目的：开发细胞级高分辨率、结构与功能一体化的双模态全视场光学相干层析系统(full-field optical coherence tomography,FFOCT)，实现角膜缘组织的双模态FFOCT成像。方法：基于Linnik干涉成像原理，利用高数值孔径显微物镜(NA=0.8)及高速平面互补金属氧化物半导体(complementary metal oxide semiconductor,CMOS)相机，设计搭建高分辨率的组织静态结构和内源动态功能成像一体化双模态FFOCT系统；构建基于四相位调制结构影像提取及时域干涉信号动态频谱分析的功能影像重建算法；对人供体角膜缘组织开展各深度层的双模式FFOCT成像有效性验证。结果：搭建的双模态FFOCT成像系统可实现横向分辨率0.5 μ m，轴向分辨率1.7 μ m，成像视野320 μ m×320 μ m，相机采集速度100 Hz。系统实现角膜缘组织无外源标记情况下的细胞级分辨率三维结构和内源功能成像，FFOCT静态结构影像清晰显示角膜缘上皮、Vogt栅栏、隐窝、基质、血管及淋巴管等结构，FFOCT动态功能影像突出显示了代谢活跃细胞(角膜缘上皮细胞、免疫细胞等)。结论：双模态FFOCT高分辨率成像系统可提供角膜缘微观结构和活细胞无标记内源功能可视化信息，将为角膜缘疾病的研究及临床诊疗提供全新的成像分析技术。
Objective: To develop a cellular-level, high-resolution, integrated dual-modal full-field optical coherence tomography (FFOCT) system capable of simultaneously imaging the structure and function of limbus tissue. Methods: Utilizing the Linnik interference imaging principle, a high-resolution dual-modal FFOCT system was designed and constructed using a high numerical aperture (NA=0.8) microscope objective and a high-speed flat CMOS camera. A functional imaging reconstruction algorithm based on four-phase modulation structure image extraction and dynamic frequency spectrum analysis of temporal interference signals was developed. The effectiveness of dual-mode FFOCT imaging at various depth layers of human corneal limbal tissue was validated. Results: The constructed dual-modal FFOCT imaging system achieved lateral resolution of 0.5 μ m, axial resolution of 1.7 μ m, imaging field of view of 320 μ m × 320 μ m, and camera acquisition speed of 100 Hz. The system enabled cellular-level resolution three-dimensional structural and intrinsic functional imaging of corneal limbal tissue without exogenous labeling. Static structural FFOCT images clearly displayed limbal epithelium, palisades of Vogt, crypts, stroma, blood vessels, and lymphatic vessels, while dynamic functional FFOCT images highlighted metabolically active cells (limbal epithelial cells, immune cells, etc.). Conclusion: The dual-modal FFOCT high-resolution imaging system provides visualization of corneal limbal microstructural and live cell intrinsic functional information without labeling, offering a novel imaging analysis technique for research and clinical diagnosis and treatment of limbal diseases.
角膜缘的细胞，特别是角膜缘干细胞，对于维持角膜的透明和健康至关重要。基于影像技术对角膜缘进行高精度可视化评价是相关疾病诊疗的重要手段。眼科临床使用的裂隙灯显微镜、共聚焦显微镜、眼前节光学相干断层扫描仪(optical coherence tomography,OCT)等成像技术，因低分辨、低对比度、侵入性等原因，限制了其在角膜缘细胞结构及功能影像评估中的应用。本团队创新研发新型双模态全视场光学相干断层扫描仪(full-field OCT，FFOCT)，成功实现了无标记的角膜缘细胞级分辨率结构及功能成像。FFOCT基于空间非相干光平面干涉原理提取组织内部散射光，获得微米级分辨率三维结构成像；通过FFOCT原始相干信号的高时空分辨率采集及动态特征解析，实现源于活细胞新陈代谢运动的无标记细胞功能影像可视化。双模态FFOCT创新性地整合了高分辨率、无标记的结构及功能成像模态，不仅清晰获取角膜缘组织的高精结构特征如Vogt栅栏、角膜缘隐窝、血管壁等，同时还能捕捉不同角膜缘细胞内的代谢活性动态变化，无需使用外源荧光染料或标记剂，为角膜缘生物学及疾病机制研究提供全新细胞水平结构及功能成像方法，具有广泛应用前景。
角膜缘的细胞，特别是角膜缘干细胞，对于维持角膜的透明和健康至关重要。基于影像技术对角膜缘进行高精度可视化评价是相关疾病诊疗的重要手段。眼科临床使用的裂隙灯显微镜、共聚焦显微镜、眼前节光学相干断层扫描仪(optical coherence tomography,OCT)等成像技术，因低分辨、低对比度、侵入性等原因，限制了其在角膜缘细胞结构及功能影像评估中的应用。本团队创新研发新型双模态全视场光学相干断层扫描仪(full-field OCT，FFOCT)，成功实现了无标记的角膜缘细胞级分辨率结构及功能成像。FFOCT基于空间非相干光平面干涉原理提取组织内部散射光，获得微米级分辨率三维结构成像；通过FFOCT原始相干信号的高时空分辨率采集及动态特征解析，实现源于活细胞新陈代谢运动的无标记细胞功能影像可视化。双模态FFOCT创新性地整合了高分辨率、无标记的结构及功能成像模态，不仅清晰获取角膜缘组织的高精结构特征如Vogt栅栏、角膜缘隐窝、血管壁等，同时还能捕捉不同角膜缘细胞内的代谢活性动态变化，无需使用外源荧光染料或标记剂，为角膜缘生物学及疾病机制研究提供全新细胞水平结构及功能成像方法，具有广泛应用前景。
目的：针对活体共聚焦显微镜(in vivo confocal microscopy，IVCM)和传统光学相干层析技术(optical coherence tomography，OCT)在人眼角膜成像各自存在成像视野小或无法细胞成像的限制，开发具有高分辨率的非接触全视场光学相干层析系统(full-field optical coherence tomography，FFOCT)，实现活体人眼角膜细胞结构FFOCT成像。方法：FFOCT系统采用高数值孔径干燥显微物镜及高速面阵相机，使用双相位调制图像处理方法，实现系统高速高分辨率非接触成像。利用系统对健康人眼进行角膜各深度层的活体FFOCT成像验证其可行性。结果：本研究团队研发了FFOCT的新型活体人眼角膜高分辨率成像系统，实现理论平面成像分辨率1.7 μm，成像视野1.26 mm×1.26 mm，成像速率达275帧/s。利用该系统对正常活体人眼角膜成像实验，在非接触情况下获取了角膜各主要结构层的高分辨率结构影像。结论：FFOCT高分辨率活体人眼角膜成像系统兼具了传统OCT的非接触、大成像视野及IVCM的细胞级别平面分辨率的优势，将为角膜疾病的研究及临床诊疗提供全新的成像分析技术。
Objective: Due to the limitations of small imaging field of view of in vivo confocal microscopy (IVCM) or the incapability of cellular imaging of traditional optical coherence tomography (OCT) in human corneal imaging, this study was designed to develop a novel high-resolution in vivo human corneal imaging system based on full-field OCT (FFOCT). Methods: The FFOCT system utilized a high numerical aperture air immersion microscope objective and a high-speed area array CMOS camera with two-phase modulation image processing algorithm to achieve high-speed high-resolution non-contact imaging of human cornea. To verify its feasibility, in vivo cornea imaging at different depth was performed on a healthy human subject. Results: The FFOCT system achieved a theoretical lateral imaging resolution of 1.7 μm, an imaging field of view of 1.26 mm×1.26 mm, and an imaging rate of 275 Hz/s. High-resolution FFOCT images of the main structural layers of cornea were achieved by imaging a healthy human cornea in vivo with this system in a non-contact way. Conclusion: The FFOCT human corneal imaging system combines the advantages of the non-contractness and the large imaging field of view of traditional OCT with the cellular lateral resolution of IVCM, potentially providing a new imaging system for the research and clinical diagnosis and treatment of corneal diseases.
