Supplementary document for Characterization of Pathological Stomach Tissue Using Polarization-Sensitive Second Harmonic Generation Microscopy - 6516038.pdf

Derivation assuming trigonal symmetry, example ? values assuming cylindrical and trigonal symmetries, ? and DOLP values, and polarization-sensitive SHG data for cancerous and normal tissue

Supplementary document for Characterization of Pathological Stomach Tissue Using Polarization-Sensitive Second Harmonic Generation Microscopy - 6574372.pdf

Derivation of Trigonal Symmetry Equation, Kappa and DOLP Results, Enlarged Figures, and Data Set

Molecular Organization of Crystalline β‑Carotene in Carrots Determined with Polarization-Dependent Second and Third Harmonic Generation Microscopy

Polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and third harmonic generation (THG) microscopy was used to study the crystalline organization of β-carotene molecules within individual aggregates contained in the chromoplasts of orange carrots <i>in vivo</i>. Multimodal PIPO SHG and PIPO THG studies of the aggregates revealed one dominant SHG and THG dipole signifying that β-carotene molecules are oriented along a single axis. Three-dimensional visualization of the orientation of β-carotene molecules with respect to the aggregate axis was also performed with both microscopy modalities and revealed organization of the aggregates as ribbon-like structures consisting of twists and folds. Therefore, PIPO SHG and PIPO THG microscopy provides information on the crystalline organization and the orientation of ordered biological structures <i>in vivo</i> where multimodal polarization dependent SHG and THG investigations are particularly advantageous as both noncentrosymmetric and centrosymmetric crystalline organizations can be probed

PIPO SHG and PIPO THG data of an aplanospore.

<p>An aplanospore visualized by (a) SHG, (b) THG, and (c) structural image cross-correlation analysis between SHG and THG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107804#pone.0107804-Barzda1" target="_blank">[16]</a>. The images consist of a summation of images recorded at various polarizations. Specific regions seen in (a, b, c) labeled as (i, ii, iii, iv, v, vi) were analyzed to produce (d, e) PIPO data contour plots and fits. Fitting of (a) PIPO SHG data for regions (i, ii, iii, iv, v, vi) were performed with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107804#pone.0107804.e003" target="_blank">equation (1)</a> while fitting of (b) PIPO THG data for regions (i, ii, iii, iv, v, vi) were performed with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107804#pone.0107804.e008" target="_blank">equation (2)</a>. The contour plots shown of regions i and ii represent areas of intense SHG signal and no THG signal, regions iii and iv represent areas of intense THG signal and no SHG signal while regions v and vi represent areas of correlated SHG and THG signals.</p

PIPO THG data of an H-aggregate of astaxanthin.

<p>(a) THG image of an astaxanthin H-aggregate along with (b) the measured PIPO THG data and (c) the PIPO THG fit performed with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107804#pone.0107804.e008" target="_blank">equation (2)</a>.</p

Normalized absorbance spectra of astaxanthin and astaxanthin H-aggregates.

<p>The UV-Vis spectra of astaxanthin dissolved in methanol and astaxanthin dissolved in methanol in the presence of distilled water in a ratio of 1∶3 was recorded. The absorbance peak of astaxanthin dissolved in methanol and mixed with water resulted in a blue spectral shift which is indicative of the formation of H-aggregates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107804#pone.0107804-Khn1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107804#pone.0107804-Giovannetti1" target="_blank">[35]</a>.</p

PIPO SHG and PIPO THG fitting parameters of a red aplanospore of <i>Haematococcus pluvialis</i>.

<p>The fitting parameters pertaining to specific regions highlighted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107804#pone-0107804-g003" target="_blank">Fig. 3</a> of a red aplanospore were found. <i>R</i>² values for SHG fits were at least 0.94 and <i>R</i>² values for THG fits were at least 0.92.</p>a<p><i>R_THG1</i> was fixed between -2 and 2, and <i>R_THG2</i> was set as a free fitting parameter.</p><p>PIPO SHG and PIPO THG fitting parameters of a red aplanospore of <i>Haematococcus pluvialis</i>.</p

The orientations of coordinate frames in the experiment.

<p>The cylindrical axis (Z′) of a crystal (black cylinder) within a cell is oriented in the laser scanning plane (XZ) at an angle, <i>δ</i>, away from the laboratory Z-axis. The laser is directed perpendicularly (into the page) with the linear polarization oriented along <i>E</i>, at an angle, <i>θ</i>, from the Z-axis. The analyzer is oriented along <i>A</i>, at an angle, <i>φ</i>, from the Z-axis.</p

Nonlinear optical microscopy images of <i>Haematococcus pluvialis</i> at varying stages of light-stress.

<p>(a through h) palmella non-induced cells, (i through p) palmella induced cells and (q through x) aplanospores visualized by (a, e, i, m, q, u) MPF, (b, f, j, n, r, v) SHG, (c, g, k, o, s) THG and (d, h, l, p, t, x) structural image cross-correlation analysis between MPF, SHG and THG <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107804#pone.0107804-Barzda1" target="_blank">[16]</a>. The numbers in the upper left corner indicate the maximum photon counts in a pixel and the grey scale bar on the left side of the image ranges from zero to the maximum signal intensity. In general, a large THG intensity difference is seen between green-colored palmella non-induced cells and red aplanospores where, for example, the red aplanospore (s) is about 20 times more intense than the green-colored palmella non-induced cell (c) due to increased concentration of astaxanthin.</p

THG power dependency plot.

<p>THG images of osteocytes obtained at 1550 nm (a) and 1700 nm (b) excitation <i>in vivo</i> as well as a logarithmic plot (c) of the THG intensity of an osteocyte with fundamental laser power for both 1550 nm and 1700 nm excitation. The data was fit with a line corresponding to a slope of 2.8 ± 0.2 for 1550 nm excitation and 2.9 ± 0.2 for 1700 nm excitation.</p