Steinman Joe, Koletar Margaret M, Stefanovic Bojana, Sled John G
Mouse Imaging Centre, The Hospital for Sick Children, Toronto, Ontario, Canada.
Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
PLoS One. 2017 Oct 20;12(10):e0186676. doi: 10.1371/journal.pone.0186676. eCollection 2017.
Ex vivo 2-photon fluorescence microscopy (2PFM) with optical clearing enables vascular imaging deep into tissue. However, optical clearing may also produce spherical aberrations if the objective lens is not index-matched to the clearing material, while the perfusion, clearing, and fixation procedure may alter vascular morphology. We compared in vivo and ex vivo 2PFM in mice, focusing on apparent differences in microvascular signal and morphology. Following in vivo imaging, the mice (four total) were perfused with a fluorescent gel and their brains fructose-cleared. The brain regions imaged in vivo were imaged ex vivo. Vessels were segmented in both images using an automated tracing algorithm that accounts for the spatially varying PSF in the ex vivo images. This spatial variance is induced by spherical aberrations caused by imaging fructose-cleared tissue with a water-immersion objective. Alignment of the ex vivo image to the in vivo image through a non-linear warping algorithm enabled comparison of apparent vessel diameter, as well as differences in signal. Shrinkage varied as a function of diameter, with capillaries rendered smaller ex vivo by 13%, while penetrating vessels shrunk by 34%. The pial vasculature attenuated in vivo microvascular signal by 40% 300 μm below the tissue surface, but this effect was absent ex vivo. On the whole, ex vivo imaging was found to be valuable for studying deep cortical vasculature.
采用光学透明技术的离体双光子荧光显微镜（2PFM）能够对深层组织中的血管进行成像。然而，如果物镜与透明材料的折射率不匹配，光学透明技术也可能产生球差，同时灌注、透明和固定过程可能会改变血管形态。我们比较了小鼠体内和离体2PFM，重点关注微血管信号和形态的明显差异。在体内成像后，对四只小鼠灌注荧光凝胶并对其大脑进行果糖透明处理。对体内成像的脑区进行离体成像。使用自动追踪算法对两张图像中的血管进行分割，该算法考虑了离体图像中空间变化的点扩散函数（PSF）。这种空间变化是由用水浸物镜对果糖透明处理的组织成像所引起的球差导致的。通过非线性扭曲算法将离体图像与体内图像对齐，能够比较表观血管直径以及信号差异。收缩率随直径变化，毛细血管在离体时缩小了13%，而穿通血管缩小了34%。软脑膜血管系统在体内使组织表面以下300μm处的微血管信号衰减了40%，但在离体时这种效应不存在。总体而言，发现离体成像对于研究深层皮质血管系统很有价值。
