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In vivo quantification of the structural changes of collagens in a melanoma microenvironment with second and third harmonic generation microscopy.

Wu PC, Hsieh TY, Tsai ZU, Liu TM - Sci Rep (2015)

Bottom Line: The corresponding GLCM traces showed oscillation features and the sum of squared fluctuation VarGLCM increased with the tumor sizes.In addition, the THG intensities of the extracellular matrices increased, indicating an enhanced optical inhomogeneity.We believe these indices have the potential to help the diagnosis of skin cancers in clinical practice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Engineering, National Taiwan University, Taipei 10617, Taiwan.

ABSTRACT
Using in vivo second harmonic generation (SHG) and third harmonic generation (THG) microscopies, we tracked the course of collagen remodeling over time in the same melanoma microenvironment within an individual mouse. The corresponding structural and morphological changes were quantitatively analyzed without labeling using an orientation index (OI), the gray level co-occurrence matrix (GLCM) method, and the intensity ratio of THG to SHG (RTHG/SHG). In the early stage of melanoma development, we found that collagen fibers adjacent to a melanoma have increased OI values and SHG intensities. In the late stages, these collagen networks have more directionality and less homogeneity. The corresponding GLCM traces showed oscillation features and the sum of squared fluctuation VarGLCM increased with the tumor sizes. In addition, the THG intensities of the extracellular matrices increased, indicating an enhanced optical inhomogeneity. Multiplying OI, VarGLCM, and RTHG/SHG together, the combinational collagen remodeling (CR) index at 4 weeks post melanoma implantation showed a 400-times higher value than normal ones. These results validate that our quantitative indices of SHG and THG microscopies are sensitive enough to diagnose the collagen remodeling in vivo. We believe these indices have the potential to help the diagnosis of skin cancers in clinical practice.

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Related in: MedlinePlus

The (a) OI, (b) GLCM trace, (c) VarGLCM, and (d) RTHG/SHG of harmonic generation images after subcutaneous injection of HA.The areas with dense blue lines indicate the range of baseline values. The bars indicate the variation among different depth.
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f7: The (a) OI, (b) GLCM trace, (c) VarGLCM, and (d) RTHG/SHG of harmonic generation images after subcutaneous injection of HA.The areas with dense blue lines indicate the range of baseline values. The bars indicate the variation among different depth.

Mentions: In the control study, for each normal mouse, we acquired SHG images over six different zones (zone1-6), in which we obtained 10 layers (0–18 μm) of imaging stacks at three different locations. In the quantitative analysis of the SHG and THG images, we avoided sebaceous glands (Fig. 3), vessel cavities (Fig. 3), and the bulky tumor regions where collagens were absent. Among the different normal mice, the overall average OI values were approximately 38% (Fig. S2a, p > 0.05). However, the standard deviations for the OIs in each mouse were large. This large deviation was caused by the large regional variation of the OI, which ranged from 20% to 45% (Fig. S2b). We found that zones 5 and 6 had significantly lower OI values than the other zones (p < 0.05). Even within the same zone, some of the standard deviations for particular zones were still large. From the skin surface down to 18 μm deep, there is no significant depth variation in the OI value (Fig. S2c). These results indicate that regional variation predominately produces the baseline variation of the OI values in the normal control mice. The relative variation of the means among different zones was approximately ±16%. To represent the range of the baseline values, we plotted an area with dense blue lines in the analysis (Fig. 7a).


In vivo quantification of the structural changes of collagens in a melanoma microenvironment with second and third harmonic generation microscopy.

Wu PC, Hsieh TY, Tsai ZU, Liu TM - Sci Rep (2015)

The (a) OI, (b) GLCM trace, (c) VarGLCM, and (d) RTHG/SHG of harmonic generation images after subcutaneous injection of HA.The areas with dense blue lines indicate the range of baseline values. The bars indicate the variation among different depth.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352861&req=5

f7: The (a) OI, (b) GLCM trace, (c) VarGLCM, and (d) RTHG/SHG of harmonic generation images after subcutaneous injection of HA.The areas with dense blue lines indicate the range of baseline values. The bars indicate the variation among different depth.
Mentions: In the control study, for each normal mouse, we acquired SHG images over six different zones (zone1-6), in which we obtained 10 layers (0–18 μm) of imaging stacks at three different locations. In the quantitative analysis of the SHG and THG images, we avoided sebaceous glands (Fig. 3), vessel cavities (Fig. 3), and the bulky tumor regions where collagens were absent. Among the different normal mice, the overall average OI values were approximately 38% (Fig. S2a, p > 0.05). However, the standard deviations for the OIs in each mouse were large. This large deviation was caused by the large regional variation of the OI, which ranged from 20% to 45% (Fig. S2b). We found that zones 5 and 6 had significantly lower OI values than the other zones (p < 0.05). Even within the same zone, some of the standard deviations for particular zones were still large. From the skin surface down to 18 μm deep, there is no significant depth variation in the OI value (Fig. S2c). These results indicate that regional variation predominately produces the baseline variation of the OI values in the normal control mice. The relative variation of the means among different zones was approximately ±16%. To represent the range of the baseline values, we plotted an area with dense blue lines in the analysis (Fig. 7a).

Bottom Line: The corresponding GLCM traces showed oscillation features and the sum of squared fluctuation VarGLCM increased with the tumor sizes.In addition, the THG intensities of the extracellular matrices increased, indicating an enhanced optical inhomogeneity.We believe these indices have the potential to help the diagnosis of skin cancers in clinical practice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedical Engineering, National Taiwan University, Taipei 10617, Taiwan.

ABSTRACT
Using in vivo second harmonic generation (SHG) and third harmonic generation (THG) microscopies, we tracked the course of collagen remodeling over time in the same melanoma microenvironment within an individual mouse. The corresponding structural and morphological changes were quantitatively analyzed without labeling using an orientation index (OI), the gray level co-occurrence matrix (GLCM) method, and the intensity ratio of THG to SHG (RTHG/SHG). In the early stage of melanoma development, we found that collagen fibers adjacent to a melanoma have increased OI values and SHG intensities. In the late stages, these collagen networks have more directionality and less homogeneity. The corresponding GLCM traces showed oscillation features and the sum of squared fluctuation VarGLCM increased with the tumor sizes. In addition, the THG intensities of the extracellular matrices increased, indicating an enhanced optical inhomogeneity. Multiplying OI, VarGLCM, and RTHG/SHG together, the combinational collagen remodeling (CR) index at 4 weeks post melanoma implantation showed a 400-times higher value than normal ones. These results validate that our quantitative indices of SHG and THG microscopies are sensitive enough to diagnose the collagen remodeling in vivo. We believe these indices have the potential to help the diagnosis of skin cancers in clinical practice.

Show MeSH
Related in: MedlinePlus