| Background:The diagnosis of colorectal adenoma and carcinoma was performed by colonoscopy and endoscopic biopsy of suspicious lesions.It can easily lead to bleeding in the biopsy site.In addition,the biopsy may cause the submucosa fibrosis and adhesion,so that subsequent endoscopic treatment is prone to perforation,incomplete tumor dissection and other complications.Therefore,it is an urgent need for the clinicians to diagnose the colorectal mucosa at the cellular level in real time.Confocal laser endomicroscopy is an advanced real-time diagnostic technology,but it is a single photon microscopy imaging,the need for exogenous fluorescent substances and the existence of light bleaching and light toxicity are its limitations.Multiphoton microscopy imaging technologyis a technology thatstimulating biological fluorescence material[nicotinamide adenine dinucleotide(NADH)and flavin adenine dinucleotide(FAD)]to generate two-photon excited fluorescence effect(two-photon excitationfluorescence,TPEF)and collagen to generate second harmonic generation(SHG),so the optical sectioning of specimensand the cellular level of tissue imaging were achieved.Itsadvantages include no external fluorescent substance,high resolution,no light toxicity and light bleaching.With the development of colonoscopy and endoscopic therapy,endoscopic submucosal dissection(ESD)can be performed in the early stage of colorectal cancer and precancerous lesions.ESD postoperative specimens need to send for accurate pathology judgment.It needs to wait several days before pathology diagnosis result comes out,unable to real-time guide endoscopic therapy.Multiphoton microscopy imaging can effectively carry out non-destructive real-time optical biopsy,to judge the cutting edge and to guide endoscopic treatment in real time.Objective:1,Establishment of multiphoton imaging diagnostic criteria of colorectal lesions.2,Comparison of the sensitivity,specificity and accuracy of the confocal laser endomicroscopy and multiphoton microscopic imaging in diagnosis of benign and malignant colorectal lesions.3,Evaluation of the sensitivity,specificity and accuracy ofthe multiphoton microscopic imaging in real-time diagnosis of ESD specimens of early colorectal cancer.Methods:1.Colonoscopy was performed in 30 patients,biopsies after colon lesions have been found,the fresh biopsy tissue was performed multiphoton microscopy imaging,then the same specimen was performed HE staining.The HE diagnosis is regarded as the gold standard,comparing multiphoton imaging and HE pictures to determine the diagnostic criteria of colorectal lesions using the multiphoton imaging technique.2.The diagnosis of colorectal cancer was performed by confocal laser endomicroscopy and multiphoton microscopy,respectively and "blinded".Compare two kinds of optical diagnosis and HE diagnosis after "unblinded",calculate diagnostic sensitivity,specificity and accuracy of confocal laser endomicroscopy and multiphoton imaging.3.The specimens of early colorectal cancer which have been dissected by ESD were performed multiphoton microscopy imaging.Whether the incisal edge has residual lesions was judged by scanning the circular incial edge.The bottom edge of the specimen was scanned and imaged to determine whether there was infiltration of cancer cells at the bottom margin.Then the specimens were examined by pathology and compared with pathological results to determine the sensitivity,specificity and accuracy of the diagnosis.Results:1.Through30 patients research,multiphoton microscopy imaging diagnostic criteria of colorectal lesions was established.Multiphoton microscopy imaging of normal colorectal mucosa:1,Normal tissue structure and cell morphology;2,Typical small concave structure and circular recess opening can be seen in the central;3,The covering epithelial cells and goblet cells arranged regularly;4,The cell gap can be clearly seen;5,The nucleus is clearly visible,configuration is the same size;6,Basement membrane is integrity.Multiphoton microscopy imaging of colorectal adenoma:1,Iirregular tissue structure and cell morphology,2,Visible villous and tubular shape gland,3,Cells were still rules,no obvious mitotic;4,Basement membrane is exition.Multiphoton microscopy imaging of colorectal cancer:1,Abnormal arrangement of tissue structure and cell morphology;2,Basement membrane completely disappeared;3,Pleomorphic morphology ofcells and nuclei;4,Cell gap junction disappeared.2.Through 79 patients research:the sensitivity of confocal laser endomicroscopy was 61.54%(24/39),with a specificity of 67.50%(27/40),an accuracy of 64.56%(51/79),a positive predictive value of 64.86%(24/37),and a negative predictive value of 64.29%(27/42);the sensitivity of multiphoton microscopy was 89.74%(35/39),the specificity was 92.5%(37/40),the accuracy was 91.14%(72/79),the positive predictive value was92.11%(35/38),and the negative predictive value was 90.24%(37/41).3.Through50 patients research,the sensitivity of multiphoton microscopy imaging was 83.33%(5/6)and the specificity was97.73%(43/44)and the accuracy was 96%(48/50).Its positive predictive value was 83.33%(5/6)and the negative predictive value was 97.73%(43/44).Conclusion:1.Multiphoton microscopy imaging is a technology that stimulating autofluorescence substances inside colon mucosa,produce two-photon fluorescence effect,can realize "optical sectioning" of colorectal lesions,which can realize the diagnosis of cellular level,consistent with HE diagnosis.2.The sensitivity,specificity and accuracy of multiphoton microscopy imaging in the diagnosis of colorectal adenoma and colorectal cancer have an obvious advantage over confocal laser endomicroscopy.3.Multiphoton microscopy imaging is an effective real-time diagnosis of early colorectal cancer which have been dissected by ESD to determine if there is residual tumor and infiltration in its circular incisal edge and bottom incisal edge,it can guide the clinical treatment.Therefore,multiphoton endoscopy after realize miniaturization will be a powerful means of real-time in vivo diagnosis. |
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