Supplementary file 2_Evaluation of the effects of clearing agents, fixation, and process durations on cardiovascular tissue imaging with second harmonic generation and multi-photon modalities.docx

IntroductionProtocols for tissue clearing have been established and optimized for the central nervous system. However, significant modifications are required for clearing different tissue types. Therefore, effective optical clearing for cardiovascular tissue remains a major challenge. The goal of this study is to better understand the responses of porcine left anterior descending artery (LADA) to label-free multiphoton imaging.
MethodsTo this end, the effects of different clearing methods (i.e., benzyl alcohol benzyl benzoate–BABB and glycerol), formalin fixation, variations in formalin fixation times (0–240 min), and extended storage in BABB (up to 14 days) are investigated. We compare tissue characteristics under different conditions (e.g., tissue clearing reagent and/or tissue fixation), particularly with regard to tissue preservation and transparency across z-stacks (i.e., imaging depths).
ResultsThe glycerol clearing method exhibited relatively lower tissue transparency, whereas BABB increased mean AF-AUC from 0.0035 ± 0.0009 to 0.1205 ± 0.0168 and SHG-AUC from 0.0003 ± 0.0002 to 0.0072 ± 0.0040 (p< 0.001), enabling robust signal intensities at deeper layers of LADA tissue. In addition, we observed that BABB preserves fluorescent signals even after extended tissue storage with no significant loss in integrity over 14 days. Finally, we found that formalin fixation in combination with the glycerol clearing method significantly improved tissue preservation compared to the glycerol clearing method alone. However, in combination with the BABB clearing method, fixation reduced tissue transparency and signal intensity compared to BABB clearing without fixation.
DiscussionThese findings establish BABB as the superior, label-free clearing agent for deep 3D multiphoton microscopy/second harmonic generation imaging of cardiovascular tissue and underscore the necessity of tailoring fixation parameters to the chosen clearing method.

引言：组织透明化方案已针对中枢神经系统建立并优化，但针对不同组织类型开展透明化时需进行大幅调整。因此，实现心血管组织的高效光学透明化仍是一项重大挑战。本研究旨在深入解析猪冠状动脉左前降支（porcine left anterior descending artery, LADA）对无标记多光子成像的响应特性。
方法：为此，本研究考察了不同透明化方案（即苯甲醇-苯甲酸苄酯（benzyl alcohol benzyl benzoate, BABB）与甘油）、福尔马林固定、福尔马林固定时长变化（0~240 min）以及在BABB中长时间储存（最长14天）对组织的影响。我们对比了不同条件（如组织透明化试剂和/或组织固定方式）下的组织特性，重点关注组织保存效果与Z堆叠扫描（即成像深度）层面的组织透明度。
结果：甘油透明化方案的组织透明度相对较低；而BABB可使平均自发荧光曲线下面积（AF-AUC）从0.0035±0.0009提升至0.1205±0.0168，二次谐波产生曲线下面积（SHG-AUC）从0.0003±0.0002提升至0.0072±0.0040（p<0.001），从而在LADA组织的深层实现稳定的信号强度。此外，研究发现即便经过14天的长时间储存，BABB仍可保留荧光信号，组织完整性无显著下降。最后，相较于单独使用甘油透明化方案，福尔马林固定联合甘油透明化可显著改善组织保存效果。但相较于未固定直接使用BABB透明化的样本，福尔马林固定联合BABB透明化会降低组织透明度与信号强度。
讨论：本研究结果证实，BABB是用于心血管组织深层三维多光子显微镜/二次谐波成像的最优无标记透明化试剂，同时强调了需根据所选透明化方案定制固定参数的必要性。