基于Mirau干涉结构的全场光学相干层析系统

Full⁃Field Optical Coherence Tomography System Based on Mirau Interference Structure

基于Linnik结构的全场光学相干层析系统以高分辨率、深穿透性、无损非接触、数百万通道并行层析等优点被广泛应用于生物医学成像领域。在该系统中,参考臂和采样臂采用两个完全相同的显微物镜,二者的对准精度均为微米级,无法通过机械结构的约束使其满足成像条件。在不同的应用场景下成像时,需要同时更换两个物镜并需要重新调校,过程比较繁琐。在环境振动以及温湿度和结构应力变化的工况下,系统性能会降低甚至失效。针对上述问题,本课题组提出了基于Mirau干涉结构的全场光学相干层析系统,将参考臂和采样臂集成到一个方向上,同时,针对样品的折射率特性,在物镜结构中填充相应的介质进行折射率匹配。该系统性能稳定,并且可以随意切换倍率。采用该系统对非生物样本(PCB板)和生物样本(洋葱表皮和绿萝叶片)进行了成像实验,验证了该系统的可行性。

Objective Full-field optical coherence tomography(FFOCT)is a contactless,high-resolution,real-time imaging method based on OCT technology.It utilizes the backscattering ability of the internal structure of the tissue to obtain a light signal with structural information about the tissue through the detector and then restores the real internal structure through a computer.With the introduction of the Linnik interferometer structure with two identical large numerical aperture microscope objectives into the FFOCT system,the imaging resolution of the system has been improved to the submicron level.Therefore,scholars have begun to conduct further research on aspects such as system effectiveness,stability,practicality,cost,and efficiency.Because the reference and sample arms use two identical microscope objectives,their alignment accuracy is at the micron level,which cannot meet the imaging conditions by the constraints of the mechanical structure.The presence of these two microscope objectives also increases the cost of the system.For different application scenarios,both objectives should be replaced and recalibrated simultaneously,which is a cumbersome process.In this study,we built an FFOCT system based on a Mirau interference structure,integrated the reference arm in the original structure into a self-made Mirau attachment,and assembled it between the objective lens of the sample arm and the sample.This makes the system more compact and stable and reduces costs.The microscope objective at different magnifications can be replaced according to requirements,and different media can be used for index matching.Methods Based on the imaging principle of the FFOCT system,we first simulated the Mirau interference structure using the optical simulation software VirtualLab Fusion.In the preliminary verification stage,only the Mirau-attachment structure was designed;thus,the microscope objective was simplified to an ideal lens that satisfied the software parameters.The feasibility of the solution was verified through light-field tracing and imaging experiments on the test surfaces at different positions.Based on the parameters obtained from the simulation,we designed and processed the mechanical structure,which included two pieces of glass with reflective and beam-splitting functions as well as a structure for the adjustment and filling of the medium.The obtained structure was assembled into a system for imaging experiments,and a USAF standard-resolution target and plane mirror were imaged to measure the lateral and axial resolutions of the system,respectively.Using PCB,onions,and plant leaves as imaging objects,a four-step phase-shift method was employed as the solution method.The imaging results were used to verify the tomographic capabilities of the system.Results and Discussions The feasibility of this principle is verified through simulations.The light-field tracing diagram conforms to the imaging principle,and the interference fringe pattern obtained by imaging the test surface at different positions is also in line with expectations(Fig.2).To meet the actual conditions,we attached the reflector to a piece of glass with the same material and thickness as the beam splitter,adjusted the parameters,and performed simulations again.The results obtained still meet expectations(Fig.3).A Mirau attachment was designed and fabricated(Fig.4).Based on optical principles,the theoretical lateral and axial resolutions are calculated to be 1.73 and 7.56μm,respectively.By imaging the USAF standard-resolution test target,the actual measured value of the lateral resolution is determined to be 2.19μm(Fig.5).An interference fringe intensity distribution diagram is obtained by imaging the plane mirror.Based on the resulting diagram,the actual measured value of the axial resolution is calculated to be 9.1μm(Fig.6).A certain position on the PCB contains two structures with different heights.The height difference is greater than the coherence length of the light source.When interference fringes appear on one side,no interference fringes appear on the other.Therefore,the calculated image contains only one side of the structural information,reflecting the tomographic capability of the system(Fig.7).When imaging biological samples,their internal structures are restored.As the depth increases,the structural morphology changes and tomographic features are observed(Figs.8‒9).Conclusions An FFOCT system based on the Mirau interferometer is developed.This structure is more compact and stable than an FFOCT system based on a Linnik interferometer.The previous cross structure was changed to the current T-shaped structure,and the system complexity was reduced.The previously used FFOCT system uses two identical water-immersion objectives;however,this system requires only one microscope objective and does not require a water-immersion objective,which reduces system costs.Using a self-made Mirau interference objective structure,the parameters can be adjusted according to different requirements,and the microscope objective magnification and filling medium can be changed according to the imaging object.The tomographic capabilities of the system are verified by imaging abiotic and biological samples,and imaging with cell-level resolution is achieved.However,the resolution and imaging quality of the system are affected by the presence of environmental noise and noise generated by the operation of the system itself.For the four-step phase-shift method used in this system,mechanical vibrations cause a deviation between the actual phase shift and the set value,resulting in insufficient signal demodulation,thus retaining some interference fringes in the resulting image and affecting the imaging effect.Moreover,because the microscope objective is a commercial version and cannot be directly simplified into a lens,the self-made Mirau interference objective structure causes certain aberrations that affect the imaging quality and depth.