用于大景深单分子定位显微的多功能全息相位片的设计及数值模拟

Design and numerical simulation demonstration of multi-functional holographic phase plate for large depth of field single molecular localization microscopy

发展具有大轴向定位范围的单分子定位技术对于实现厚样品的超分辨成像具有重要的价值.基于波前编码技术,将变形多值纯相位光栅与双螺旋点扩散函数相位片相结合,提出一种可以通过空间光调制器实现的具有高衍射效率的新型全息相位片的设计方法.这种全息相位片可以将样品内多个层面的分子信息以双螺旋的形式成像在同一个探测面的不同位置,在无需扫描的情况下提高双螺旋点扩散函数工程的轴向定位范围和分辨率,解决活细胞内单分子定位和示踪技术中的大景深探测难题.数值模拟表明,设计的5×5全息相位片可以将样品内25个层面上的分子信息以双螺旋的形式成像在同一探测面上的不同位置,相邻两个层面的间隔为0.5μm,实现了轴向12μm的探测范围,证明了设计的可行性.

The development of nanoscale single-molecule localization and tracking technology for multiple bio-molecules in intact cells has important significance for studying the dynamic process in life process. Since most of cells are several microns in depth, but the focal depth of traditional optical microscopes are less than one micron, the limited depth of field is the main drawback of conventional single molecular localization microscopy that prevents observation and tracking of multiple molecules in intact cells. In this paper, based on the wavefront coding technique, a new type of holographic phase plate with high efficiency is proposed and designed to extend the depth of field of single molecular localization microscopy, which combines the distorted multi-value pure-phase grating（DMVPPG） with the double-helix point spread function（DH-PSF）. The DMVPPG can be used to realize multiplane imaging of several tens of layers of a sample in a single detection plane. And the DH-PSF is an engineered point spread function which encodes the lateral and axial position with high precision of a molecule in the center of its two lobes and the angle between them respectively.Using the combined holographic phase plate, the molecules in dozens layers of a whole cell can be simultaneously imaged on the same detection plane with DH-PSF. Not only can the axial resolving power be improved, but the imaging depth can also be extended without scanning. Adding such a holographic phase plate to the imaging path, the limited imaging depth problem in single-molecule-localization microscopy can be solved without sacrificing the localization accuracy. The proposed new type of holographic phase plate can also be implemented with a spatial light modulator. In the following numerical simulation experiments, the designed holographic phase plate is composed of 600 × 600 pixels with a pixel size of 10 μm. The distance between two adjacent focal planes is designed to be 0.5 μm. Such a holographic phase plate is placed on the Fourier transform plane of the detection light path. When an emitter is located on the focal plane, it can be imaged as two lobes without rotation in a center area of the field of view. If an emitter is-6 μm away from the focal plane, the DH-PSF appears in the upper-left area of the field of view. Simulation results demonstrate that a total of 25 sample layers can be simultaneously imaged on the single detection plane and the 12 μm detection range can be achieved, thus proving the feasibility of this method.