Design And Application Of Biological Microscope Autofocus System Based On Piezoelectric Actuator

In many biological experiments,it is often necessary to perform microscopic imaging at high resolution and to ensure that the image of the experimental sample remains in a positive focus for hours or days,which is challenging for high magnification biological microscopy: biological microscopy thermal instability of the mechanical structure and thermal fluctuations in the air all contribute to focus drift.In addition,for uneven experimental samples,it is necessary to refocus each observed position.It is not realistic to rely on artificial time to keep the experimental sample out of focus.Therefore,based on the above requirements and microscope autofocus scheme for depth from focus,this paper designs and implements a biological microscope autofocus control system with a high-magnification and high-focus adjustment precision.This system is based on a home-built biological microscope,in which the main hardware includes a high-resolution CCD camera,a piezoelectric controller and a 3-axis stage.The customized software operating system synchronously controls the above hardware devices manifest itself as a simple and clear human-computer interaction interface,it follows the basic principles of high cohesion and low coupling in software design,which improves the stability and maintainability of the system.The implementation of the autofocus system in this paper is as follows: the image of the sample different positions along the Z axis is acquired by a CCD camera.Then these images are analyzed by the sharpness evaluation function to calculate the Z value which is most close to the position corresponding to the focused position of the sample.The voltage value corresponding to the position is then fed back to the piezoelectric controller,and the Z axis of the stage is driven by the piezoelectric controller to move the sample to the position,realizing the function of the microscope autofocus.We found that in the above implementation process,the following problems exist: the Z axis of the stage moves at a fixed value.When the value is small,the CCD camera collects more sample images,and the sharpness between adjacent images is relatively close,which may cause the image sharpness evaluation function to be trapped at a local extreme point,prolonging the processing time of the system,and even the focus fails.When the value is large,the adjustment accuracy of the focusing system will be insufficient.In order to effectively solve the above problems,this paper proposes an improved Laplace sharpness evaluation function and a search optimization algorithm,which are combined into a biological microscope autofocus system.Our experimental results show that the improved sharpness evaluation function in this three sets of biological experimental specimens,the normalized function evaluation value is better,which is conducive to the realization of the search optimization algorithm and reduce the repeated movement of the 3-axis stage.Under the imaging of 100 times objective lens,the focusing time of our setup is controlled within 1.23 s with high repeating precision,the repeatability is high,and the focus adjustment accuracy can reach 200 nm,which can basically meet the needs of optical microscope in applications.