Two-photon Microscopic Imaging Technology For Noninvasive Detection Of Dermatofibrosarcoma Protuberans

Objective To investigate the potential of two-photon microscopy used in the noninvasive examination of dermatofibrosarcoma protuberans(DFSP)in order to provide a new laboratory examination method for the diagnosis of DFSP.Methods Paraffin-embedded sections of the diseased and normal skin tissues from 5 cases of patients with DFSP selected from Fujian Medical University Union Hospital from January 2016 to December 2016 were further investigated by two-photon microscopic imaging technique.Firstly,we described the TPEF images of epidermis from the normal and diseased skin tissues of DFSP,compared and analyzed the structure and composition of them;secondly,we described the TPEF images of superficial dermis from the normal skin,superficial and deep dermis from diseased skin tissue of DFSP and subcutaneous tissue from the normal skin and diseased skin tissue of DFSP,compared and analyzed the morphology and quantity of collagen fibers of the above four tissues;thirdly,quantitative analysis was promoted to analyze the volume density of collagen fibers by MATLAB software,and the data was processed by SPSS 21software;finally,in order to identify the DFSP and keloid,we refer to past scholars,data by quantitative analysis of he volume density of collagen fibers.Results1.The epidermis of normal and diseased skin tissue of DFSP and the keratin and keratinocytes of the epidermis could be significantly distinguished by the two-photon microscopic imaging technique.The cytoplasm showed red fluorescence,nucleus showed no fluorescence(dark),and the nucleolus showed visible red fluorescence in the image of two-photon microscopy.And all cells appeared in the same size and regularmorphology.There was no significant difference in structure and composition between the two groups which indicated that the lesions did not affect the epidermis.2.From the TPEF images of superficial dermis from normal skin tissue,we could see that the collagen fibers were bunchy with no certain direction,the elastic fibers were wavy or funicular winding among the collagen fibers and they were thinner than collagen fibers.From the TPEF image of superficial dermis from the diseased skin tissues of DFSP,we could still see the collagen fibers and elastic fibers which were the same as in the images of superficial dermis from normal skin tissue.Considering the morphology,the collagen fibers in the superficial dermis of DFSP lesions were similar to those in the normal superficial dermis.From the TPEF images of deep dermis from normal skin tissue,we could see the normal collagen fibers and elastic fibers.The TPEF signal in the deep dermis of DFSP lesions mainly came from the collagen fibers and spindle cell cytoplasm.From the image we could see that the collagen fibers in deep dermis of DFSP lesions lose the bunchy shape and were arranged loosely in a certain direction and there was no elastic fibers.The nucleus showed no fluorescence(dark)and was elongated as that in spindle cell.From the subcutaneous tissue of the normal skin,we could see that the fat cells arranged closely with the same size,and the shape was regular.From the TPEF image of subcutaneous tissue from the diseased skin tissue of DFSP,we could see the fat cells with no fluorescence(dark in the image).They were round or oval and arranged loosely between each other,their size was different,and the shape was regular.3.In the first group,the volume density of collagen fiber in superficial dermis of normal skin tissue was 27.02±4.47%.And in the second group,the volume density of collagen fiber in superficial dermis of diseased skin tissue of DFSP was 26.07 ± 3.79%.In the third group,the volume density of collagen fiber in deep dermis of normal skin was 25.60±6.47%.In the fourth group,the volume density of collagen fiber in deep dermis of diseased skin tissue of DFSP was 43.57 ± 3.67%.In the comparison of the first and second group,there was no statistically significant difference between two groups(Mann-Whitney test,P>0.05).In the comparison of the third and fourth group,there was significant difference between two groups(Mann-Whitney test,P?0.05).4.Quantitative analysis was promoted to analyze the volume density of collagen fibers of the superficial and deep dermis of DFSP and keloid,we could found that there was significant difference between two groups(Mann-Whitney test,P?0.05).Conclusion Two-photon microscopic imaging technology can be used for the noninvasive examination of DFSP which can provide a new laboratory method for the diagnosis of DFSP.The volume density of collagen fiber can be used for quantitative analysis of collagen fibers,an argument for laboratory method of DFSP and a target for differential diagnosis of DFSP and keloid.