基于数字微镜器件的高时空分辨选择性光刺激系统

High Spatiotemporal Resolution System for Selective Optical Stimulation Based on DMD

利用数字微镜器件将目标图案投影在样品面的方法相比于传统光照明方式大大提高了光刺激系统的空间选择性,但其光能利用率较小。全息光照射通过调制频谱面实现光能量的重新分布,在实现复杂的选择性光刺激同时,还具备较高的空间分辨率和光能利用率。构建了基于数字微镜器件的高时空分辨率的选择性逐点光刺激系统,通过在数字微镜器件上加载结构变化的二维达曼光栅相位图实现对视场内任意位置和任意时序的选择性逐点光刺激。研究表明,系统不仅能够以不同轨迹(如方形点阵、圆、螺旋线等)对全视场进行逐点的二维扫描,还能够选取感兴趣区域以自定义路径进行逐点扫描。该系统的最大扫描视场可达到400μm,最小扫描步长为0.204μm,单个扫描光斑的峰值半高宽最小可达到1.5μm,单点扫描速度可达10 kHz。本系统适用于需要对样品进行高分辨光刺激或需要对感兴趣区域进行实时刺激的光遗传研究中。

The optical stimulation system provides an important tool for light regulation,which modulates stably the optical waves to stimulate the target under investigation.In order to regulate vital movement precisely on cellular scale,scientists have developed various high-resolution optical stimulation systems.Traditional light stimulation methods include full-field light illumination,optical fiber illumination and galvanometer scanning illumination,etc.These methods can not accurately stimulate a single neuron in a specific area due to the lack of flexibility in spatial selection.Using Digital Micromirror Device(DMD)to project the target pattern on the sample plane greatly improves the spatial selectivity of the light stimulation system,but it has the disadvantage of low light energy utilization.Holographic light scanning allows not only complex selective light stimulation,but also achieves high spatial resolution and efficient light utilization.In this article,we embarked from computational holography and Dammann grating generation,and proposed a method of selective point-by-point stimulation with high spatiotemporal resolution based on a DMD.First,the structure transformed Dammann gratings generated by computational holography were loaded on the DMD.Then,the light modulated by the Dammann gratings stimulated the 2D target area point-by-point.The wide spectrum light from a halogen source was filtered by a 650/40 nm bandpass filter.An annular iris was placed in front of a condenser to realize dark field microscopy.We used LabView to complete the synchronization control and user interface display.The control algorithm first performs threshold to the dark-field images acquired by the sCMOS.Dammann grating phase diagrams was then calculated from stores the pixel coordinates with value of 1.Due to the working characteristics of DMD,the collimated 473 nm laser needs to be incident on the chip surface at 24°.DMD located on the front focal plane of the Fourier transform lens(f=200 mm)can produce a diffraction spot on the rear focal plane of the lens,and then a square diaphragm with adjustable aperture placed on the Fourier plane retains only one first-order diffraction spot as the scanning point.The tube lens(f=200 mm)and the objective lens(M=20×)form a 4f system to make the scanning point conjugated to the sample surface for scanning light stimulation.We use Rhodamine A indicator to image the scanning path.The maximum scanning field is 400μm×400μm,the minimum scanning step is 0.204μm,and the minimum half-height width of a single point peak is 1.5μm.We proved that the system can not only scan the full field of view point by point in different scanning mode(such as square lattice,circle,spiral,etc.),but can also scan point by point through a custom path in Region of Interest(ROI).The maximum scanning speed is 10 kHz.We also proved that the system can clearly scan complex patterns on homogeneously-stained cancer cells in a transcellular manner under 10×objective.In this article,we present a selective light stimulation system,which used DMD to deliver light to specified targets with high spatiotemporal resolution.We also completed the experimental verification of the system functions:1)it can perform optical stimulation on the imaging plane with arbitrary scanning mode;2)it can perform point-by-point light stimulation on the ROI;3)it can scan complex patterns on homogeneously-stained cells.The system is suitable for biomedical scientific research that requires optical stimulation on samples with high spatial resolution or real-time stimulation on ROI.For example,in optogenetics,the study of the contribution of single neuron in the entire neural circuit.On the basis of this system,adding a motorized stage can realize the real-time optogenetic research in freely moving organism.In terms of holographic projection,feedback algorithms such as G-S iteration can be applied to the system to realize the integration stimulation of regional light mode and selective point-by-point mode.In addition,combined with calcium imaging,two-photon microscopic imaging and other technologies,it can also obtain more microscopic neuronal activity information in real time,and analyze organism behaviors more effectively.