Figure 6

<p>A. Measurement of depth of the remaining viable cell surface. Objective measurement of the depth (Dp) of the remaining viable cell surface layer in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes of phonation. Measurement bar represents 4 µm. B. Objective examination of depth of the remaining viable cell surface using TEM. Average depth (µm) of the remaining viable cell surface in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions for 30, 60, and 120 minutes. * Denotes a significant difference between groups (p<0.0167).</p

Damage severity descriptions used for visual evaluation of microprojections.

<p>All images were categorized into four groups according to the degree of surface damage. (1) extensive – the cell surface was destroyed and cytoskeleton was exposed; (2) moderate – all microprojections were damaged and appeared flat or normal; (3) minimal – microprojections were slightly damaged; and (4) normal – microprojections showed no damage.</p

SEM image of a rabbit vocal fold.

<p>Scanning electron microscopy image of a rabbit vocal fold. Large box represents the central portion of the middle one-third region of the vocal fold. Small box represents a 2×2 µm image used for SEM analysis.</p

Figure 5

<p>A. Measurement of epithelial surface microprojection density and height. Objective measurement of the density (Dn) and height (H) of vocal fold epithelial surface microprojections in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes of phonation. Measurement bar represents 1 µm. B. Objective examination of epithelial surface microprojection density using TEM. The number of microprojections per 10 µm field in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes. * Denotes a significant difference between groups (p<0.0167). C. Objective examination of epithelial surface microprojection height using TEM. Average height (µm) of microprojections in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes. * Denotes a significant difference between groups (p<0.0167).</p

Figure 4

<p>A. SEM representative images for control, modal intensity, and raised intensity phonation. Representative 2×2 µm SEM images for the control, modal intensity, and raised intensity phonation conditions after 30, 60, and 120 minutes of phonation. Measurement bar represents 0.5 µm. B. Visual examination of epithelial surface microprojections. Classification of images by damage severity using routine visual examination of epithelial surface microprojections. Four categories of damage severity: extensive (black fill), moderate (dark gray fill), minimal (light gray fill), and normal (white fill) in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes of phonation. C. Objective examination of epithelial surface microprojection density using SEM. The percentage of vocal fold surface covered by microprojections in the control (C), modal intensity phonation (M), and raised intensity phonation (R) conditions after 30, 60, and 120 minutes of phonation. * Denotes a significant difference between groups (p<0.01).</p

HE-stained section of case 1.

<p>(A) Thyroid papillary cancer tissue was localized on the left, and normal thyroid tissue was localized on the right (original magnification 40×). The stromal region was excluded. The ROI was determined from the corresponding HE-staining results. The black boxes indicate the representative region of cancer and normal thyroid tissue. (B) Magnified representative regions of cancer and normal tissue (original magnification 200×). The cancer cells had a high cytoplasmic ratio and displayed nuclear features characteristic of papillary thyroid cancer. Histologic findings of thyroid papillary cancer consisted of columnar thyroidal epithelium set in papillary projection. The normal thyroid tissue is composed of many spherical hollow sacs called thyroid follicles.</p

Overview of the <i>m/z</i> values that had higher expression levels in cancer regions of all cases.

<p>Overview of the <i>m/z</i> values that had higher expression levels in cancer regions of all cases.</p

Visualization of molecular distribution of <i>m/z</i> values that were expressed to higher levels in cancer tissue from all cases.

<p>The ROI of each case is defined by a dashed line in HE-staining images. The intensity of all values in the cancer region was higher than in normal regions. The distribution of intensity in <i>m/z</i> 741.5 was different from the distribution of intensity in the other <i>m/z</i> values.</p

Peak picking and statistical analysis of mass spectra in case 1.

<p>Peak picking and statistical analysis of mass spectra in case 1.</p

Averaged spectrum for case 1.

<p>(A) Spectrum of the cancer region and (B) spectrum of normal region. Each spectrum was averaged from the ROI of cancer and normal tissue in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048873#pone-0048873-g001" target="_blank">Figure 1</a>. Each number shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048873#pone-0048873-t001" target="_blank">Table 1</a> was assigned using these spectra.</p