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.
Highlights
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
Through quantitative analyses of the SHG and THG in vivo images, we found that the collagen structures have an obvious regional variation in the normal control mice
After implanting hyaluronic acid (HA) in zone 5, there was a significant increase in the orientation index (OI) at the same location in the first 2 weeks, which may have been caused by inflammation
Summary
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. We used the following three different methods (See Methods section) to quantitatively analyze the collagen remodeling in vivo: the orientation index (OI)[34,43,44], the gray level cooccurrence matrix (GLCM)[35,45,46,47,48], and the ratio of the THG to the SHG (RTHG/SHG) We found these indices have significant changes over baseline variations, which together can potentially provide an effective diagnosis of the developmental stages of melanoma-associated collagens
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