale bar shown in panel A is 100.0 µm and is common to panels A–D.</p

NBCe1^+/+ (panels A–D), NBCe1+/− (panels E–H) and NBCe1^−/− panels (I–L) teeth at age E11.5+70 in host kidney capsules.

<p>Micro-CT three-dimensional (3D) images (panels A, E and I) and cross-sectional slices (panels B–D, F–H and J–L) of three typical examples of teeth grown underneath kidney capsule. Avizo software was used to analyze µCT data. Scale bars shown in panels A, E and I are 100.0 µm and are common to panels A–D, E–H and I–L respectively.</p

Enamel and dentin relative density measurements.

<p>The densities of teeth grown in the host kidney capsule, indicated by mean intensity value from µCT scanning, were compared across NBCe1^+/+, NBCe1^+/− and NBCe1^+/− groups. Teeth imaged and used for analysis were NBCe1^+/+ (n = 5), NBCe1^+/− (n = 3) and NBCe1^−/− (n = 5). The averages and standard deviations (error bars) were plotted. Two-tailed Student’s t tests were run and no statistically significant difference in either enamel or dentin density was identified between any of the groupings. Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097318#pone-0097318-t001" target="_blank">Table 1</a> for raw data.</p

Characterization of the shot noise profiles for PMT detector.

<p>Plot of the variance as a function of the mean intensity for uniform illumination acquired with CLSM. Only the initial part of the data was taken into account for linear fitting.</p

SpIDA measurements of NBCe1-A monomer-dimer density in expression systems (CHO-K1 cells) and native tissues.

<p>SpIDA was applied to CLSM images assuming a monomer-dimer mixture model (Equation 4). The values of monomeric, dimeric and total subunit number density were obtained for NBCe1-A in heterologous expression systems and tissues. The recovered number density of monomeric NBCe1-A in tissues is expected to be overestimated based on the presence of non-specific antibody binding. The error bars represent the standard error of the mean obtained from multiple cells. All of the measurements were carried out under identical collection conditions.</p

Sample CLSM images of CHO-K1 cells transiently transfected with NBCe1-A.

<p>(A) NBCe1-A-EGFP. (B) NBCe1-A-α-bungarotoxin mutant expressed in cells, and labeled with Alexa 488-α-bungarotoxin conjugate. In both cases, basal membranes of highly adherent cells was imaged. The cells were chemically fixed prior to imaging. The pixel size is 0.046 µm. The images were taken under the identical acquisition condition so that valid comparisons between different images could be made. The image contrast was enhanced in both panel A and B for visualization purposes.</p

Sample CLSM images of native NBCe1-A on basolateral cell membranes of rat kidney.

<p>Post fixation, tissues were immunolabeled with the primary anti-wt-NBCe1-A antibody (I) and the secondary (II) Alexa Fluor 488 conjugated antibody. Panel A shows non-specific secondary antibody staining in the absence of the primary. Panel B shows kidney cells immunostained with both antibodies. The pixel size is 0.0921 µm. The images were taken at identical acquisition conditions so valid comparisons between different images could be made. The image contrast was enhanced for visualization and comparison purposes.</p

Quantal brightness of Alexa Fluor 488 measured for 2D samples prepared with different values of concentration in 3D solution.

<p>(A) Mean intensity measured from ROIs. (B) The values of the quantal brightness measured with fluorescent image moment analysis and SpIDA as a function of the dye concentration in solution. The error bars were calculated as the standard error of multiple images taken at different locations in the sample.</p

The measurements of quantal brightness of monomeric EGFP for two distinct sets of imaging conditions (referred as “Set I” and “Set II” throughout the text).

<p>Set I corresponds to pixel size of 0.046 µm, with the scan speed set to “fast” (18.1 µm/µs), and Set II - pixel size of 0.0921 µm, with the scan speed set to “slow” (10.1 µm/µs). The values of ε were recovered with both fluorescence moment image analysis and SpIDA for comparison purposes. The error bars were calculated as the standard error of images of multiple cells. A) measured quantal brightness for various values of the mean intensity; B) average values (with error bars) for sets I & II.</p