一种大视野快速光学衍射层析成像技术

Large Field-of-View Fast Optical Diffractive Tomographic Microscopy

光学衍射层析成像技术是一种新兴的对细胞和组织进行非侵入、无创伤、无标记快速三维成像的技术,在细胞代谢、病理和肿瘤诊断等方面都有很大的应用前景。传统的光学衍射层析成像技术视场范围较小,视野内仅有单个或数个细胞,难以直接观察到细胞间的相互作用,无法对散射较强的厚、大细胞进行成像,这在一定程度上限制了其进一步应用。针对视场范围较小的不足,提出了一种用满采集物镜的视场数的大视野光学衍射层析成像技术,可以获得更高的散射光子通量,提高成像质量,减弱伪影、振铃等边缘效应,视野内可以观测到多种状态的细胞和细胞间相互作用。结果表明,大视野光学衍射层析成像技术在兼具亚细胞分辨率和无标记活细胞长时程三维观测能力的同时,具有更大的视野、更小的边缘效应,生物应用前景更加广泛。

Objective Optical diffractive tomographic microscopy is a new widefield,noninvasive and labelfree threedimensional(3D)imaging technology for cells and tissues,which has great application prospects in cell metabolism,pathology and tumor diagnosis.However,with the continuous development of modern biological research,the fieldofview(FOV)of traditional optical diffractive tomography(ODT)cannot meet the needs of observation any more.The invention of large fieldofview ODT technology,while maintaining subcellular resolution,is in an increasingly urgent need.At present,various quantitative phase imaging technologies require higher spatialbandwidth product.For example,the sampling rate of interference streaks acquisition in offaxis holographic imaging is more than three times that of intensity imaging.In the condition of a certain number of camera pixels,only a small FOV can be acquired.In order to conduct largeFOV quantitative phase imaging,the number of pixels in the single image is doubled,and the data flux of images is too large.It leads to the facts that the image storage becomes more difficult,the complexity of recovery algorithm is aggravated,and the time of settlement increases.Methods The traditional method to realize large FOV is to scan different areas and then splice the images.However,the method is not suitable for living cells since they constantly move,which limits the further application of the traditional ODT method in biology.To solve the problem,we propose a new ODT technology which can realize the large FOV.Based on the MachZehnder transmission holographic imaging system,we make some unique designs for largeFOV imaging requirements.The main innovations are described here.Firstly,we design a nondestructive pupil holographic beam binding scheme.We use the Dshaped mirror instead of beam splitters for beam combining.It can achieve zero loss of intensity and unlimited size.Secondly,we achieve largeFOV oblique plane illumination under the large numerical aperture(NA).Finally,we improve the image acquisition system.We choose a 21 megapixels camera and the faster CoaXPress12 card as the data acquisition card,and achieve the 50 Gbit/s data flux.The optical elements and galvanometer of the system are reselected to ensure that there is no aperture limitation.In addition,we rewrite the data processing program,considering the large amount of data in the largeFOV ODT system.We reconstruct the software of images acquisition to realize the highspeed image acquisition and storage.Then,we edit the new multithread ODT recovery algorithm based on C++for 64-bit system,which can automatically recover all the collected data synchronously.Using the above system and algorithm,we image 5μm polystyrene microspheres to verify the feasibility of the system.Then,Hela cells are imaged,which verifies that the method has longterm 3D observation ability for dense tissue cells and living cells.Results and Discussions In this paper,a largeFOV optical diffraction tomography technique is proposed.The largeFOV ODT uses all the FOV of the objective lens to reach the limit of the imaging range.At the same time,it has both high resolution and longterm 3D imaging capability for living cells.Compared with the traditional ODT system,the imaging range of the proposed system is larger[Figs.3(a)and 5(a)];more photons scattered by complex samples can be obtained,so that the signaltonoise ratio(SNR)is better[Figs.3(b)and 3(c)].Moreover,the ringing and artifacts effects of the edge are smaller[Figs.3(b),3(c),5(b),and 5(c)].The interaction between cells,as well as more cells in different states,can be observed simultaneously in a FOV(Figs.5 and 6).Conclusions The results show that the largeFOV optical diffraction tomography technology has both subcellular resolution and longterm 3D observation ability of labelfree living cells.Compared with the traditional system,the largeFOV ODT system has smaller edge effects and obtains more information of cells,so it is beneficial to observing the interaction between cells,and is helpful to realizing the longterm 3D observation of huge living cells such as oocytes.It will have more biological applications.