Research On Key Technology Of Large Field-of-view Biological Imaging System And Its Application

Optical microscopy is the most important observation method in life science,clinical medicine,pharmacy,et al.Nowadays,optical microscopy imaging technology has been developing towards obtaining more detailed information(high resolution)and wider information(large field-of-view).Field-of-view and resolution are two important parameters of an optical system,and its total amount of information is determined by both field-of-view and resolution.At present,in biomedical research,the field-of-view is developing from micro nano scale to larger scale.Commercial high-resolution microscopic imaging technology can not meet the demand.In life science research,such as rare cells and embryo detection,it is necessary to achieve micron resolution in centimeter level field-of-view,and obtain sample images containing hundreds of millions of pixels of information,which can greatly improve the detection efficiency.One of the telescope system,Off-axis Imaging System,has high spatial resolution imaging ability and large imaging field-of-view.So,in this thesis,we explored a method to apply unconventional optical imaging technology to microscopy imaging research.A series of basic research work has been carried out from optical system design,system performance test and evaluation,high-performance fluorescent probe development and application development.Including:1.Research on the bioimaging method and system designing.Firstly,the polychromatic fluorescence imaging methodology was designed,and the core technical parameters of the large field-of-view biological imaging system are analyzed and refined.A two-step imaging method combining large field-of-view preliminary positioning and small field-of-view accurate analysis was adopted.Secondly,taking the coaxial three mirror optical system as the initial structure,under the design principle of NA=0.1 and MTF reaching diffraction limit,a large field-of-view high-resolution off-axis three mirror reflective optical system with wide spectrum and achromatic imaging was designed,which has a resolution of 2.8μm,a field-of-view of 150 mm × 20 mm.Finally,the mutually independent arrangement scheme was accepted between the large and small field-of-view system,based on the analysis of the overall size,mechanical structure statics,ergonomics and other aspects.2.Research on preparation of the standard microspheres.It was used for testing the large field imaging system performance and evaluation.Monochromatic and polychromatic fluorescent microsphere calibrators were prepared by swelling method.The properties of the fluorescent microspheres were characterized by scanning electron microscope,flow cytometry,fluorescence spectrometer and fluorescence microscope,et al.Based on the above self-made standard fluorescent microspheres,the performance evaluation of the whole large field-of-view biological imaging system was finished.3.Research on exploring the application of large field-of-view biological imaging technology.Firstly,a new type of carbon quantum dot fluorescent probe with high biocompatibility and high brightness was designed and developed,and the synthesis conditions were optimized.The performance of the probe,combined with cytotoxicity experiment,was verified.Combined with cascade hybridized chain reaction,the high sensitivity detection of miRNA in cells based on image recognition was realized.This research can expand the application of the large field-of-view biological imaging system.Secondly,the large field-of-view biological imaging system was used to screen and select the large sample of abnormal embryos.Furthermore,based on zebrafish embryonic fibroblast model,the stress changes of transporters and metabolic enzymes under the simultaneous treatment of Ag+and Pb2+,as well as the regulatory role of nuclear transcription factors(Pxr and Nrf2)and miRNA(126 and 122)in this process were preliminarily studied,which laid the foundation for further research.This paper provided a valuable biological imaging technology which can simultaneously achieve large field-of-view and high resolution for life science research.It finished the preliminary application and lays the foundation for further application.