组织光透明技术结合双光子显微成像(TPM)技术能够有效提升生物样品的显微成像深度, 然而现有的光透明剂与常用显微物镜的浸润介质折射率并不匹配, 会引入球差从而降低深层组织成像的荧光强度和分辨率。针对该问题分析了球差对TPM荧光强度和分辨率的影响, 建立了由物镜特性(数值孔径和浸润介质)、聚焦深度和物体折射率等参数构成的球差补偿模型, 进而指导空间光调制器进行球差补偿。对荧光小球仿体样品和光透明脑组织样品的双光子成像结果显示, 该球差补偿方法能显著提升样品信号量和系统纵向分辨率。另外, 该方法在校正过程中无需多次成像, 操作简单且耗时短, 对光透明剂和显微物镜无特殊要求, 具有较强的通用性。

Tissue optical clearing technique combined with two-photon microscopy (TPM) can improve the imaging depth of biological samples. However, the refractive index mismatch between objective medium and optical clearing agent will cause spherical aberration which degrades the fluorescence intensity and axial resolution. To solve this problem, we analyzed the effect of SA at the focus, and built a spherical aberration compensation model based on objective characteristics (numerical aperture and immersion media), the imaging depth and the sample refractive index. Then, we corrected the spherical aberration by incorporating a spatial light modulator into the TPM system. The TPM images of fluorescent bead phantom and optical clearing brain tissues show considerable improvement of fluorescence intensity and axial resolution. The proposed correction process is simple and fast since it does not require repeated imaging. More importantly, it is suitable for different objectives and optical clearing agents.