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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

In vivo (a) SHG, (b) THG, and (c) combined imaging of a normal mouse ear.Fields of view: 240 μm × 240 μm.
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f3: In vivo (a) SHG, (b) THG, and (c) combined imaging of a normal mouse ear.Fields of view: 240 μm × 240 μm.

Mentions: Our nonlinear optical microscopy system can perform in vivo sectioning images of mouse skin without a label505152. The imaging depth in skin tissues can be as deep as 350 μm. In general, SHG microscopy reveals structural proteins like collagen fibers (Fig. 3a). The intensity is dependent on the density of the collagen, the orientation of the fibers, and the laser polarization. In contrast, THG contrasts are sensitive to optical inhomogeneities1653. They have been used to reveal adipocytes51, immune cells52, keratinocytes, basal cells49, elastin fibers54, and red blood cells (RBCs)55. The modality of THG is also sensitive to lipids56. An ultra-strong THG contrast can be found with sebaceous glands due to the high lipid content of these tissues (Fig. 3b). In blood vessels, the SHG signal is absent, and we can observe circulating RBCs with the THG contrast (Fig. 3).


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)

In vivo (a) SHG, (b) THG, and (c) combined imaging of a normal mouse ear.Fields of view: 240 μm × 240 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: In vivo (a) SHG, (b) THG, and (c) combined imaging of a normal mouse ear.Fields of view: 240 μm × 240 μm.
Mentions: Our nonlinear optical microscopy system can perform in vivo sectioning images of mouse skin without a label505152. The imaging depth in skin tissues can be as deep as 350 μm. In general, SHG microscopy reveals structural proteins like collagen fibers (Fig. 3a). The intensity is dependent on the density of the collagen, the orientation of the fibers, and the laser polarization. In contrast, THG contrasts are sensitive to optical inhomogeneities1653. They have been used to reveal adipocytes51, immune cells52, keratinocytes, basal cells49, elastin fibers54, and red blood cells (RBCs)55. The modality of THG is also sensitive to lipids56. An ultra-strong THG contrast can be found with sebaceous glands due to the high lipid content of these tissues (Fig. 3b). In blood vessels, the SHG signal is absent, and we can observe circulating RBCs with the THG contrast (Fig. 3).

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