1] Department of Bioengineering, Stanford University, Stanford, California, USA. [2] Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.
1] Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA. [2] Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.
Nat Methods. 2014 May;11(5):572-8. doi: 10.1038/nmeth.2888. Epub 2014 Mar 16.
A method for non-invasive visualization of genetically labeled cells in animal disease models with micrometer-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the 'optical window' above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence than mNeptune, whereas the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts into myocytes in living mice with high anatomical detail.
一种具有亚微米级分辨率的非侵入性可视化动物疾病模型中基因标记细胞的方法将极大地促进基于细胞的治疗方法的发展。使用 600nm 以上的“光学窗口”中的红色激发光来对荧光蛋白（FPs）进行成像，是一种潜在的可视化植入细胞的方法。然而，之前为了将激发峰设计到 600nm 以上而对 FPs 进行工程改造的尝试，导致了亮度的不可取降低。在这里，我们报告了通过对 mNeptune 进行结构引导的突变获得的三种新的可被红光激发的单体 FP，其中两种，mNeptune2 和 mNeptune2.5，表现出比 mNeptune 更好的成熟度和更亮的荧光，而第三种，mCardinal，则具有红移的激发光谱，而亮度没有降低。我们表明，mCardinal 可用于非侵入性和纵向可视化活小鼠中肌母细胞向心肌细胞的分化，具有高解剖细节。
