Research On Confocal Microscopy By Array Structured Detection Based On Serial Beam Scanning

Optical microscopy imaging and measuring technique has been widely applied in the development frontier of modern physics,material science,molecular biology,microelectronics and other advanced disciplines because of its noninvasive,nondestructive and high efficiency characteristics.However,the resolution of conventional wide-field microscopy is restricted by the diffraction limit.The highest lateral and axial resolution are about 200 nm and 500 nm,respectively,which means the optical microscopy can not give full play to its advantage and be used in more extensive applications.Therefore,the urgently needed development direction of optical microscopy in nowadays is to breakthrough the diffraction limit to realize the supper-resolution imaging,so that it could satisfy more extensive applications.Aiming at the problems of improving the lateral resolution and measurement efficiency of existing confocal microscopy,a method of confocal microscopy by array structured detection based on serial beam scanning is proposed in this study.The method combines confocal microscopy,structured detection,virtual pinhole and array scanning.On the one hand,it learns from the idea of structured illumination and realizes structured detection by applying spatial modulation into the detection opticalarm.In addition,an area CCD is used to displace the point detector for image sampling and the virtual pinhole technique is used to process the sampled image.On the other hand,the array scanning of the measured object is realized by using light beam scanning technique while improving the lateral resolution of the confocal microscopy,which means that the measurement efficiency of the microscopy is highly improved.Therefore,the fast and super-resolution imaging is realized.In this study,a model of structured detection based scanning confocal microscopy is established and its coherent imaging process is analyzed.Simulations are done to analyze and compare the coherent transfer functions and intensity point spread functions of different kinds of microscopes,thus to verify the super-resolution characteristics of structured detection based confocal microscopy.The parameters which have impacts on its lateral resolution is also analyzed.And a structured detection function which can be actually applied in real system is obtained.Then,aiming at the problem of low scanning efficiency in the point-by-point scanning confocal microscopy,a method of structured detection based array scanning confocal microscopy is proposed.In the method,a galvanometer is controlled to realize the light beam scanning and the area CCD is synchronously controlled for image sampling.At the same time,the laser diode is also synchronously controlled to match with the galvanometer to eliminate the trailing phenomenon in image sampling by shut-down the laser diode while shifting the light spot.Three kinds of methods for determining the light spot positions in the array scanning image are proposed and the centroid method is chosen after comparison.Finally,the resolution evaluation method in experiment is determined according to the half width at half maximum(HWHM).Simulation result indicates that the lateral resolution of structured detection based confocal microscopy is 1.3 times of ideal confocal microscopy and 1.8 times of conventional wide-field microscopy while the size of pinhole is chosen to equal with which of the Airy spot.An experimental structured detection based array scanning confocal microscopy is established and the 8th and 9th line-group of identification board is imaged using the established system.The imaging results are compared to which of the conventional point-by-point scanning confocal microscopy.Experimental results indicate that scanning speed of the established system in this study is 48 times higher than which of the conventional system.