新型快速自动宽场显微光切片控制系统

摘要

目的:介绍了采用精密步进电机、数码相机和宽场显微镜相结合所研发出的新型快速自动宽场显微光切片系统.方法:为了提高宽场显微光切片快速断层成像速度,准确得到所感兴趣的各焦平面上的图像信息,通过对显微镜Z轴精密操作、摄像系统和荧光激发光的曝光量同步自动控制,一方面可使切层变得更加均匀、精细,同时使摄像系统和荧光激发光的曝光量智能控制成为可能:另一方面,可大大缩短曝光时间,减少样品的荧光衰减和光漂白损伤,使得所获取的荧光图像信息更为可靠和丰富.结果:采用本系统不仅能使切片的层厚变得更加均匀、精细,使得三维重构后的样品三维结构更能接近真实状态,而且使图像存储自动化成为可能.结论:该系统解决了自动调节显微镜物镜Z轴的纵向运动使其断层扫描的进行获得足够的精度和足够薄的切层的问题,并实现了对数码摄像系统图像的自动摄录与存储,以及荧光激发光曝光量的自动控制.预期可广泛应用于各种需要进行三维断层观测的领域如生物医学与材料领域,具有重要的使用价值.%Objective:A fast wide-field auto-optical sectioning microscopic system is described.The system uses accurate step-motor,digital camera and a wide-field microscope,is able to perform high speed optical sectioning on wide-field microscope.Methods:In order to enhance the optical slice imaging speed of wide-field microscopic and get accurately the imaging information on different focal plane,the system can self-regulate the z-axial motion of the objective lens,and synchronically control the fluorescence excitation light exposure and image capturing sub-system,it is also able to obtain accurate,uniform thin sectioning slices on one hand,and on the other hand it can reduce the fluorescent bleaching.Results:The auto-control system for the Z-axis motion of the microscope objective,which can be controlled by a computer using the connect of serial port and produce PWM wave by the microcontroller to accurately control a 5-phase step motor,and can also accurately fulfill auto control for the Z-axis motion of the microscope objective to as precise as 2nm ; The computer-controlled electronic shutter switch system which is used to control the excited light exposure of fluorescence to reduce the fluorescent bleaching ; The system of auto digital imaging which is controlled by computer and the corresponding software system is based on windows and controls the microcontroller using the MSComm.Its functions is to saving the image,setting the mode,adjusting the ISO value,adjusting the focus and so on.Conclusions:The system can help to obtain more reliable and rich imaging information,it is expected to be extensively applied to the fields that require three-dimensional sectioning observation,such as biomedical material and medical imaging.