Hato Takashi, Winfree Seth, Day Richard, Sandoval Ruben M, Molitoris Bruce A, Yoder Mervin C, Wiggins Roger C, Zheng Yi, Dunn Kenneth W, Dagher Pierre C
Departments of Medicine,
Departments of Medicine.
J Am Soc Nephrol. 2017 Aug;28(8):2420-2430. doi: 10.1681/ASN.2016101153. Epub 2017 Mar 1.
In the live animal, tissue autofluorescence arises from a number of biologically important metabolites, such as the reduced form of nicotinamide adenine dinucleotide. Because autofluorescence changes with metabolic state, it can be harnessed as a label-free imaging tool with which to study metabolism Here, we used the combination of intravital two-photon microscopy and frequency-domain fluorescence lifetime imaging microscopy (FLIM) to map cell-specific metabolic signatures in the kidneys of live animals. The FLIM images are analyzed using the phasor approach, which requires no prior knowledge of metabolite species and can provide unbiased metabolic fingerprints for each pixel of the lifetime image. Intravital FLIM revealed the metabolic signatures of S1 and S2 proximal tubules to be distinct and resolvable at the subcellular level. Notably, S1 and distal tubules exhibited similar metabolic profiles despite apparent differences in morphology and autofluorescence emission with traditional two-photon microscopy. Time-lapse imaging revealed dynamic changes in the metabolic profiles of the interstitium, urinary lumen, and glomerulus-areas that are not resolved by traditional intensity-based two-photon microscopy. Finally, using a model of endotoxemia, we present examples of the way in which intravital FLIM can be applied to study kidney diseases and metabolism. In conclusion, intravital FLIM of intrinsic metabolites is a bias-free approach with which to characterize and monitor metabolism , and offers the unique opportunity to uncover dynamic metabolic changes in living animals with subcellular resolution.
在活体动物中，组织自发荧光源于多种具有重要生物学意义的代谢物，如还原型烟酰胺腺嘌呤二核苷酸。由于自发荧光会随代谢状态而变化，因此可将其用作一种无标记成像工具来研究代谢。在此，我们结合使用活体双光子显微镜和频域荧光寿命成像显微镜（FLIM）来绘制活体动物肾脏中细胞特异性代谢特征图谱。使用相量法分析FLIM图像，该方法无需事先了解代谢物种类，并且可以为寿命图像的每个像素提供无偏代谢指纹。活体FLIM显示，S1和S2近端小管的代谢特征在亚细胞水平上是不同且可分辨的。值得注意的是，尽管S1和远端小管在形态和传统双光子显微镜下的自发荧光发射存在明显差异，但它们表现出相似的代谢谱。延时成像揭示了间质、尿腔和肾小球区域代谢谱的动态变化，而这些区域是传统的基于强度的双光子显微镜无法分辨的。最后，我们以内毒素血症模型为例，展示了活体FLIM可用于研究肾脏疾病和代谢的方式。总之，对内在代谢物进行活体FLIM是一种无偏差的方法，可用于表征和监测代谢，并提供了以亚细胞分辨率揭示活体动物动态代谢变化的独特机会。
