Montague Jenna E, Hutchens Gabrielle V, Howard Caitlin C, Rice Photini F S, Besselsen David G, Slayton Michael, Utzinger Urs, Barton Jennifer K, Sawyer Travis W
James C Wyant College of Optical Sciences, University of Arizona, Tucson, Arizona, USA.
Department of Biomedical Engineering, University of Arizona, Tucson, Arizona, USA.
Lasers Surg Med. 2025 Feb;57(2):204-218. doi: 10.1002/lsm.23871. Epub 2024 Dec 19.
To study the healing response of rat Achilles tendon when lacerated or treated with intense therapeutic ultrasound (ITU) via utilization of multiphoton microscopy (MPM) imaging and histology.
The right Achilles tendon of each Sprague Dawley rat within a cohort was partially lacerated. 1 to 2 days post-surgery, each rat received ITU treatment of the Achilles tendon on either the right or left leg. Rats were euthanized in groups at 1, 3, 7, 14, or 28 days posttreatment and their tendons were explanted, formalin fixed, paraffin embedded, sectioned, and placed on slides for imaging. Slides from each time point were imaged using a laboratory built MPM with a 780 nm Ti:Sapphire laser. The resulting second harmonic generation (SHG) and two-photon excited fluorescence (2PEF) signals were captured, assessed, and compared to brightfield microscopy images of the same section subsequently stained with hematoxylin and eosin.
At early timepoints, 2PEF images show the presence of red blood cells, infiltration of inflammatory cells and formation of a fibrin clot at laceration sites, and attraction of fibroblasts to ITU coagula. SHG images indicate an absence of organized collagen in both types of lesions. At later timepoints, new organized collagen can be seen at the laceration sites, and the concentration of inflammatory cells has noticeably decreased. Automated detection of red blood cells and infiltrative cells, as well as analysis of SHG signal intensity and homogeneity was performed at laceration locations. Results show that all quantities except SHG signal intensity approach normal values by day 28. Thus, combined analysis of 2PEF and SHG images elucidates tendon healing processes that align with and complement histological findings.
These results indicate that multiphoton imaging can effectively visualize the healing response to mechanical (laceration) and thermal (ITU) injury, including the organization of new collagen which is more difficult to visualize with histology.
通过多光子显微镜（MPM）成像和组织学方法，研究大鼠跟腱在撕裂或接受高强度治疗超声（ITU）治疗后的愈合反应。
将一组Sprague Dawley大鼠的右跟腱部分撕裂。术后1至2天，每只大鼠的右或左腿跟腱接受ITU治疗。在治疗后1、3、7、14或28天对大鼠进行分组安乐死，并取出其跟腱，用福尔马林固定，石蜡包埋，切片，置于载玻片上进行成像。使用配备780 nm钛宝石激光器的实验室自制MPM对每个时间点的载玻片进行成像。采集、评估所产生的二次谐波产生（SHG）和双光子激发荧光（2PEF）信号，并与随后用苏木精和伊红染色的同一切片的明场显微镜图像进行比较。
在早期时间点，2PEF图像显示撕裂部位存在红细胞、炎性细胞浸润和纤维蛋白凝块形成，以及成纤维细胞向ITU凝固物的聚集。SHG图像表明两种损伤类型中均不存在有组织的胶原蛋白。在后期时间点，可在撕裂部位看到新的有组织的胶原蛋白，炎性细胞浓度明显降低。在撕裂部位对红细胞和浸润细胞进行自动检测，并对SHG信号强度和均匀性进行分析。结果显示，除SHG信号强度外，所有指标在第28天时均接近正常值。因此，2PEF和SHG图像的联合分析阐明了与组织学结果相符并互补的肌腱愈合过程。
这些结果表明，多光子成像可以有效地可视化对机械（撕裂）和热（ITU）损伤的愈合反应，包括新胶原蛋白的组织化，而这在组织学上更难可视化。
