Research On Fast Autofocus Method Of Microscope Based On The Principle Of Chromatic Dispersion Difference

When testing some large samples with micro-nanostructure,it is often inevitable to scan the samples continuously and stitch the sub-apertures to obtain a complete scanned image of samples due to the limitation of the field of microscopic imaging view.The slight movement of the stage may cause the sample to exceed the depth of field of the system during scanning,resulting in image blur.Therefore,the scanning system is required to focus in real time.Due to the existing scanning technology,the axial focusing process of each field of view will occupy additional transverse scanning time,resulting in low scanning efficiency of system.In order to improve the efficiency and quality of sample scanning,a fast autofocus method is proposed based on the existing confocal microscope.Combining dispersion principles with the differential technique,the multi-spectral camera is used to obtain the gray images of samples in different bands and calculate the gray difference.The defocus signal is calculated by mapping relationship between the gray difference and the defocus amount.The major research contents of this subject are as follows:Firstly,the focusing principle of chromatic aberration method is analyzed as well as the theoretical formula derivation and the simulation of the defocus measurement curve are carried out,and the influence of the spectral band on the working range and the sensitivity and that of pinhole size on axial resolution are researched by simulation,which guide the selection of experimental parameters in later stage.Then,an experimental platform of multispectral autofocus is built based on the principle of chromatic aberration focusing.According to the overall structure design of the system,some major devices are selected.Besides,the calibration process of the corresponding relationship between gray difference and defocus is described.Thirdly,experiment is carried out to verify the measurement principle of chromatic aberration method.Under the condition of three different magnification objective lens of 4,10 ×,20 ×,the working range is 350?m,70?m and 20?m respectively,the sensitivity is 6?m,1.5?and 0.5?m respectively,and the maximum standard deviation under normalized condition is 0.0839,0.0584 and 0.0611 respectively the system can achieve.Then focusing performance of the system is tested on the multispectral focusing experimental platform.Under the condition of 10× objective lens,the maximum defocus amount detection error of the system is 2.5?m.which within the allowable rang of the system,the detection efficiency is as high as 0.0296s,it can meet the needs of real-time focusing of most samples in the scanning process.Finally,the error source of the system is analyzed qualitatively,and the corresponding solutions are proposed.In this paper,a new focusing method is proposed,and it also provides a new application direction for multispectral camera,the real-time focusing of sub aperture continuous transverse scanning process is realized based on the principle of chromatic aberration by combining with the dispersive tube mirror.Compared to the traditional focusing method based on image processing,not only can the proposed reduce the complexity of the system,but decrease the amount of calculation,which improve the scanning efficiency of the system significantly.