| With the rapid development of the interdisciplinary field of medicine and industry,micromanipulation for all kinds of micro-organisms,including micro-imaging and micro-injection technology,has been widely used in life science research.However,the performance of traditional micro-imaging and micro-injection techniques will be severely degraded or not applicable in many micromanipulation experiments with complex environmental conditions and object states.Therefore,it is necessary to make intelligent improvement on the shortcomings of these traditional methods to make them capable of dealing with all kinds of complex situations.As a typical vertebrate model organism,zebrafish has been widely used in biological research.But on the other hand,its operation method is also relatively complex.Its unique advantages include its high genetic similarity to humans,the optical transparency of embryos and larva body,and its rapid developmental process.In experiments,it is often necessary to utilize the transparency property of the larva body to conduct imaging to observe the changes in its development,so as to analyze the influence of drugs.In high-throughput studies,large numbers of larvae need to be observed and analyzed.In addition,most biological agents cannot be absorbed directly from the water by larvae,and consequently microinjection is needed.For the above two problems,traditional manual observation and injection are difficult to meet the needs of efficiency and accuracy,so it is necessary to develop automated imaging and injection systems to replace manual methods.Although some automated techniques have been proposed in recent years,the existing ones still have some shortcomings in terms of stability and automation due to the influence of various unavoidable complex conditions.Specifically,the complex conditions in the above operations mainly include the following three aspects.The first is the complicated object posture.Compared with spherical objects such as cells and embryos that can be rotated arbitrarily,the control of different degrees of freedom and overlap between different individuals should be considered in the operation of zebrafish larvae.The second is the unknown initial states of the system,including the initial compression state of the air pump and the initial roll angle of the larvae in the imaging experiment.The third is environmental changing,including changes in water surface height during the injection experiment and disturbances caused by the life activities of larvae.In this paper,solutions are proposed to solve the problems in the automated zebrafish larva imaging and injection systems under these three complex conditions.The main contents of this paper include:To solve the problem of automated zebrafish larva loading under complex object attitude,in this paper,a larva orientation adjustment method based on microfluidic technology is proposed,which can adjust the larvae entering the microfluidic chip to be tail-first one by one for subsequent operation.In addition,considering that larvae can stick to each together,in this paper,a scheme is proposed to automatically load multiple randomly placed zebrafish larvae in the system with specific orientation one by one.The scheme can identify and separate the aggregated larvae,and the isolated ones can be aspirated into the pipette,thus improving the stability of automated larva loading.For another scheme of rotating larvae in an open-ended container,this paper determined the desired trajectory of the larva through force analysis,and the larva could be accurately rotated to the desired angle with the help of visual feedback.To solve the problem of automated orientation control of zebrafish larva in the microimaging system under unknown initial state,this paper firstly built a dynamic model for the movement of larvae controlled by air pump,and designed an adaptive robust controller to accurately move the larva to the desired observation position.In addition,this paper proposes a method to automatically identify the current rotation angle of the larva and rotate it to the desired orientation,thus improving the imaging efficiency.To solve the problem of contact detection for automated injection of zebrafish larvae under changing environment,a method was proposed to accurately move the needle tip to the injection position on the epidermis of the zebrafish larva.Considering that there is a certain inclination of the line of sight when using stereomicroscope to capture images,the refraction at the water surface cannot be ignored,and the relationship between the image coordinate and the manipulator coordinate will also change when the water depth changes,which makes it difficult to move the tip to the injection site accurately.In this paper,an adaptive calibration method is proposed,which can adjust coordinate transformation matrix automatically with water depth.In addition,an edge detection method based on curve evolution is proposed to detect the deformation caused by the contact between the larva skin and the tip.This method has been applied to contact detection with good success rate and efficiency.Finally,the proposed schemes and algorithms are verified by experiments,and the advantages of the proposed schemes and algorithms in performance are proved by comparing with the traditional method through various indicators(success rate,efficiency,error,etc.). |
