Research On The System Structure Engineering Design Of Multispectral Fluorescence Endomicroscopy

Purpose The incidence of gastrointestinal cancer remains high,and early detection and early treatment are the key strategies to improve the survival rate of cancer patients.Digestive endoscopy provides an effective method for diagnosing gastrointestinal tumors.However,the existing digestive endoscopy system still has difficulties in detecting small lesions and unclear recognition of lesion boundaries,the intraoperative immediate microscopic imaging technology is helpful to realize the immediate intraoperative pathological diagnosis and is expected to improve the accuracy of diagnosis.Our research team built a prototype prototype of Multispectral Fluorescence Endomicroscopy(MFE)based on the independently developed High-resolution Microendoscopy(HRME),the purpose of this study is to further engineer and upgrade on the basis of the MFE principle prototype,optimize existing MFE equipment,and explore the MFE engineering prototype function and the feasibility of multi-target molecular imaging.Methods 1.Based on the principle of MFE imaging,through the design of light source module,multi-spectral module,image acquisition and other modules,the design of mechanical and electrical control structures,carry out the engineering design research on MFE to realize the development of MFE from principle prototype to engineering prototype.2.Ten nude mice were selected and anesthetized with chloral hydrate.Among them,5nude mice were injected with 100 ?L sodium fluorescein solution through the tail vein.After 5 minutes,they were dissected for MFE imaging,and the other 5 were subjected to confocal laser endomicroscopy(CLE)imaging after the same operation.Observe and compare the imaging results of gastric mucosa,gastric antrum mucosa,small intestine mucosa,colon mucosa,liver tissue.3.Immunohistochemistry(ICH)method was used to detect the expression levels of CD44,Matrix Metalloproteinases 9(MMP 9),Vascular Endothelial Growth Factor(VEGF),Epidermal Growth Factor Receptor(EGFR)in SGC 7901 gastric cancer cells,after the software evaluates the respective H-score(Histochemical Score,histochemical score),two of the highest scores were selected to construct the fluorescent probes with fluorescent dyes AF680 and Fluorescein Isothiocyanate(FITC).The probes are:FITC-anti-EGFR group,AF680-anti-MMP 9 group,and the control group is Phosphate Buffered Saline(PBS)group.After the probe was incubated with SGC 7901 gastric cancer cells,the MFE and fluorescence microscopic imaging were performed to evaluate the feasibility of multi spectral molecular imaging of the engineering prototype at the cellular level;in order to evaluate the feasibility of multi spectral molecular imaging of the engineering prototype at the animal living level,construct a nude mouse subcutaneous xenograft tumor model,spray the probe on the surface of the subcutaneous xenograft tumor with MFE for imaging,and evaluate the feasibility of the multi spectral molecular imaging of the engineering prototype.Results 1.Through the module design,appearance design,opto-mechanical integration design of MFE,the engineering prototype of MFE was successfully constructed,the operation steps were simplified,and the engineering upgrade was completed.2.MFE imaging results are similar to the results of CLE imaging,and endoscopic microscopic imaging can be achieved.3.The results of H-score indicate that MMP 9 and EGFR are highly expressed in SGC7901 gastric cancer cells and can be used as targets for molecular imaging.Cell experiments showed that there were obvious fluorescence signals on the surface of tumor cells in AF680-anti-MMP 9 group and FITC-anti-EGFR group.The mean fluorescence intensity(Mean Fluorescence Intensity,MFI)was 69.591±5.699 ?61.174±8.821,respectively;there was no obvious signal in the PBS group,the results were 27.311 ± 3.78?32.087 ± 5.214 respectively.Under the fluorescence microscope,the blue stained tumor cell nucleus and the green or red fluorescent signals on the cell surface can be seen,but there is no obvious signal on the cell surface of the corresponding PBS group.The MFE image after spraying the probe on the surface of the subcutaneous xenograft tumor is characterized by a bright fluorescent signal,which shows the general shape of the tumor.After local amplification,the signal is star shaped and evenly distributed on the surface of the tumor.In the FITC-anti-EGFR group and AF680-anti-MMP 9 group,the MFI of the subcutaneous xenograft tumor was 76.886 ±6.748 and 79.831±4.43 respectively;the corresponding PBS group had no clear signal,the results were 42.976±4.592 and 36.086±2.042 respectively.The results of hematoxylin eosin staining(HE)showed that the subcutaneous xenograft tumor was gastric cancer,and the expression of EGFR and MMP-9 was also detected in ICH.The ratio between the above two probe groups and the corresponding two PBS groups are different,and has statistical significance(P <0.05).Conclusions 1.Successfully constructed the MFE engineering prototype imaging system and tested its imaging function to realize the optimization and upgrade of MFE from the principle prototype to the engineering prototype.2.Select two targets of EGFR and MMP 9 to construct fluorescent probes,and use MFE engineering prototype to achieve molecular imaging of different targets of SGC 7901 gastric cancer cells at the cellular level,it proves the feasibility of MFE engineering prototype at the cellular level for multispectral molecular imaging.3.Successfully implemented the MFE engineering prototype to image fluorescent molecules of two targets of subcutaneously transplanted tumors of gastric cancer,the results show that the MFE engineering prototype has the feasibility of simultaneous multispectral molecular imaging in vivo and real-time immunohistochemistry in vivo.