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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

Two-photon fluorescence spectra of (a) melanoma cells, (b) medium before culture, (c) medium after culture, and (d) pure melanin.The narrow peaks around 417 nm and 625 nm are the THG of the glass-solution interfaces and the SHG of aggregated materials on glass, respectively.
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f2: Two-photon fluorescence spectra of (a) melanoma cells, (b) medium before culture, (c) medium after culture, and (d) pure melanin.The narrow peaks around 417 nm and 625 nm are the THG of the glass-solution interfaces and the SHG of aggregated materials on glass, respectively.

Mentions: In order to locate melanoma cells in vivo and analyze surrounding microenvironment, we need a fluorescence contrast specific to melanoma cells. Using the nonlinear optical microscopy to observe, the melanoma cells revealed an intrinsic THG49 and TPF contrasts with a granular appearance (Fig. 1). The TPF spectrum of the granules peaked at approximately 680 nm (Fig. 2a). This granular red autofluorescence was much weaker in the absence of melanoma cells (Fig. 2b, serum only). Collecting the culture media right above the melanoma cells, the secreted granules also had the same TPF spectrum (Fig. 2c). Both of these melanoma-related TPF spectra had peak wavelengths close to that of commercial melanin (Fig. 2d). These results suggest the granules might be melanosomes encapsulating melanin. In the following experiments, we exploited this characteristic TPF of melanin to track the melanoma cells in vivo.


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)

Two-photon fluorescence spectra of (a) melanoma cells, (b) medium before culture, (c) medium after culture, and (d) pure melanin.The narrow peaks around 417 nm and 625 nm are the THG of the glass-solution interfaces and the SHG of aggregated materials on glass, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Two-photon fluorescence spectra of (a) melanoma cells, (b) medium before culture, (c) medium after culture, and (d) pure melanin.The narrow peaks around 417 nm and 625 nm are the THG of the glass-solution interfaces and the SHG of aggregated materials on glass, respectively.
Mentions: In order to locate melanoma cells in vivo and analyze surrounding microenvironment, we need a fluorescence contrast specific to melanoma cells. Using the nonlinear optical microscopy to observe, the melanoma cells revealed an intrinsic THG49 and TPF contrasts with a granular appearance (Fig. 1). The TPF spectrum of the granules peaked at approximately 680 nm (Fig. 2a). This granular red autofluorescence was much weaker in the absence of melanoma cells (Fig. 2b, serum only). Collecting the culture media right above the melanoma cells, the secreted granules also had the same TPF spectrum (Fig. 2c). Both of these melanoma-related TPF spectra had peak wavelengths close to that of commercial melanin (Fig. 2d). These results suggest the granules might be melanosomes encapsulating melanin. In the following experiments, we exploited this characteristic TPF of melanin to track the melanoma cells in vivo.

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