Preliminary Researches On Histomorphology Of Female Reproductive Organs And Diseases With Diffractive Optical Techniques

Objective:Women’s health is the cornerstone of national health.The development of gynecology is also closely related to gynecology histopathology.At present,the auxiliary examination methods used in gynecology mainly include gynecological ultrasound,computer tomography（CT）and magnetic resonance imaging（MRI）.The"gold standard"of disease diagnosis mainly depends on the results of pathological examination of postoperative tissues.With the progress and development of science and technology,the diagnosis of the disease only through pathological diagnosis after surgery is slightly single and weak.Nowadays,gynecology and obstetrics urgently need a technology that can quickly and intuitively display the tissue structure without staining to observe the tissue structure and diagnose diseases.Adenomyosis refers to the invasion of endometrial glands and stroma into the myometrium and the maintenance of functional changes such as periodic hyperplasia,exfoliation and bleeding that can be divided into two types:focal and diffuse.The uterus is uniformly enlarged in diffuse adenomyosis.Focal lesions,known as adenomyomas,grow locally and have no obvious boundary with surrounding tissue which makes them difficult to resect during surgery.If a non-invasive diagnostic tool can be found to clearly identify the adenomyosis focus with surrounding tissues,the Uterine-sparing surgery with adenomyosis focus stripping may be realized.In recent times,utilizing engineering technology alongside medicine has proven popular.Among them,the application of optical microscopy in medicine is emerging.Compared with the time-consuming traditional H&E（Hematoxylin-eosin）staining method,the biggest advantage of optical microscopy lies in its convenience,efficiency and label-free.In this experiment,we try to observe the microstructure of normal cervix,myometrium,endometrium,fallopian tube lumen and fimbria oviduct using the second-harmonic generation（SHG）microscope and Coherent Anti-Stokes Raman scattering（CARS）microscope.Combined with Raman microscope,the diagnosis of adenomyosis was carried out to identify the lesion boundary and provide the possibility for adenomyosis exfoliation.Methods:We randomly selected patients who underwent surgery in the First Affiliated Hospital of Dalian Medical University.After cutting off the uterus during the operation,Normal patients need to provide cervical,myometrium,endometrium,fallopian tube lumen and fimbria oviduct,while patients with adenomyosis need to provide samples of adenomyosis focus tissue and normal tissue outside the focus.The tissues will be placed in liquid nitrogen tanks for cold storage,in order to preserve the cell activity for the convenience of subsequent experiments.All patients signed the informed consent form under the informed consent of the research process after surgery,allowing us to conduct experiments on their in vitro tissues.This experiment was certified by the ethics Association of the First Affiliated Hospital of Dalian Medical University.After sample preparation,the tissues were sliced about 1x1 cm in size and made continuous frozen sections for each tissue.From each tissue,three 10μm slices were cut.The three slices of the same tissue were numbered（1）,（2）,and（3）.All“Slices（1）”underwent H&E staining,then were diagnosed by two experienced pathologists and photographed under the microscope.And Slices（2）and（3）were directly observed under a nonlinear optics microscope without any staining treatment.Results:In the first phase of the experiment,SHG and CARS microscopes were used to explore the imaging effect of the microstructure of the cervix,myometrium,endometrium,fallopian tube lumen and fimbria oviduct.We found that SHG microscopes were good at imaging the microstructure of the tissues rich in collagen fibers such as cervix,myometrium and fallopian tube,clearly showing the microstructure outline and the distribution of collagen fibers in the tissues.On the contrary,CARS signals are strongly reflected in uterine glands,endometrium and blood vessels.In the second phase of the experiment,the diagnostic effect of SHG,CARS microscope and Raman microscope on adenomyosis was discussed.Second-harmonic generation（SHG）microscope presents the outline of the ectopic uterine gland perfectly,and clearly shows the distribution of collagen around the lesion.It can confirm the proliferation of fibro-collagen caused by bleeding around adenomyosis lesions.unlike SHG imaging,CARS microscopy showed a stronger intensity in the glandular portion of the uterus.This also proves that the part of the uterine gland not shown in SHG is not a blank cavity,but there is a non collagen fiber material,such as mucus secreted by the gland.Raman microscope showed the characteristic spectrum of pathological tissues and normal tissues,in the range of 900–1600 cm-1,the characteristic wave number of adenomyosis is more obvious,and allows the clear distinction of the Raman spectrum curve of normal tissue and adenomyosis.In the following experiment,we used adenomyosis tissues from different patients,and the characteristic peaks appeared at 1155cm^-1 and 1519 cm^-1.Conclusion:In this study,SHG and CARS microscopes were used to investigate the imaging effect of the microstructure of cervix,myometrium,endometrium,fallopian tube lumen and fimbria oviduct,and to clarify the exploration value of diffraction optical microscopy in gynecological histology.Then,a new way to study adenomyosis by combining SHG,CARS and Raman microscope is proposed,which proves the role of nonlinear optics in the diagnosis of adenomyosis and the differentiation of lesion boundaries.