Reseach On High Speed And Resolution Planar Flow Cytometric Microimaging Technology

With the increasing demand for higher detection speed and more inspection items of automated urinary analyzer,planar flow cytometric microimaging technology is developing towards high speed and resolution.In this paper,the detection speed of plane flow microscopic imaging system is increased from 60 to 120 samples per hour,and the test items of urine sediment are increased from 12 to 25 categories.It is easy to smear blur with the increase of detection speed,the acceleration of cell movement and the decrease of dynamic modulation transfer function(DMTF)value.With the increase of test items,the resolution of the micro imaging system is improved,but the depth of field becomes smaller,so it can't image all the formed elements in the sample laminar flow thickness clearly.At the same time,the tiny object distance change may cause defocusing,which will reduce the temperature adaptability of the system.This paper focuses on the high speed and clear imaging technology of low cost and high power LED light source,equivalent focusing focus technology based on focused particles,reference image method temperature compensation technology and depth of field expansion technology of dual sensor common optical path,to solve the problems of smear blur,difficult to focus,temperature defocusing and depth of field shortage caused by high speed and resolution.The composition and principle of high speed and resolution planar flow cytometry imaging system are described.The design of micro imaging optical system,illumination optical system and particle imaging room are completed.The design of micro imaging optical system,which is composed of objective lens and tube lens.According to the requirements of the measured object size and algorithm identification,the objective selection,tube lens optical design and image quality evaluation are carried out to ensure the high resolution imaging.The illumination optical system adopts Kohler illumination light path structure,to ensure the uniform high brightness illumination,both collection and condenser lens group's optical systems are designed and simulated.The laminar flow channel of particle imaging chamber adopts asymmetric structure,which is composed of flat channel wall,curved channel wall and two side channel walls.According to the depth of field,processing technology level and law of mass conservation,the fluid channel sizes of the particle imaging chamber are calculated.Through analysis and simulation,the arc-shaped surface with small curvature change of acceleration curve is selected as the curved channel wall surface,which makes the acceleration process of the sample smoother and ensures the formed elements to flow stably through the imaging area of the particle imaging chamber.The DMTF(Dynamic Modulation Transfer Function)model of high speed and resolution planar flow cytometry imaging system is established,and the dynamic imaging characteristics of the system are analyzed.According to the fact that DMTF of the system must be greater than 0.9 at Nyquist frequency,it is deduced that the exposure time of the system must be less than or equal to 1.15?s to meet the requirements of high speed dynamic clear imaging.The reasons why low cost and high power LED's response speed is slow,both LED constant brightness and stroboscopic exposure modes can not meet the requirement of short exposure are analyzed.A short exposure control method is proposed.The exposure time is determined by the interval between the rising edge of LED light emitting pulse and the falling edge of camera exposure pulse,and the exposure time of 1?s is achieved,which makes DMTF reach 0.93 at 90.9lp/mm.The method improves the dynamic imaging clarity,and realizes high speed and clear imaging with low cost and high power LED light source.The method based on focusing particles is used to realize equivalent focusing focus,which solves the problem that the high speed sample sequence images can not be used for auto focus by focusing depth method.The clear position of the focused particles in the focusing solution can be equivalent to the clear position of the sample.The image segmentation algorithm is used to extract the focused particle image in each frame,and only the focused particle image is evaluated,so as to reduce the impact of background information and improve the focus evaluation sensitivity.In order to reduce the influence of the shape and number difference of focused particles in different positions,10 photos are taken at each position in the search process,and the median value of multiple focused particle image focus evaluation values is taken as the final focus evaluation value,so as to improve the accuracy and repeatability of focus judgments.The ratio of high-frequency coefficient to low-frequency coefficient after wavelet transform is taken as the focus evaluation function.Because the edge shape of focused particles is easily affected by the liquid disturbance in the direction of motion,only the horizontal component of wavelet is extracted to eliminate the interference of vertical component,and the focus evaluation function based on wavelet transform is optimized.A low cost and easy to realize temperature compensation technology based on reference image is proposed to solve the problem of temperature defocusing caused by the small depth of field of micro imaging system.Firstly,the optical system design ensures that the imaging quality,focal length and image surface position meet the design requirements when the temperature changes,except for the object surface.Secondly,the influence of object distance is analyzed from two aspects when the temperature changes.The change of object distance is caused by the heat expansion and cold contraction of materials,and the change of optical path is caused by the change of refractive index of liquid with temperature.The mechanical passive method is used for preliminary temperature compensation to ensure that the defocus distance is reduced when the temperature changes.Finally,the reference image method is used to accurately compensate the change of object distance.Only a reference image for auto focus is attached to the particle imaging chamber,and the distance between the reference image and focusing particles' clearest positions is taken as the calibration length.When the system detects the temperature change,the focus depth method is used to automatically focus and find the clearest position of the reference image again,and the motor moves the calibration length to the focus position,so as to realize the temperature compensation of the plane flow cytometry imaging system.The laminar flow thickness of the measured sample is 3?m,while the depth of field of the system is only 1.85?m.The depth of field can not cover all imaging targets within the laminar flow thickness of the sample.The method of dual sensors common optical path is used to extend the depth of field,which uses dual sensors to collect images of different depth of field in two image positions,and then fuses two depth of field images into one large depth of field image.The implementation method is to add prism to the rear end of the tube lens optical system.After splitting the beam,the light beams are received by different sensors.The distance between the sensor and prism is adjusted to make the two sensors receive a slightly overlapped double depth of field image at the same time.The algorithm based on image multi scale decomposition and visual saliency detection is used to fuse the two images with different depth of field into one image with clear targets,which realizes large depth of field image reconstruction.The method can enlarge the depth of field to 3.3 ?m,realizes the clear imaging of all the formed elements in the whole laminar flow thickness,and solves the problem of insufficient depth of field caused by high resolution.This paper has completed the research on the key technologies of short exposure,auto focus,temperature compensation and depth of field expansion needed for the development of planar flow cytometry imaging system to high speed and resolution direction,which provides technical support for the production of high speed and resolution planar flow cytometry imaging system,and provides research direction and method for the future development to higher speed and resolution.It has important theoretical research value and practical guiding significance.