基于波前编码的大像面荧光显微成像系统景深延拓研究

Research on depth of field extension of large image area fluorescence microscopy imaging system based on wavefront coding

大像面荧光显微成像系统可以对大尺寸dPCR生物基因芯片进行一次性成像,但其景深(DOF)较小,限制了系统的物方检测深度。文章采用波前编码技术在不降低分辨率的前提下,有效扩展了大像面荧光显微成像系统的景深,为后续高通量多层堆叠式dPCR生物芯片实现一次性成像提供了技术支持。但考虑到显微成像系统正向设计仿真时不适合直接进行景深延拓分析,故对其倒置翻转后在像方(原物方)分析焦深延拓范围(即景深)。为确定最优的相位板参数值,编写遗传算法与ZEMAX的API接口链接,以动态交互的方式对相位板调制系数进行优化、筛选、定值。在大像面荧光显微成像系统孔径光阑前的平行光路处添加特定系数的相位板,对光学系统的波前相位进行调制,使得在焦点附近一段范围内产生成像一致的中间模糊像。采用维纳滤波对中间模糊像进行复原,由模拟的结果可见,在传统光学系统的±10倍景深内,编码复原图像具有较高的分辨率(达到6.5μm),能够实现系统的景深延拓。装配相位板的大像面荧光显微成像实际系统,其延拓倍数在±7倍景深范围内有较好的图像复原分辨率(6.5μm)。

The large image area fluorescence microscopy imaging system can perform one-time imaging of large-size dPCR biological gene chips,but its depth of field(DOF,Depth of field)is small,which limits the object detection depth of the system.The wavefront coding technique is used to effectively expand the depth of field of the large image area fluorescence microscopy imaging system without reducing the resolution,which provides technical support for the subsequent one-time imaging of high-throughput multilayer stacked dPCR chips.However,considering that the forward design and simulation of the microscopic imaging system is not suitable for direct depth of field extension analysis,so it is inverted and flipped to analyze the focus depth extension range(i.e.,depth of field)on the image side(original object side).In order to determine the optimal phase plate parameter values,agenetic algorithm was written to link with ZEMAX's API interface,and the phase plate modulation coefficients were optimized,screened,and set in a dynamic interactive manner.A phase plate with a specific coefficient is added to the parallel light path in front of the aperture diaphragm of the large image area fluorescence microscopy imaging system to modulate the wavefront phase of the optical system,so that a consistent intermediate blurred image is produced within a range near the focus.The Wiener filter is used to restore the intermediate fuzzy image,and the simulation results show that the encoded image has a high resolution(up to 6.5μm)within the range of±10times depth of field of the traditional optical system,which can realize the depth of field extension of the system.The actual large image area fluorescence microscopy imaging system equipped with a phase plate has a good image restoration resolution(6.5μm)within the range of±7times the depth of field.