| Light sheet fluorescence microscopy uses a light sheet to excite samples with good optical layer selection ability and large excitation area,which replaces the slow point scan imaging methods.It has the advantages of fast imaging acquisition rate,minimal phototoxicity and photodamage effect,high signal-to-noise ratio,high threedimensional spatial resolution and so on.It is suitable for long-term three-dimensional imaging in vivo and has been widely used in biomedical research.With the development of biological research,the imaging function of the microscope is becoming more and more demanding.Combining a variety of microscopic imaging results makes the study and analysis of samples more comprehensive and effective.In addition,due to the drawbacks of living anesthesia imaging and the limitations of traditional large-scale microscopic imaging devices,miniaturized microscopes for in vivo animal imaging,endoscopic imaging,etc,are also being developed.Focusing on the light sheet fluorescence microscopy and combining with the specific application requirements,this paper carried out related research work in the design and construction of the microscopy imaging systems,test analysis and imaging experiments.The main content and contribution of this paper are as follows:(1)Study on widefield and light sheet bimodal fluorescence microscopy imaging system.In order to realize wide field excitation and light sheet excitation,a widefield and light sheet bimodal fluorescence microscopy imaging system was designed and built,and the two excitation paths were integrated into the same imaging system.Based on the imaging principle of wide field illumination and light sheet illumination,and using the optical imaging theory of beam expander and spatial filter,the optical path of the imaging system was designed and constructed into four parts:laser generation and beam expanding and shaping light path,wide field excitation light path,light sheet excitation light path,imaging light path,and the imaging test and analysis of the system were carried out.The system tests and imaging experiments show that the imaging system with a 10× imaging objective lens has a 7.28μm thick excitation light sheet,an effective field of view of 844μm×706μm,a high lateral resolution of 1.23 ± 0.02μm and a high contrast light sheet excitation image,which is 65%better than wide field excitation image.(2)Study on miniature light sheet fluorescence microscopy imaging system.In order to miniaturize the light sheet fluorescence microscopy,a high resolution and contrast light sheet fluorescence microscopy imaging system was designed and manufactured.Based on the theory of light sheet generation and the imaging principle of light sheet illumination,and using the 4f imaging system and other optical imaging theories,the imaging light path was designed and simulated.The whole imaging system is designed and built into five units,including control unit,laser and coupling unit,illumination unit,detection unit,and movement unit.The imaging probe housing was designed and 3D-printed,and the illumination unit and detection unit were integrated into a lightweight,compact and easy to implement miniature imaging probe with a weight of about 3.28g and a volume of 23.50mm × 9.00mm × 38.70mm using miniature lens.The experimental results show that the imaging system can perform high resolution and high contrast tomographic imaging with large imaging field of view.Among them,the magnification of the system is 6.74 times,the lateral resolution is 1.11±0.01μm,the maximum imaging field of view is 245μm×245μm,the imaging frame rate is 15fps,and the excitation light sheet thickness is 5.35μm.The contrast of the light sheet excitation imaging is 295%higher than the wide field excitation imaging. |
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