Research On High-throughput Gene Sequencing Fluorescence Microscopic Imaging Optical System

With the rapid development of life science,computer science and information technology,bioinformatics came into being and became one of the most valuable,beneficial and focused research topics in the fields of human health,disease prevention,drug research,and life exploration.Bioinformatics is based on the collection,processing,storage,analysis and interpretation of massive biological information.The most important means of biological information collection is microscopic imaging technology,among which high-throughput fluorescence microscopic imaging system is the most powerful tool.High-throughput fluorescence microscopic imaging system relies on high-density biochip,high-throughput optical system,and high-throughput image sensor.Biochip fabrication technology is now highly mature.Millions of information points can be stored in each square millimeter on a biochip.High-throughput imaging sensor is also in rapid progress.However,commercial microscope develops slowly in the field of high-throughput technique.Optical systems have become the bottleneck of high-throughput fluorescence microscopic imaging technology,which cannot meet the needs for high-throughput collection of biological information in bioinformatics applications,such as high-throughput screening and high-throughput sequencing.This thesis takes high-throughput gene sequencing fluorescence microscopic imaging optical system as the research object,and conducts the research from the aspects of the requirements of high-throughput technology to the optical system,high-throughput microscope objective,high-precision focusing optical unit,laser illuminating optical unit and fluorescence imaging simulation.The main research results are shown as follows:1.High-throughput implementation methods and requirements on the optical system.Proceeded from basic conception of information throughput at first,five ways to improve the system throughput are discussed: increasing the numerical aperture,enlarging the field of view,extending the fluorescence channel,improving the sample rate and optimizing the image sampling;Then high-throughput fluorescence microscopic imaging requirements to optical systems are discussed,including microscope objectives,focusing unit and illuminating systems.2 High-throughput microscope objective.The liquid-solid immersion optical structure based on a doublet lens is proposed to solve the problem that large refractive index deviation of the immersion liquid of the high-throughput gene chip without the cover glass affects the image quality.A high-throughput optical system of TDI imaging mode and one with staring imaging mode are designed with this optical structure,and their Lagrange invariants are all over 2.In the staring imaging mode,optical structure with the exit pupil outside of the objective is designed which reduces the requirements on the size of filters and tube lens for high numerical aperture and large field of view imaging.3 High accuracy autofocus method.Based on the requirements of the autofocusing unit for high-throughput fluorescence microscopic imaging,by applying laser differential confocal principle,an extremely fast and high accuracy autofocus method is proposed which provides autofocus accuracy superior to 1/10 objective depth of focus.And a multi-position laser differential confocal method is proposed to extend the working range up to several times of the depth of focus.The degradation of optical system aberrations to autofocus shows that aberration compensation is needed to reduce spherical aberration,coma and astigmatism.Then an athermal collimating lens is designed to adapt with the temperature variation in working environment.4.Laser illuminating system.Based on the requirements of the illuminating system for high-throughput fluorescence microscopic imaging,the critical epi-illumination path is designed to achieve high uniformity for large field of view.Aiming at the characteristics of high reflectivity of polished substrate of the gene chip,a new illuminating method based on total internal reflection is proposed by use of a customized prism,which can effectively suppress the laser background and overcome the structural constraints of high numerical aperture and short working distance microscopic objective.The design and analysis results are given.5.Multi-channel fluorescence imaging simulation and illumination laser power optimization.Spectral crosstalk exists in the optical system of multi-channel fluorescence microscopy imaging.Firstly,the calculation method of multi-channel fluorescence energy link is introduced,and the simulation model of fluorescence imaging is established.By analyzing the characteristics of multi-channel fluorescence image,the power optimization method of multi-wavelength laser is established based on the signal-to-noise ratio of simulation image.The method is applied to optimize the power of a four-color laser in high-throughput sequencer.And the correctness of the method is verified by fluorescence imaging test.