Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis.
Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis.
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DOI：
10.1016/j.kint.2016.06.030
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发表时间：
2016-11
影响因子：
19.6
通讯作者：
Furgeson S
中科院分区：
文献类型：
--
作者：
Ranjit S;Dobrinskikh E;Montford J;Dvornikov A;Lehman A;Orlicky DJ;Nemenoff R;Gratton E;Levi M;Furgeson S
所有形式的进行性肾脏疾病发展为肾小管间质纤维化和肾小球硬化的最终途径。肾纤维化通常使用组织学染色来量化，这是一个耗时且依赖于病理学家的过程。这里描述的工作显示了一种快速和操作员独立的方法来测量纤维化的发展。采用单侧输尿管梗阻（UUO）模型研究肾纤维化。小鼠的梗阻肾脏出现时间依赖性增加;对侧肾脏用作对照。在UUO后，在三个时间点分析肾脏：7天、14天和21天。使用FLIM（荧光寿命成像）和SHG（二次谐波发生）在称为DIVER（通过增强光子恢复的深度成像）的深层组织成像显微镜中研究纤维化。该显微镜专为深层组织和SHG和THG（三次谐波发生）成像而开发，对谐波发生具有非凡的灵敏度。SHG数据表明在患病肾脏中存在更多的纤维状胶原。短波长FLIM和SHG分析的组合产生了独立于观察者解释的稳健分析程序，并使我们能够创建直接从图像量化纤维化程度的标准。使用这种新的FLIM-SHG技术研究了UUO模型中的纤维化进展，与标准组织学技术相比，它在纤维化定量方面显示出显著改善。
All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. The work described here shows the development of a fast and operator-independent method to measure fibrosis. To study renal fibrosis, the unilateral ureteral obstruction (UUO) model was chosen. Mice develop a time-dependent increase in obstructed kidneys; contralateral kidneys are used as controls. After UUO, kidneys were analyzed at three time points: 7 days, 14 days, and 21 days. Fibrosis was investigated using FLIM (Fluorescence Lifetime Imaging) and SHG (Second Harmonic Generation) in the deep tissue imaging microscope called DIVER (Deep Imaging via Enhanced photon Recovery). This microscope was developed for deep tissue and SHG and THG (Third Harmonic Generation) imaging and has extraordinary sensitivity towards harmonic generation. SHG data suggests the presence of more fibrillar collagen in the diseased kidneys. The combinations of short wavelength FLIM and SHG analysis results in a robust analysis procedure independent of observer interpretation and let us create a criterion to quantify the extent of fibrosis directly from the image. The progression of fibrosis in UUO model has been studied using this new FLIM-SHG technique and it shows remarkable improvement in quantification of fibrosis compared to standard histological techniques.